Biomedical subjects

M Ackermann

Publications and source records attributed to M Ackermann.

At least 55 records · Page 3Linked to original sources

PubMed1999

zfNLRR, a novel leucine-rich repeat protein is preferentially expressed during regeneration in zebrafish.

zfNLRR is a novel transmembrane protein that is most prominently expressed during regeneration of the zebrafish central nervous system. Retinal ganglion cells and descending spinal cord neurons strongly increase zfNLRR mRNA levels after axotomy in the adult. In contrast, during development expression is hardly detectable and is restricted to a few sensory systems. In the adult brain, zfNLRR mRNA is found at low levels in several motor and premotor systems. Sequence analysis reveals that zfNLRR contains in its extracellular region 12 leucine-rich repeats, 1 immunoglobulin-like domain and 1 fibronectin type III-like domain. The same protein binding motifs were identified in transmembrane proteins from frog, mouse, and human. Together, they constitute a novel family of vertebrate neuronal leucine-rich repeat proteins. Three distinct isoforms are identified so far. On the basis of its structural features and expression pattern, we propose that zfNLRR functions as a neuronal-specific adhesion molecule or soluble ligand binding receptor, primarily during restoration of the nervous system after injury.

Aging↗

PubMed1999

Step-by-step description of a rotary root canal preparation technique.

CLINICAL TECHNIQUE: Since the introduction of nickel-titanium in endodontics, several canal preparation techniques involving the use of rotary instruments have become popular. Such engine-driven rotary instruments rotate between 150 and 2000 r.p.m. and may be high or low torque orientated. This paper describes one such engine-driven system called the ProFile technique. The instruments are of a different specification to that used for conventional endodontic files and reamers. This paper describes a technique employed by the Division of Endodontology, Zurich Dental School, in a step-by-step procedure using primarily photographs of radiographs. The intention is to give any interested clinician a better idea of the technique using radiographs taken from both the buccolingual (clinical) perspective and the mesiodistal projection. Basically, the technique involves preparing the coronal portion of the root canal using Gates-Glidden burs and the ProFile instruments. Only when any constricting coronal parts of the canals have been removed is the working length established using conventional files. Finally, the apical part of the canal is prepared using only the ProFile instruments. Three clinical cases are also briefly described, in order to illustrate the potential of the technique in cases treated generally by clinicians.

Dental High-Speed Technique↗

PubMed1999

Immortalized cell lines derived from mice lacking both type I and type II IFN receptors unify some functions of immature and mature dendritic cells.

Cells with dendritic morphology obtained from several organs of mice lacking both type I and II IFN receptors were immortalized by a retrovirus and analysed for their phenotype and for their function to induce cognate immune responses in vitro and in vivo. Two cell lines called AG101 (skin) and AG116 (brain) were cloned and analysed in more detail. They constitutively expressed the cell surface markers CD45, CD11b, MHC class II, F4/80, N418, B7-2 and ICAM1 but were CD8- and B220-negative. Cells from both lines were capable of taking up ovalbumin (OVA). The processed protein was presented to the OVA-specific T cell hybridoma BO97.105 which responded specifically with the production of IL-2. AG101 and AG116 cells were able to induce a mixed lymphocyte reaction as shown by a 50-fold increase of IL-2 production over background. Naive T cells were stimulated by antigen-primed AG101 and AG116, resulting in a T cell proliferation which was 20-30 times over background, and in IL-2 production it was 10 times the background. The capacity of AG101 or AG116 cells to prime naive T cells was directly compared with freshly isolated and cultured cutaneous dendritic cells (DC) from 129 Sv/Ev mice (wtDC). After cognate T cell interaction, IL-6 (20-100-fold) and IL-12 p40 (100-1000-fold) were similarly up-regulated in either AG101, AG116 or mature wtDC. To analyse the capacity of the immortalized DC to induce antibodies in vivo, cell line AG116 was permanently infected with Borna disease virus (BDV) which is unable to replicate in adult mice. One hundred and twenty-nine Sv/Ev mice injected with different cell numbers of AG116 carrying BDV (but not control cells) produced antibodies against the viral BDVp40 and BDVp24 protein. Therefore, the cell lines AG101 and AG116 appear to unify some functions of immature and mature DC. They are able to pick up antigen and process it. In the absence of externally added cytokines, the antigen presented on AG101 or AG116 cells drives T cells with an efficiency similar to mature DC. The cloned cell lines may prove to be useful to study both immune response and replication of infectious agents in the absence of functional interferon receptors.

Animals↗

PubMed1999

Role of the individual interferon systems and specific immunity in mice in controlling systemic dissemination of attenuated pseudorabies virus infection.

The importance of each of the two interferon (IFN) systems in impeding herpesvirus replication and in stimulating virus-specific lymphocytes to control an acute systemic infection is not completely understood. To further our knowledge, pseudorabies virus, attenuated by deletion of the glycoprotein E gene to impair its neurovirulence and by deletion of the thymidine kinase gene (gE-TK-PRV), was used to infect wild-type 129Sv/Ev and congenic mice with immune system-associated genetic deficiencies. Mice with mature B and T lymphocytes but lacking either one or both functional receptors for members of each of the two IFN families were infected with gE-TK-PRV. At 3 and 7 but not 14 days after infection, replicating gE-TK-PRV could be isolated only from livers or spleens of mice lacking the receptors for both IFN families, and these mice survived the infection. Therefore, functional IFN receptors were not required to induce a protective immune response against an acute infection with gE-TK-PRV. Furthermore, PRV-specific antibodies of all immunoglobulin G isotypes were produced in these mice. Mice without mature B and T lymphocytes and lacking either one or both functional receptors for members of each of the two IFN families were also infected with gE-TK-PRV. Three days after infection, replicating virus could be isolated only from mice lacking both mature B and T lymphocytes and functional IFN receptors, and these mice were not able to clear the virus. We present evidence that mice with an intact gamma IFN system but without mature B and T cells were able to prevent systemic dissemination of gE-TK-PRV.

Animals↗

PubMed1998

Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors.

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only approximately 1% of the 152-kb HSV-1 genome, and consequently, replication and packaging into virions depends on helper functions. These helper functions have been provided conventionally by a helper virus, usually a replication-defective mutant of HSV-1, or more recently, by a set of five cosmids that overlap and represent the genome of HSV-1 deleted for DNA cleavage/packaging signals (pac). In the absence of pac signals, potential HSV-1 genomes that are reconstituted from the cosmids via homologous recombination are not packageable. The resulting amplicon stocks are, therefore, virtually free of contaminating helper virus. To simplify this packing system, the HSV-1 genome was cloned and maintained stably as a single-copy, F plasmid-based bacterial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 genome deleted for the pac signals (fHSV delta pac) did not generate replication-competent progeny virus on transfection into mammalian cells, but rather, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks had titers of up to 10(7) transducing units per milliliter of culture medium and efficiently transduced neural cells in the rat brain, as well as hepatocytes in the rat. The capacity of generating infectious and replication-competent HSV-1 progeny following transfection into mammalian cells was restored after insertion of a pac signal into fHSV delta pac.

Animals↗

PubMed1998

Indirect immobilization of recombinant proteins to a solid phase using the albumin binding domain of streptococcal protein G and immobilized albumin.

Immobilization of proteins to a solid phase leads to denaturation of the adsorbed molecules which may subsequently affect biological interactions. However, for many applications maintenance of the native structure is desired. Therefore, an indirect immobilization system was developed, based on binding of the albumin binding domain (ABP) of streptococcal protein G to rat serum albumin (RSA) precoated on a solid phase (RSA-microtiter plates). Escherichia coli vectors were adapted for production of recombinant protein fused to ABP and the 6 X His-tag. The expressed ABP tag was found to form homodimers. Plasmon resonance was used to study the interaction between an ABP fusion protein and immobilized RSA. Apparent on- and off-rates were calculated using a model for a bivalent analyte (k(a1) = 3.37 x 10(4) M(-1) s(-1), k(d1) = 1.23 x 10(-4) s(-1)). Thus, the stability of the ABP-RSA interaction can be explained by a slow off-rate. This was confirmed by chase experiments in an ELISA format. The ABP-RSA interaction remained stable after addition of different albumins. This immobilization system was used for the development of an ELISA to detect antibodies against Borna disease virus protein p40. The use of RSA-microtiter plates for indirect immobilization of ABP fusion protein was shown to be superior to direct adsorption on plastic. To obtain maximal antibody binding ten times less antigen was needed for indirect immobilization compared to direct adsorption. The binding capacity of the RSA-microtiter plates was determined to be about 0.8 pmol of monomeric ABP protein.

Adsorption↗

PubMed1998

Rabbit monoclonal Fab derived from a phage display library.

Rabbit monoclonal antibodies (RmAb) are not routinely obtained by eukaryotic cell fusion techniques. Therefore, we have applied phage display technology to produce a recombinant rabbit Fab molecule directed against the KLH model antigen. The Fab fragments selected from the rabbit phage display library were subcloned in an expression vector to permit the production of a fusion protein comprising a dimer of bacterial alkaline phosphatase (phoA). This fusion protein was directly produced into the periplasmic space of Escherichia coli. We show that a crude extract containing these conjugates can be used in a direct enzyme immunoassay, as exemplified in the case of the KLH antigen.

Alkaline Phosphatase↗

PubMed1998

Prokaryotic expression of porcine epidemic diarrhoea virus ORF3.

Wild type (wt) and cell culture adapted (ca) strains of the coronavirus PEDV differ in their ability to cause diarrhea in neonate piglets: the wt strains are virulent; the ca strains are attenuated. Comparison of the available nucleotide sequences obtained from the different viral isolates revealed almost complete sequence identity with the exception of variations and truncations in open reading frame 3 (ORF3) observed exclusively in ca-PEDV isolates. In order to study the biological function(s) of the putative ORF3 product, the molecule was expressed as a heterodimeric fusion protein in E. coli. ORF3 was fused in frame to the alkaline phosphatase gene. Simultaneously, the construct was designed to form specific heterodimers by inclusion of the well known leucine zipper motiv of Jun and Fos. The heterodimerization partner contained the E. coli heat-labile enterotoxin subunit B (LTB) to allow specific binding to the eukaryotic cell receptor GM1. Our results indicate that heterodimeric fusion protein containing a truncated form of ORF3 was produced in high amounts, carried the expected ORF3 epitope, showed phosphatase activity, and was able to bind to the GM1 receptor. In contrast, a fusion protein containing the entire sequence of the ORF3 product was produced in minute amounts, indicating that it may have biological activity in prokaryotes, which led to the reduction of the amounts of proteins expressed.

Animals↗

PubMed1998

Further analysis of the genome of porcine epidemic diarrhoea virus.

We report here the continued determination and analysis of the nucleotide sequence of both wild type (wt) and cell culture adapted (ca) porcine epidemic diarrhoea coronavirus (PEDV). These studies were undertaken with two objectives in mind: the identification of common and divergent features in the genomic sequences of wt and ca PEDV which can explain the differences in virulence of these isolates and the further exploration of the relationship of PEDV to other coronaviruses.

Animals↗

PubMed1998

Comparison of the di- and trinucleotide frequencies from the genomes of nine different coronaviruses.

As an alternative to protein alignments for the comparison of sequences, the reiterations of mono- di- and trinucleotide frequencies were used for the comparison of coronavirus sequences. The relative abundance of the di- and trinucleotide frequencies within the 3' part from nine coronavirus genomes were determined. The patterns of dinucleotide frequencies and the trinucleotide frequencies showed some common features for all coronaviruses but also differences between the groups formerly defined on the base of antigenic relatedness. The normalised dinucleotide frequencies were further used to calculate the distances between coronavirus sequences. Based on the dinucleotide frequency distances, coronaviruses can be divided into two groups which roughly reflect the taxonomic groups. In this kind of evaluation, however, IBV occupies a position different to the one that it would take based on most protein sequence comparisons. Based on similarities within coding sequences and antigenic properties IBV occupies a place outside of both groups. Based on the dinucleotide frequencies IBV gained a position in between of the TGEV-related and the MHV-clustered coronaviruses.

Animals↗

PubMed1998

Construction and characterization of a stably transformed HeLa cell line in which the expression of bovine herpesvirus 1 ICP0 (BICP0) is induced by tetracycline.

To explore the effects BICP0 (a principal transactivator of BHV-1 gene expression) on viral promoter elements, we established a cell line in which the expression of BICP0 is regulated by tetracycline. A hybrid promoter containing reiterated copies of the tet-operator (tet-O) and a minimal herpesviral alpha gene transinducing factor (alpha TIF) responsive element (minimal human cytomegalovirus immediate early promoter) was fused to the BICP0 gene and used to transform a HeLa cell line which expressed a fusion protein consisting of the repressor of the tet-O and the transactivating domain of alpha TIF. Simultaneously, the hygromycin resistance gene was transfected to select cells in media containing either hygromycin alone or both hygromycin and tetracycline. Immunofluorescent assays indicated that BICP0 was synthesised in the transformed cell lines solely upon induction of the gene by tetracycline removal. Only cells which had been kept constantly in medium containing tetracycline were able to synthesise BICP0 upon induction. Induced cell lines transactivated the native BICP0 promoter as well as the herpes simplex virus thymidine kinase promoter and the long terminal repeat sequences of human immunodeficiency virus in a dose dependent manner. These cell lines may help to further explore the functions of BICP0 as well as to investigate the molecular basis of interactions between herpes- and retroviruses.

Cell Line, Transformed↗

PubMed1998

A system for stable indirect immobilization of multimeric recombinant proteins.

BACKGROUND: To perform an ELISA or for panning phage particles which display recombinant proteins, one of the reactants is immobilized on solid phase. Immobilization in ELISA is generally performed by passive adsorption of ligands to plastic. However, protein is denatured during the adsorption process. This may result in low efficiency interaction between ligands and receptors which depend on native structures. In contrast, indirect immobilization has been shown to prevent protein denaturation. OBJECTIVES: The aim was to develop a system that allows efficient and stable indirect immobilization of a variety of recombinant multimeric proteins to solid phase. RESULTS: A new vector was constructed which allows the expression of up to three proteins linked by the Jun/Fos leucine zipper. Purification of the resulting protein was achieved by Ni+ affinity chromatography utilizing the 6xHis-ABP (albumin binding protein) protein fused to the N-terminus of the Jun polypeptide. The high binding affinity of ABP to rat serum albumin (RSA) was exploited for indirect immobilization of recombinant proteins to solid phase. In an enzyme linked assay, the binding of ABP to immobilized RSA was shown to be 10-1000 times more efficient than other immobilization systems. Using the ZZ IgG binding domain of staphylococcal protein A as bait, the RSA-ABP immobilization system was successfully used to screen and enrich IgG Fc encoding DNA fragments from a cDNA phage library. CONCLUSION: The newly designed vector termed pJuFoexpress allows production and purification of multimeric protein complexes linked by the Jun/Fos leucine zipper. Without chemical modifications, the recombinant proteins can be immobilized indirectly to solid phase. The immobilization results in the stable display of native, biologically active proteins which can be used in ELISA and phage display systems.

Animals↗

PubMed1998

Immunohistochemical detection of porcine epidemic diarrhea virus compared to other methods.

An immunohistochemistry method using formalin-fixed tissues, a direct immunofluorescence method using cryostat sections, an enzyme-linked immunosorbent assay (ELISA), and a PCR method were compared for diagnosis in a litter of weaned pigs that had been experimentally inoculated with wild-type porcine epidemic diarrhea virus (PEDV) and killed between 6 and 60 h after onset of diarrhea. The immunohistochemistry method proved to be as reliable as direct immunofluorescence for diagnosis of PEDV in tissues collected postmortem. The good reliability of ELISA for investigating clinical samples was confirmed, whereas the PCR method used was ineffective.

Animals↗

PubMed1998

Detection of anionic antimicrobial peptides in ovine bronchoalveolar lavage fluid and respiratory epithelium.

Three small antimicrobial anionic peptides (AP) were originally isolated from an ovine pulmonary surfactant. However, their presence in bronchoalveolar lavage (BAL) fluid and tissues of the respiratory tract is unknown. In this study, we made affinity-purified rabbit polyclonal and mouse monoclonal antibodies to synthetic H-DDDDDDD-OH. Antibody specificity was assessed by a competitive enzyme-linked immunosorbent assay (ELISA), and the exact epitope binding sites were determined with analog peptides synthesized on derivatized cellulose. These antibodies were used to detect AP in BAL fluid by ELISA and in respiratory tissues by Western blot analysis and immunocytochemistry. BAL fluid from 25 sheep contained 0.83 +/- 0.33 mM AP (mean +/- standard deviation; range, 0.10 to 1.59 mM) and was antimicrobial. The presence of AP in BAL fluid was confirmed by reverse-phase high-pressure liquid chromatography fractionation followed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry on those fractions which were positive by competitive ELISA and demonstrated antimicrobial activity. In Western blots, polyclonal antibody PAB96-1 and monoclonal antibody 1G9-1C2 (5.0 micrograms/ml) detected four bands in solubilized turbinate and tracheal epithelial cells (53.7, 31.2, 28.0, and 25.7 kDa) and five bands in lung homogenates (53.5, 37.1, 31.2, 28.0, and 25.7 kDa). Only a single band was seen in solubilized liver and small-intestine homogenates, and no bands were seen in blots containing BAL fluid, albumin, or kidney or spleen homogenates. In pulmonary-tissue sections, both antibodies PAB96-1 and 1G9-1C2 identified accumulated protein in the apical cytoplasm of the bronchial and bronchiolar epithelia, in the cytoplasm of pulmonary endothelial cells, and in an occasional alveolar macrophage. As a first step in identifying a candidate AP precursor gene(s), degenerate oligonucleotides representing all possible coding combinations for H-GADDDDD-OH and H-DDDDDDD-OH were synthesized and used to probe Southern blots of sheep genomic DNA. Following low-stringency washes and a 2-day exposure, strongly hybridizing bands could be identified. One degenerate oligonucleotide, SH87, was used as a hybridization probe to screen a sheep phage genomic library. Two independent phage contained the H-GADDDDD-OH coding sequence as part of a larger predicted protein. AP may originate as part of an intracellular precursor protein, with multistep processing leading to the release of the heptapeptide into mucosal secretions. There it may interact with other innate pulmonary defenses to prevent microbial infection.

Amino Acid Sequence↗

PubMed1998

Field validation of laboratory tests for clinical diagnosis of sheep-associated malignant catarrhal fever.

Until recently, sheep-associated malignant catarrhal fever (SA-MCF) was diagnosed mainly on the basis of clinical presentation and histopathological changes. Using clinically diagnosed field cases, we have evaluated a seminested PCR and a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) and compared these assays in the diagnosis of SA-MCF in cattle with histopathology as a provisional "gold standard." Samples from 44 cattle with clinical signs suggestive of SA-MCF were examined by histopathology, PCR, and CI-ELISA. In addition, samples from healthy cattle were evaluated by PCR (n = 96) and CI-ELISA (n = 75). Based on histopathology, 38 of the 44 clinical cases were classified as SA-MCF positive, 3 were classified as inconclusive, and 3 were classified as SA-MCF negative. The sensitivity of PCR was 95 to 97%, whereas the specificity ranged between 94 and 100%. The CI-ELISA showed a sensitivity of 56 to 87% and a specificity between 91 and 100%. In the field, there is good correlation between the diagnoses of SA-MCF by histopathology, PCR, and CI-ELISA. These data also confirm the close association of ovine herpesvirus 2 with SA-MCF in Switzerland.

Animals↗

PubMed1998

[Transfusion of plasma of endotoxin tolerant rats improves survival of hemorrhagic shock in the rat model].

To evaluate the protective effect of endotoxin tolerant plasma after hemorrhagic shock, plasma of endotoxin tolerant animals was used for resuscitation of normal rats. The transfusion of plasma of endotoxin tolerant rats improved the survival rate from 20 to 60 per cent. In the sera of endotoxin tolerant animals a TNF inhibitory activity was detected, which could be transferred by the plasma. This effect may be responsible for the protection to hemorrhagic shock.

Animals↗

PubMed1998

Production and characterization of canine osteosarcoma cell lines that induce transplantable tumors in nude mice.

OBJECTIVE: To produce and characterize cell lines from canine primary appendicular osteosarcomas that induce transplantable tumors in athymic nude mice. ANIMALS: 57 six- to 8-week-old female athymic nude mice. PROCEDURE: Canine primary appendicular osteosarcoma tumors were harvested and cell lines were produced. Canine osteosarcoma (COSCA)-Toby (COSCA-T; 10 mice), COSCA-Princess (COSCA-Pr; 16) or canine osteosarcoma D-17 (ATCC CCL-183; 31) cells were injected into the proximal portion of the left tibia of nude mice to evaluate tumor production from each cell line; the right tibia served as the control. Tibial measurements were taken on alternating days to evaluate tumor growth during a 6-month period. Student's t-tests were used to determine whether size of the proximal portion of the left and right tibias differed significantly during the observation period. RESULTS: 88% of mice receiving COSCA-Pr and 50% of mice receiving COSCA-T cells developed a tumor at the injection site by 9 days after implantation. The D-17 cells induced tumors in 50% of injected tibias; however, tumors were not detected for 79 days. Tumors generated from COSCA-Pr and COSCA-T cells in nude mice were histologically similar to the canine tumor from which they were developed. CONCLUSION: New osteosarcoma cell lines that can reliably and rapidly induce transplantable tumors in nude mice were developed. CLINICAL RELEVANCE: Use of cell lines will allow evaluation of new treatments of canine primary appendicular osteosarcoma in a nude mouse model.

Animals↗

PubMed1997

Population dynamics, demographic stochasticity, and the evolution of cooperation.

A basic evolutionary problem posed by the Iterated Prisoner's Dilemma game is to understand when the paradigmatic cooperative strategy Tit-for-Tat can invade a population of pure defectors. Deterministically, this is impossible. We consider the role of demographic stochasticity by embedding the Iterated Prisoner's Dilemma into a population dynamic framework. Tit-for-Tat can invade a population of defectors when their dynamics exhibit short episodes of high population densities with subsequent crashes and long low density periods with strong genetic drift. Such dynamics tend to have reddened power spectra and temporal distributions of population size that are asymmetric and skewed toward low densities. The results indicate that ecological dynamics are important for evolutionary shifts between adaptive peaks.

Biological Evolution↗