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Biomedical subjects

M Fabry

Publications and source records attributed to M Fabry.

At least 19 recordsLinked to original sources

Reptile trade and the risk of exotic tick introductions into southern South American countries.

Ticks exotic for the Neotropical region were found on Python regius imported into Argentina and Chile. All ticks (7 males and 3 females) were classified as Amblyomma latum Koch, 1844 ( = Aponomma latum). Additionally, four lots comprising 18 males of the Argentinean tortoise tick, Amblyomma argentinae Neumann, 1904, were found on a terrestrial tortoise, Chelonoidis chilensis, and on three terrestrial tortoises (probably C. chilensis) imported to Uruguay presumably from Argentina). These findings alert us to the risk of expanding the distribution of reptile parasites and their diseases into regions previously free of these parasites.

Animals↗

Hormone-triggered conformational changes within the insulin-receptor ectodomain: requirement for transmembrane anchors.

Interaction between two alphabeta half-receptors within the (alphabeta)(2) holoreceptor complex is required for insulin binding with high affinity and for insulin-triggered changes of size and shape. To understand the underlying structure-function relationship, two truncated receptor constructs have been characterized. Reduction in the Stokes radius and increase in the sedimentation coefficient, which are characteristic for wild-type receptors, were entirely lacking for the recombinant human insulin receptor (HIR) ectodomain (HIR-ED). Stokes radii of about 5.8 nm and sedimentation coefficients of 10.2 S were found for both insulin-bound and free HIR-EDs. However, attaching the membrane anchors to the ectodomain, as with the recombinant membrane-anchored ectodomain (HIR-MAED) construct, was sufficient to restore not only high-affinity hormone binding but also the marked insulin-inducible alterations in hydrodynamic properties. The Stokes radii of HIR-MAED complexes, as assessed by non-denaturing PAGE, decreased upon insulin binding from 9.5 nm to 7.9 nm. In parallel, the sedimentation coefficient was increased from 9.0 S to 9.8 S. CD and fluorescence spectroscopy of HIR-MAED revealed only minor insulin-induced changes in the secondary structure. Similarity with wild-type receptors has also been demonstrated by the differential insertion of insulin-bound and free HIR-MAED complexes into artificial bilayer membranes of Triton X-114. The results are consistent with a model of receptor function that ensures a global insulin-triggered reorientation of subdomains within the ectodomain moieties while the secondary structure is essentially retained. For the rearrangement of such subdomains, the transmembrane anchors confer essential structural constraints on the receptor ectodomain.

Chromatography, Gel↗

Structural basis of HIV-1 and HIV-2 protease inhibition by a monoclonal antibody.

BACKGROUND: Since the demonstration that the protease of the human immunodeficiency virus (HIV Pr) is essential in the viral life cycle, this enzyme has become one of the primary targets for antiviral drug design. The murine monoclonal antibody 1696 (mAb1696), produced by immunization with the HIV-1 protease, inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides that include the N terminus of the enzyme, a region that is highly conserved in sequence among different viral strains and that, furthermore, is crucial for homodimerization to the active enzymatic form. RESULTS: We report here the crystal structure at 2.7 A resolution of a recombinant single-chain Fv fragment of mAb1696 as a complex with a cross-reactive peptide of the HIV-1 protease. The antibody-antigen interactions observed in this complex provide a structural basis for understanding the origin of the broad reactivity of mAb-1696 for the HIV-1 and HIV-2 proteases and their respective N-terminal peptides. CONCLUSION: A possible mechanism of HIV-protease inhibition by mAb1696 is proposed that could help the design of inhibitors aimed at binding inactive monomeric species.

Antibodies, Monoclonal↗

Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human beta-globin in engrafted mice.

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human beta-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked beta-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human beta-globin and the green fluorescent protein in 20-95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of beta-locus control region hypersensitive site 2 alone, human beta-globin mRNA expression levels ranged from 0.15% to 20% with human beta-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

Animals↗

Photoreactive insulin derivatives for the detection of the doubly labeled insulin receptor.

Two different photoinsulins, the radioactive N(epsilonB29)-(4-azidosalicyloyl) insulin and the novel biotinylated N(epsilonB29)-(4-azidotetrafluorobenzoyl-biocytinyl) insulin, were synthesized in order to study the binding stoichiometry of insulin and the insulin receptor in a direct approach. Both derivatives were cross-linked simultaneously to the (alphabeta)(2) receptor. Insulin-receptor conjugates were formed that carry a radioactive label as well as a biotin label. The doubly labeled complexes were isolated by streptavidin-affinity chromatography. Analysis of both markers, the radioactive (125)I marker and the biotin marker, proved the existence of a covalent complex of one receptor molecule and two ligands. Thus, orthogonal photocross-linking is introduced as a method for the isolation and analysis of bivalent receptor complexes.

Azides↗

Differently labeled peptide ligands for rapid investigation of receptor expression on a new human glioblastoma cell line.

The neuropeptides vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and substance P (SP) as well as insulin and insulin-like growth factor 1 (IGF-1) were labeled with biotin, fluorescent dyes, and radioactivity to characterize the expression of peptide receptor of a novel cancer cell line, established from a human glioblastoma multiforme. Thus, not only binding sites could be detected but advantages and disadvantages of the different labels could be compared, too. With all three markers, the presence or absence of the receptors could be answered rapidly and sensitively. The glioblastoma cells express receptors for VIP (IC(50) = 9 nM +/- 30%), insulin (K(d) = 0.66 nM +/- 14%, B(max) = 0.028 nM +/- 13%), and IGF-1 (K(d) = 21 nM +/- 25%, B(max) = 1.65 nM +/- 24%), but there are no binding sites for NPY and SP. As especially VIP and IGF-1 receptors are expressed in huge amounts, these receptors might be an interesting target for tumor diagnostics and therapy.

Binding Sites↗

Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK-N-MC.

Neuropeptide Y (NPY) is an important neuromodulator in the central and peripheral nervous system. The peptide acts through different NPY receptor subtypes (Y1-Y5, y6) that belong to the family of G protein-coupled receptors. In general, cellular responses to prolonged exposure to agonists of G protein-coupled receptors are attenuated, often through internalization of the receptors and their bound ligands. In this study, a fluorescent labeled NPY derivative was synthesized and characterized to investigate the internalization of NPY in the human neuroblastoma cell line SK-N-MC. Internalization was proven by binding experiments and subsequent acidic washing as well as by direct visualization by means of confocal laser scanning microscopy. Approximately 20-30% of the fluorescent labeled NPY and a tritium-marked NPY were resistant to acid removal of cell surface-bound ligands indicating internalization. Extracellular fluorescent labeled NPY was found to be distributed heterogeneously in a clustered pattern, which suggests that the ligand-receptor complex is collected in pits and caveolae followed by endocytosis.

Amino Acid Sequence↗

Quantitative Assessment of Protein Adsorption by Combination of the Enzyme-Linked Immunosorbent Assay with Radioisotope-Based Studies.

Protein adsorption at polymer surfaces has been investigated by means of both ELISA and radiolabeling techniques. Most of the data obtained are linearly related to each other for protein concentrations between 0.01 and 1 µg/ml, i.e., the concentration range in which the maximum amount of adsorbed active protein (ELISA) is achieved. The correlation of ELISA data with radioisotope-based measurements allows quantification of the former. Specific correlation factors are described. Adsorption is shown to be strongly dependent on the polymer/protein system. Copyright 1999 Academic Press.

Journal Article↗

Inhibition of the HIV-1 and HIV-2 proteases by a monoclonal antibody.

The monoclonal antibody 1696, directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates. This antibody cross-reacts with peptides that include the N-terminus of the enzyme, a region that is well conserved in sequence among different viral strains and which, furthermore, is crucial for homodimerization to the active enzymatic form. This observation, as well as antigen-binding studies in the presence of an active site inhibitor, suggest that 1696 inhibits the HIV protease by destabilizing its active homodimeric form. To characterize further how the antibody 1696 inhibits the HIV-1 and HIV-2 proteases, we have solved the crystal structure of its Fab fragment by molecular replacement and refined it at 3.0 A resolution. The antigen binding site has a deep cavity at its center, which is lined mainly by acidic and hydrophobic residues, and is large enough to accommodate several antigen residues. The structure of the Fab 1696 could form a starting basis for the design of alternative HIV protease-inhibiting molecules of broad specificity.

Amino Acid Sequence↗

Structure of HOE/BAY 793 complexed to human immunodeficiency virus (HIV-1) protease in two different crystal forms--structure/function relationship and influence of crystal packing.

Human immunodeficiency virus 1 (HIV-1) protease is a prime target in the search for drugs to combat the AIDS virus. The enzyme functions as a C2-symmetric dimer, cleaving the gag and gag-pol viral polyproteins at distinct sites. The possession of a twofold axis passing through the active site, has led to the design of C2-symmetrical inhibitors in the form of substrate-based transition-state analogs. One of the most active compounds of this class of inhibitors is HOE/BAY 793, which contains a vicinal diol central unit [Budt, K.-H., Hansen, J., Knolle, J., Meichsner, C., Paessens, A., Ruppert, D. & Stowasser, B. & Winkler, I. (1990) European Patent application EP0428,849; Budt, K.-H., Hansen, J., Knolle, J., Meichsner, C., Ruppert, D., Paessens, A. & Stowasser B. (1993) IXth International Conference on AIDS; Budt, K.-H., Peyman, A., Hansen, J., Knolle, J., Meichsner, C., Paessens, A., Ruppert, D. & Stowasser, B. (1995) Bioorg. Med. Chem. 3, 559-571.] The structure of this inhibitor bound to HIV-1 protease, in two different crystal forms, has been solved at 0.24-nm resolution using X-ray crystallography. In both forms, the details of the inhibitor-protease interactions revealed an overall asymmetric binding mode, especially between the central diol unit and the active-site aspartates. The main binding interactions comprise several specific H-bonds and hydrophobic contacts, which rationalize many of the characteristics of the structure/activity relationship in the class of vicinal diol inhibitors. In a general analysis of the mobility of the flap regions, which cover the active site and participate directly in binding, using our structures and the HIV protease models present in the Brookhaven databank, we found that in most structures the flexibility of the flaps is limited by local crystal contacts. However, in one of the structures presented here, no significant crystal contacts to the flap regions were present, and as a result the flexibility of the inhibitor bound flaps increased significantly. This suggests that the mobility and conformational flexibility of the flap residues are important in the functioning of HIV-1 protease, and must be considered in the future design of drugs against HIV protease and in structure-based drug design in general.

Crystallography, X-Ray↗

Three-dimensional structure of an Fab-peptide complex: structural basis of HIV-1 protease inhibition by a monoclonal antibody.

F11.2.32, a monoclonal antibody raised against HIV-1 protease (Kd = 5 nM), which inhibits proteolytic activity of the enzyme (K(inh) = 35(+/-3)nM), has been studied by crystallographic methods. The three-dimensional structure of the complex between the Fab fragment and a synthetic peptide, spanning residues 36 to 46 of the protease, has been determined at 2.2 A resolution, and that of the Fab in the free state has been determined at 2.6 A resolution. The refined model of the complex reveals ten well-ordered residues of the peptide (P36 to P45) bound in a hydrophobic cavity at the centre of the antigen-binding site. The peptide adopts a beta hairpin-like structure in which residues P38 to P42 form a type II beta-turn conformation. An intermolecular antiparallel beta-sheet is formed between the peptide and the CDR3-H loop of the antibody; additional polar interactions occur between main-chain atoms of the peptide and hydroxyl groups from tyrosine residues protruding from CDR1-L and CDR3-H. Three water molecules, located at the antigen-antibody interface, mediate polar interactions between the peptide and the most buried hypervariable loops, CDR3-L and CDR1-H. A comparison between the free and complexed Fab fragments shows that significant conformational changes occur in the long hypervariable regions, CDR1-L and CDR3-H, upon binding the peptide. The conformation of the bound peptide, which shows no overall structural similarity to the corresponding segment in HIV-1 protease, suggests that F11.2.32 might inhibit proteolysis by distorting the native structure of the enzyme.

Amino Acid Sequence↗

Mutagenesis, biochemical characterization and X-ray structural analysis of point mutants of bovine chymosin.

Chymosin B point mutants, A115T and G243D (chymosin A), were expressed in Escherichia coli and Trichoderma reesei respectively, characterized biochemically, crystallized and studied by X-ray analysis at 2.3 and 2.8 angstroms resolutions respectively. The three-dimensional structures showed that the mutations gave rise to local conformational changes only when compared with that of chymosin B. Kinetic analysis of the A115T mutant with a six residue synthetic peptide revealed a reduction in Km with respect to the wild type, possibly caused by the small local changes in the vicinity of S1 and S3. Although, kinetic analyses of the G243D mutant using the short substrate showed reduced catalytic activity, use of a 15 residue substrate based on residues 98-112 of kappa-casein, the natural substrate, revealed an increase in the kcat compared with chymosin B, probably a consequence of the charge introduced that may interact with the substrate between P4 and P8.

Amino Acid Sequence↗

Preliminary crystallographic studies of an anti-HIV-1 protease antibody that inhibits enzyme activity.

F11.2.32, a monoclonal antibody directed against the HIV-1 protease, displays strong inhibitory effects toward the catalytic activity of the enzyme. The antibody cross-reacts with peptides 36-46 and 36-57 from the protease. Crystals of the Fab have been obtained both in the free state and as complexes formed with the protease peptide fragments, 36-46 and 36-57. Diffraction data have been collected for the free and complexed forms of Fab F11.2.32 and preliminary models for the crystal structures were obtained by molecular replacement.

Animals↗

Potent photoaffinity labelled and iodinated antagonists of bradykinin.

Continuing the studies on photoaffinity labelled analogues of the peptide hormone bradykinin (BK), several labelled antagonists were synthesized and characterized regarding their biological activities on rat uterus (RUT) and guinea pig ileum (GPI). The photoreactive amino acid p-benzoyl-phenylalanine (Bpa) was incorporated in potent, iodinated BK analogues at positions -2, -1, 0 and 7. The newly synthesized BK antagonists were derived from HOE 140 ([DArg0, Hyp3, Thi5, D-Tic7, Oic8]-BK) or [D-Phe7]-BK. Because the application of Bpa requires an additional group for the introduction of 125I, iodinated tyrosine was inserted at different positions as a model for radioiodination. Suitable positions for incorporation of tyrosine residues are -1, 0, 3 and 7, whereas the compound with 3-I-Tyr at position 4 had only a low biological activity. The antagonists obtained by modification of HOE 140 generally retained a high antagonistic potency. In this group [D-Bpa-2, 3-I-D-Tyr-1, D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-BK (pA2 values 8.06 on RUT and 8.15 on GPI) and [Bpa-1, D-Arg0, 3-I-Tyr3, Thi5, D-Tic7, Oic8]-BK (pA2 values 7.55 on RUT and 8.07 on GPI) belong to the most active compounds. The incorporation of D-Bpa at position 7 also resulted in potent analogues. The antagonists [3-I-Tyr-1, D-Arg0, D-Bpa7]-BK (pA2 on RUT 7.69) and [3-I-Tyr-1, D-Arg0, D-Bpa7, Oic8]-BK (pA2 on GPI 7.53) are an alternative to the N-terminal modified HOE 140 analogues. Compounds with D-Bpa7 act as pure competitive antagonists, whereas the HOE 140 derivatives show a mixed antagonism. The comparison of the results between photoaffinity labelled agonists and antagonists suggests that modifications in the series of BK antagonists were better tolerated.

Affinity Labels↗

Insulin as a target antigen in autoimmune diabetes: a natural repertoire as the source of antibody response.

A solid-phase immunoenzymatic technique with B1- or B29-biotinylated insulin coupled to avidin-coated wells was used to characterize serum anti-insulin antibodies and to locate insulin antibody-producing B lymphocytes in different organs of mice. Low natural serum anti-insulin IgM and IgG antibodies were found in ten different healthy inbred strains of mice. Prediabetic non-obese diabetic (NOD) mice had significantly higher measurements than BALB/c mice (P < 0.05). Anti-insulin IgM antibody-producing B lymphocytes were found in bone marrow and spleen of NOD mice and healthy strains of mice, but not in peripheral lymph nodes, thymus, blood or pancreas. B29-fixed insulin was more frequently recognized than B1-fixed insulin. There was no relationship to the MHC or to other immune markers. IgG insulin antibody-producing cells were not detected. IgG insulin antibody-producing cells appeared in the draining lymph node and in the blood 10 days after immunization with insulin. IgM insulin-recognizing cells in the spleen were reduced in number during the same period (P < 0.05-0.01 for BALB/c, DBA2, B10.D2 and NOD), suggesting migration of these cells. This was tested by in vivo staining of spleens with the red-fluorescent membrane linker PKH-26 on day 7 after immunization. Cells from immunized lymph nodes were FACS-sorted on day 10. Insulin antibody-producing B lymphocytes with red-fluorescence were found, indicating a splenic origin. Examination of IgG subclasses showed preferential production of complement-fixing IgG2b in sera and lymph node cells of immunized NOD mice (P < 0.05 vs BALB/c).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Myeloblastosis associated virus (MAV) proteinase site-mutated to be HIV-like has a higher activity and allows production of infectious but morphologically altered virus.

We have characterized the structure and infectivity of an avian retrovirus, myeloblastosis associated virus (MAV), containing a genetically altered proteinase (PR). A site-directed mutant of MAV-PR that shows an increased proteolytic activity in vitro (about 20 times higher kcat/Km) as a consequence of substituting five amino acids from the substrate-binding pocket with those corresponding to the HIV-1 PR was cloned into a full-sized MAV plasmid. In particular, the wild-type MAV-PR gene was replaced with the mutant one. Despite encoding for an enzyme with increased PR activity, mutant plasmid-transfected turkey fibroblasts displayed an unimpaired virus production in cell cultures. Further, the mutant progeny virus was infectious and its pattern of gag processing products appeared identical to that of wild-type virus. However, by electron microscopy we found that the predominant morphology of mutant viral particles was altered. Instead of a centrally collapsed avian retroviral core, a more diffuse core was visualized for wild-type mutant virions, similar to that observed in mammalian C-type retroviruses.

Alpharetrovirus↗