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Biomedical subjects

F Dorner

Publications and source records attributed to F Dorner.

At least 73 records · Page 4Linked to original sources
PubMed1996

Overexpression of human prothrombin in permanent cell lines using a dominant selection/amplification fusion marker.

Human prothrombin was overexpressed in transformed eukaryotic cells using a dominant bifunctional selection and amplification marker. The marker consists of the murine wild-type dihydrofolate reductase (dhfr) cDNA and the Escherichia coli hygromycin phosphotransferase gene fused in frame. The gene of interest is connected by the encephalomyocarditis virus 5' untranslated region to the fusion marker gene, forming a dicistronic transcription unit. The human prothrombin gene (FII) was used to monitor expression after initial selection for hygromycin B resistance and DHFR activity. In Chinese hamster ovary (CHO) cells, 5-15 mU prothrombin/10(6) cells per 24 h was obtained; in human 293 kidney cells levels of 20-50 mU/ 10(6) cells per 24 h were obtained. Methotrexate-mediated amplification of the foreign gene in CHO cells resulted in a more than 10-fold increase in FII expression, while in the presence of methotrexate, 293 cells expressed 200-250 mU/10(6) cells per 24 h. The use of this fusion marker within a dicistronic transcription unit allowed efficient dominant selection of cell clones and amplification of the gene of interest. Stably transfected cell lines that were able to secrete high levels of processed gamma-carboxylated human prothrombin were thus obtained.

Animals↗
PubMed1996

Comparison of N-Glycan Pattern of Recombinant Human Coagulation Factors II and IX Expressed in Chinese Hamster Ovary (CHO) and African Green Monkey (Vero) Cells.

The N-glycan patterns of recombinant human coagulation factors II (rF-II) and IX (rF-IX), derived from both transfected Chinese hamster ovary (CHO) cells and African green monkay (Vero) cells produced at industrial scale, were analyzed by binding to carbohydrate-specific lectins and were compared with the glycan structure of human plasma-derived coagulation factors. Human plasma-derived coagulation factors II (hpF-II) and IX (hpF-IX) exhibited complex-type glycan structures with carbohydrate chains capped with alpha(2-6)-sialic acid. Terminal galactose-beta(1-4)-N-acetylglucosamine units were detected in hpF-IX. Both CHO cell-derived rF-II and rF-IX exhibited complex-type glycosylation and contained alpha(2-3)-sialic acid in addition to terminal galactose-beta(1-4)-N-acetylglucosamine. Vero cell-derived rF-IX exhibited a complex-type glycan structure similar to that of CHO cell-derived rF-IX. In contrast, rF-II produced by Vero cells exhibited a glycan microheterogeneity composed of hybrid-type glycosylation containing "high-mannose" structures and complex-type glycosylation containing alpha(2-3)-sialic acid. Galactose-beta(1-4)-N-acetylglucosamine structures and a low concentration of alpha(2-6)-sialic acid were detected in both microheterogeneity fractions of Vero cell-derived rF-II. Although different in their carbohydrate structures, coagulation factors II and IX obtained recombinantly from both transformed CHO cells and Vero cells exhibited coagulation activities comparable with the plasma-derived proteins.

Journal Article↗
PubMed1996

A sensitive PCR assay system for the quantitation of viral genome equivalents: hepatitis C virus (HCV).

A sensitive and reliable quantitative method has been developed for the detection and quantitation of hepatitis C virus (HCV) target sequences. In this procedure, termed "laser induced fluorescence PCR' (LIF-PCR), reverse transcriptase PCR (RT-PCR) is performed and the PCR products are detected and quantified by laser-induced fluorescence. Precise quantitation of the viral target sequences is accomplished by the use of two calibrators that are amplified by the same set of primers as the target template. A high degree of reliability is achieved by co-processing, co-amplification and co-detection of the calibrators, together with the nucleic acid to be determined. Genome equivalents of HCV containing biological samples, including samples from international test panels, were accurately quantitated with this procedure.

Calibration↗
PubMed1996

Evaluation of the thymidine kinase (tk) locus as an insertion site in the highly attenuated vaccinia MVA strain.

The highly attenuated 'modified vaccinia Ankara' (MVA) strain is a potential live vaccine vector. Insertional inactivation of the tk-gene resulted in viruses difficult to purify. Co-integration of a functional fowlpox virus tk-gene allowed easy generation of recombinants, indicating that the genetically stable tk-gene region is a suitable insertion site, if tk-gene activity is substituted.

Animals↗
PubMed1996

Differentiation between proteolytic activation and autocatalytic conversion of human prothrombin. Activation of recombinant human prothrombin and recombinant D419N-prothrombin by snake venoms from Echis carinatus and Oxyuranus scutellatus.

Recombinant human prothrombin (r-prothrombin) and recombinant mutant prothrombin with active site Asp419 substituted by Asn (D419N-prothrombin) were expressed in recombinant CHO cells, isolated and purified from the fermentation supernatant. The r-Prothrombin and D419N-prothrombin were digested by both Echis carinatus venom and Oxyuranus scutellatus venom. Prior to, during and after activation, generation of thrombin activity and the proteolytic degradation of the prothrombin polypeptide chain were analysed. Owing to the recombinant preparation and inactivity of D419N-prothrombin and its activation products, the proteolytic action of E.carinatus and O.scutellatus venoms could be studied without addition of thrombin inhibitor, without interference from autocatalytic digestion of prothrombin and in the absence of any other blood coagulation protease. The comparison between the activation of r-prothrombin and D419N-prothrombin by snake venoms permitted differentiation between proteolytic activation and autocatalytic conversion of prothrombin. Incubation of D419N-prothrombin with E.carinatus venom resulted in the generation of stable D419N-meizothrombin by hydrolysis of the peptide bond Arg320-Ile321. By contrast, O.scutellatus venom exhibited activity towards peptide bonds Arg320-Ile321 and Arg271-Thr272 and lower activity towards peptide bond Arg155-Ser156, thus converting D419-prothrombin into D419N-thrombin and also liberating Fragment-1, Fragment-2 and Fragment-1/2 activation peptide. Activation of r-prothrombin by E.carinatus and O.scutellatus venoms demonstrated the autocatalytic potential of prothrombin-derived molecules and indicated that meizothrombin hydrolysed the cleavage between Fragment-2 and thrombin A-chain in the meizothrombin molecule, but not in prothrombin, preferentially at position Arg284-Thr285. By contrast, both meizothrombin and thrombin exhibited no detectable activity towards peptide bond Arg320-Ile321 between thrombin A- and B-chain, although this site exhibits the optimum sequence for thrombin cleavage.

Amino Acid Sequence↗
PubMed1996

Production of highly homogeneous and structurally intact recombinant von Willebrand factor multimers by furin-mediated propeptide removal in vitro.

Recombinant human von Willebrand Factor (rvWF), a multimeric glycoprotein essential to haemostasis, has been developed as a potential therapeutic agent for treatment of von Willebrand disease (vWD). Permanent Chinese-hamster ovary (CHO)-rvWF cell clones co-expressing recombinant furin (rfurin) were established in order to ensure complete rvWF propeptide removal [Fischer, Schlokat, Mitterer, Reiter, Mundt, Turecek, Schwarz and Dorner (1995) FEBS Lett. 375, 259-262]. Large quantities of material are required for in vivo tests and clinical studies. This demand is commonly met by achieving high-yield expression of the desired protein via amplification. Co-amplification of rfurin, necessary to completely process increasing amounts of rvWF precursor, could not be accomplished, presumably due to lethal effects of overexpressed rfurin for the host cells [Creemers (1994) Ph.D. Thesis, University of Leuven]. Recent reports have inferred that rfurin can only mediate rvWF processing intracellularly [Rehemtulla and Kaufman (1992) Blood 79, 2349-2355; Rehemtulla, Dorner and Kaufman (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8235-8239]. We report here that rvWF-precursor processing, however, occurs predominantly extracellularly upon rfurin co-expression. Mixing experiments employing rfurin- as well as rvWF-precursor-containing conditioned media demonstrate that rvWF precursors are accessible and cleavable by rfurin in vitro. Exposure to rfurin in vitro converts the heterogeneous multimer pattern typical of incompletely processed rvWF multimers into highly homogeneous and structurally intact multimers superior to the ones exhibited by plasma-derived vWF. These findings thus demonstrate the feasibility of large-scale production of a completely processed, intact and homogeneous rvWF preparation, based on individual rvWF-precursor high-yield expression and subsequent propeptide removal by rfurin in vitro.

Animals↗
PubMed1995

Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing recombinant furin.

Thorough analysis of multimer composition and molecular structure of recombinant von Willebrand factor (r-vWF) produced by recombinant CHO cells demonstrated r-vWF to be more intact and less proteolytically degraded than plasma-derived vWF (pd-vWF) [B. Fischer et al. (1994) FEBS Lett. 351, 345-348]. In contrast to pd-vWF, r-vWF preparations consisted of pro-vWF (vWF containing covalently attached propeptide) as well as mature vWF subunits forming homo- and hetero-multimers. In order to ensure complete propeptide processing, a r-vWF-producing CHO cell clone was transfected with the cDNA of the human propeptide processing enzyme Furin. A r-vWF/r-Furin co-expressing cell clone was cultivated at industrial scale in high cell density perfusion fermenters. r-vWF obtained from these cells was fully processed. Analysis of r-vWF by multimer analysis revealed a multimer pattern equal in number of high molecular weight multimer to pd-vWF, but absence of satellite bands. Two-dimensional electrophoretic analysis of both the primary dimer and the complete multimer pattern of r-vWF showed that the recombinant coagulation factor was composed exclusively of intact and mature subunits. Since the triplet structure typical to pd-vWF is known to reflect proteolytic degradation, r-vWF thus exhibits an integrity far superior compared to pd-vWF.

Animals↗
PubMed1995

Purification of recombinant human coagulation factors II and IX and protein S expressed in recombinant Vaccinia virus-infected Vero cells.

Recombinant human coagulation factors II and IX and recombinant human protein S were expressed by the Vaccinia virus expression system in Vero cells and isolated from the serum-free media. All three recombinant proteins were purified by the same universal strategy. After concentration by anion exchange chromatography, recombinant coagulation factors were purified by anion exchange filtration in the presence of Ca2+. The addition of Ca2+ to the vitamin K-dependent coagulation factors modified their physico-chemical behavior during anion exchange chromatography and resulted in the separation of the recombinant coagulation proteins from contaminating proteins.

Animals↗
PubMed1995

Requirements for optimal expression of secreted and nonsecreted recombinant proteins in vaccinia virus systems.

Selection of an optimal promoter is necessary for efficient expression of foreign genes with vaccinia virus. Since a variety of powerful (homologous) vaccinia virus promoters and foreign (heterologous) promoter systems have been described for use in vaccinia, we have addressed the question of whether a general rule exists that allows the prediction of the optimal promoter/gene combination. We have compared the expression properties of four secreted proteins, the human blood clotting factor IX (FIX), the human blood glycoprotein Protein S (ProtS), the human von Willebrand factor (vWF), and the Hepatitis B virus (HBV) middle surface glycoprotein preS2, with proteins that were reported not to be secreted, the HBV large surface glycoprotein preS1 and the murine leukemia virus (MuLV) BM-5 Eco gag protein. In addition, we have included in our study an internal control protein, the vaccinia virus p11 protein, to monitor possible side effects of the promoter system used. Genes encoding the foreign proteins were placed either under control of a synthetic vaccinia virus early/late promoter (selP) or under control of the bacteriophage T7 promoter (T7/emc system). The secreted proteins were more efficiently expressed when fused to the homologous promoter. Direct comparison of the two promoters indicated that the expression level ranged between 1.4 (ProtS) and 3.9 (FIX)-fold higher with the selP than with the T7 promoter. In contrast, the cell-associated HBV preS1 was more efficiently expressed under the T7 promoter and the MuLV BM-5 Eco gag polypeptide was expressed equally well from both promoters. These data indicate that a careful prediction of optimal promoter/foreign gene combinations for the vaccinia virus expression system is possible. The choice of the optimal promoter/expression system is based on a simple classification scheme, discriminating secreted and nonsecreted proteins.

Animals↗
PubMed1995

Tick-borne encephalitis virus envelope protein E-specific monoclonal antibodies for the study of low pH-induced conformational changes and immature virions.

A set of ten monoconal antibodies (mabs) specific for the tick-borne encephalitis (TBE) virus envelope protein E were prepared and characterized with respect to their functional activities, the location of their binding sites on protein E and the involvement of their epitopes in acid pH-induced conformational changes and interactions with the precursor to the membrane protein (prM) in immature virions. The majority of these mabs mapped to the previously defined antigenic domain A. All of the mabs recognize parts of the E protein which undergo low pH-induced structural rearrangements believed to be necessary for the fusion activity of the virus, and six of the mabs define epitopes which are affected by the prM-E interaction in immature virions. They are therefore of potential value as specific reagents for studying the structure and function of protein E, as well as the function of the prM-E association. Five of the mabs exhibited neutralizing activity, and can therefore be used for the selection of escape mutants.

Animals↗
PubMed1995

Immunization of chimpanzees with recombinant gp160, but not infection with human immunodeficiency virus type 1, induces envelope-specific Th1 memory cells.

The human immunodeficiency virus (HIV) type 1 envelope protein (recombinant [r] gp160)-induced T cell lymphokine release pattern of chimpanzees immunized with HIVIIIB rpg160 tested and compared with rpg160-induced lymphokine releases of T cells from unimmunized, HIV-1-infected chimpanzees. The results showed that infection of chimpanzees with HIV-1 did not induce rgp160-specific memory T cells (as evidenced by the lack of Th1 and 2 type lymphokine releases after rgp160 stimulation). In contrast, T cells of rgp160-immunized chimpanzees released Th1 type lymphokines upon stimulation with rgp160 of HIVIIIB, HIVMN, and HIVRF. release was comparable whether chimpanzees were immunized with rgp160 only or also challenged with HIV-1 and protected or not protected. Thus, rgp160 immunization leads to generation of Th1 type memory cells. Whether Th1 type responses contribute to protection against HIV-1 infection has yet to be clarified.

Animals↗
PubMed1995

A novel vaccinia virus expression system allowing construction of recombinants without the need for selection markers, plasmids and bacterial hosts.

Vaccinia virus is one of the most widely applied expression systems for use in eukaryotes in molecular biology. Expression of heterologous genes in the vaccinia virus system, however, requires integration of the foreign DNA into the vaccinia virus genome by means of homologous recombination or by direct molecular cloning. In both cases, plasmid vector constructs are required that contain the gene of interest and, usually, a marker gene, both of which are controlled by suitable promoter sequences. In order to simplify the construction of recombinants and to eliminate the need for a marker gene we have developed a modified vaccinia virus genome that allows the direct targeted insertion of DNA fragments downstream of a strong vaccinia virus promoter without any further cloning steps. The gene of interest is amplified by PCR using oligonucleotide primers that provide an SfiI site at the 5' end and an RsrII site at the 3' end of the PCR product. Following digestion with these restriction enzymes, the PCR product is operationally linked to a synthetic early/late promoter within the viral genomic DNA via the unique SfiI/RsrII sites of the modified vaccinia virus genome. Using this approach, intermediate plasmid constructs and bacterial hosts are not required and time consuming screening steps can be omitted, because of 90% of the virus progeny carry the foreign DNA.

Base Sequence↗
PubMed1995

Evidence for lateral transfer and recombination in OspC variation in Lyme disease Borrelia.

The ospC gene was amplified by the polymerase chain reaction from each of 76 Lyme disease Borrelia strains. Restriction fragment length polymorphism (RFLP) analysis demonstrated 33 distinct RFLP types; two additional RFLP types were identified from published ospC sequences. For each RFLP type, at least one ospC gene was sequenced and the degree of sequence relatedness examined by construction of an ospC gene tree. The genes were extremely diverse, with sequence identity ranging from 74.4% to 99.0%; the majority of changes are localized within the central portion of the molecule. A comparison of ospC sequences suggests that recombination occurs frequently between ospC alleles; this genetic exchange is proposed to be mediated by lateral transfer of ospC sequences. Evidence indicates that recombination occurs between ospC genes from the same Borrelia species (i.e. B. afzelii and B. garinii) as well as between different Borrelia species (i.e. B. afzelii and B. garinii, B. burgdorferi and genogroup DN127).

Amino Acid Sequence↗
PubMed1995

Parenteral application of a Pseudomonas aeruginosa flagella vaccine elicits specific anti-flagella antibodies in the airways of healthy individuals.

To test the hypothesis that a Pseudomonas aeruginosa flagella (FLA) vaccine, intramuscularly administered, elicits specific antibodies in the respiratory tract, antibody titers against FLA were determined in sera and bronchoalveolar lavage (BAL) fluids of 10 healthy human adults before and after immunization. Immunization produced significantly increased anti-FLA antibody titers in all sera (mean reciprocal titers: IgG: 26,711; IgA: 2,767; IgM: 421) and on respiratory epithelial lining fluids (mean reciprocal titers: IgG: 112; IgA: 76; sIgA: 26). Significant differences in class-specific serum and BAL anti-FLA titers suggested airway antibody production. The study shows that parenteral immunization provides not only high systemic antibody titers but also local antibody production in the human respiratory tract.

Adult↗
PubMed1994

Structural analysis of recombinant von Willebrand factor: identification of hetero- and homo-dimers.

Wild-type full-length cDNA of von Willebrand factor (vWF) was expressed in CHO cells. Recombinant vWF (rvWF) was obtained and its molecular composition investigated by two-dimensional electrophoresis. Results of first dimension electrophoresis under non-reducing conditions showed that rvWF-dimer represents a mixture of three different species. Second dimension electrophoresis under reducing conditions revealed, that these protein species represent (i) homo-dimers of either two unprocessed or two fully processed, mature vWF polypeptides, and (ii) the hetero-dimer of unprocessed and mature rvWF monomers. Compared with vWF-dimers from human plasma, which contained predominantly proteolytically degraded polypeptides, recombinant vWF-dimers were shown to consist of non-proteolyzed subunits only.

Animals↗
PubMed1994

Mapping of antigenic and immunogenic sites of Haemophilus influenzae outer membrane protein P6 using synthetic lipopeptides.

The immune response to the P6 protein of Haemophilus influenzae was characterized with 24 synthetic icosapeptides and 45 dodecapeptides conjugated to the immune stimulator N-palmitoyl-S-[2,3-(bispalmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-( S)-serine . The antigenicity of these lipopeptides was investigated by enzyme-linked immunosorbent assays with rabbit anti-P6 serum. The epitopes of P6 protein were identified and localized within residues 31-46 and 59-70 and in the C-terminal part of the P6 protein. Mice were immunized with lipoicosapeptides without using additional adjuvants or carriers and the antibody titers were measured with isolated P6 protein and lipopeptides in a dot blot assay. Lipopeptides containing the sequence pattern QILDAHAA (P6 47-54) and the mouse B cell epitope GEYV (P6 43-46) induced high titers of anti P6 antibodies. These murine antibodies were able to neutralize the intact bacterium Haemophilus influenzae.

Amino Acid Sequence↗
PubMed1993

Vaccines in AIDS. Present state of knowledge.

The identification of a virus, HIV-1, as the etiological agent of AIDS has opened the door to the development of a vaccine against this disease, for both prophylactic and therapeutic purposes. A number of different vaccine strategies have been developed in pursuit of this aim. The first major advances in this direction were the reports that some candidate vaccines could prevent infection of chimpanzees with HIV-1. Human trials have now been initiated in HIV-1 seronegative and seropositive individuals and the details of the vaccine strategies involved and the results obtained to date are described.

AIDS Vaccines↗