Biomedical subjects

W Breuer

Publications and source records attributed to W Breuer.

At least 55 records · Page 3Linked to original sources

PubMed1996

Histopathological findings in pigs of various ages suffering from spontaneous infection with the porcine reproductive and respiratory syndrome virus (PRRSV).

A retrospective study was carried out to investigate the morphology of 29 routinely necropsied German Landrace pigs of various ages, namely five fetuses, eight neonates, five suckling piglets and 11 weaners, being either serologically PRRSV-positive or born or aborted from PRRSV positive sows. Virus antigen was immunohistochemically demonstrated in 12 out of 27 (44%) lungs tested. The distinguishing histopathological features of PRRSV-induced spontaneous disease are interstitial pneumonia accompanied by secondary inflammatory alterations that become more and more pronounced with increasing age, and vascular alterations hitherto not described in animals of these age groups. The latter consist of vasculitis of the medium-sized arteries and occasionally of the veins, mainly in the lungs but also in other organs, and of perivascular lymphoplasmacytic infiltrates of various degrees of frequency and organ distribution, but present above all in the CNS-including the meninges and in the lungs. These findings, which suggest vasculotropism on the part of the PRRSV, may facilitate the histopathological diagnosis of PRRSV infections in routinely examined material.

Animals↗

PubMed1996

Immunohistochemical examination of light-chain expression (lambda/kappa ratio) in canine, feline, equine, bovine and porcine plasma cells.

Five species of domestic animals were examined immunohistochemically and the light-chain ratios of the immunoglobulins produced by plasma cells analysed. Forty dogs, 11 cats, 10 horses, 11 cattle and 14 pigs were tested using the sequential indirect immunoperoxidase and immunophosphatase methods. Tissues from the tonsils, spleen and cervical lymph nodes were analysed. It could be seen that the lambda/kappa ratio in dogs, cats, horses and cattle is largely dominated by the lambda chains (lambda/kappa ratio in dogs: 91/9%, in cats; 92/8%; in horses: 96/4%; in cattle: 91/9%). A more or less balanced ratio was observed only in the case of the pigs (52/48%).

Animals↗

PubMed1995

Investigation of the active site of oligosaccharyltransferase from pig liver using synthetic tripeptides as tools.

Oligosaccharyltransferase (OST), an integral component of the endoplasmic-reticulum membrane, catalyses the transfer of dolichyl diphosphate-linked oligosaccharides to specific asparagine residues forming part of the Asn-Xaa-Thr/Ser sequence. We have studied the binding and catalytic properties of the enzyme from pig liver using peptide analogues derived from the acceptor peptide N-benzoyl-Asn-Gly-Thr-NHCH3 by replacing either asparagine or threonine with amino acids differing in size, stereochemistry, polarity and ionic properties. Acceptor studies showed that analogues of asparagine and threonine with bulkier side chains impaired recognition by OST. Reduction of the beta-amide carbonyl group of asparagine yielded a derivative that, although not glycosylated, was strongly inhibitory (50% inhibition at approximately 140 microM). This inhibition may be due to ion-pair formation involving the NH3+ group and a negatively charged base at the active site. Hydroxylation of asparagine at the beta-C position increased Km and decreased Vmax, indicating an effect on both binding and catalysis. The threo configuration at the beta-C atom of the hydroxyamino acid was essential for substrate binding. A peptide derivative obtained by replacement of the threonine beta-hydroxy group with an NH2 group was found to display acceptor activity. This shows that the primary amine is able to mimic the hydroxy group during transglycosylation. The pH optimum with this derivative is shifted by approximately 1 pH unit towards the basic region, indicating that the neutral NH2 group is the reactive species. The various data are discussed in terms of the catalytic mechanism of OST, particular emphasis being placed on the role of threonine/serine in increasing the nucleophilicity of the beta-amide of asparagine through hydrogen-binding.

Amino Acid Sequence↗

PubMed1995

Iron acquired from transferrin by K562 cells is delivered into a cytoplasmic pool of chelatable iron(II).

The release of iron from transferrin (Tf) in the acidic milieu of endosomes and its translocation into the cytosol are integral steps in the process of iron acquisition via receptor-mediated endocytosis (RME). The translocated metal is thought to enter a low molecular weight cytoplasmic pool, presumed to contain the form of iron which is apparently sensed by iron responsive proteins and is the direct target of iron chelators. The process of iron delivery into the cytoplasmic chelatable pool of K562 cells was studied in situ by continuous monitoring of the fluorescence of cells loaded with the metal-sensitive probe calcein. Upon exposure to Tf at 37 degrees C, intracellular fluorescence decayed, corresponding to an initial iron uptake of 40 nM/min. The Tf-mediated iron uptake was profoundly inhibited by weak bases, the protonophore monensin, energy depletion, or low temperatures (< 25 degrees C), all properties characteristic of RME. Cell iron levels were affected by the slowly permeating chelator desferrioxamine only after prolonged incubations. Conversely, rapidly penetrating, lipophilic iron-(II) chelators such as 2,2'-bipyridyl, evoked swift increases in cell calcein fluorescence, equivalent to sequestration of 0.2-0.5 microM cytosolic iron, depending on the degree of pre-exposure to Tf. Addition of iron(III) chelators to permeabilized 2,2'-bipyridyl-treated cells, failed to reveal significant levels of chelatable iron(III). The finding that the bulk of the in situ cell chelatable pool is comprised of iron(II) was corroborated by pulsing K562 cells with Tf-55Fe, followed by addition of iron(II) and/or iron(III) chelators and extraction of chelator-55Fe complexes into organic solvent. Virtually all of the accumulated 55Fe in the chelatable pool could be complexed by iron(II) chelators. The cytoplasmic concentration of iron(II) fluctuated between 0.3 and 0.5 microM, and its mean transit time through the chelatable pool was 1-2 h. We conclude that after iron is translocated from the endosomes, it is maintained in the cytosol as a transit pool of chelatable iron(II). The ostensible absence of chelatable iron(III) implicates the intracellular operation of vigorous reductive mechanisms.

2,2'-Dipyridyl↗

PubMed1995

Oligosaccharyl transferase is a constitutive component of an oligomeric protein complex from pig liver endoplasmic reticulum.

Oligosaccharyl transferase (OST), an intrinsic component of the endoplasmic reticulum membrane, catalyses the N-glycosylation of specific asparagine residues in nascent polypeptide chains. We have purified the enzyme from crude pig liver microsomes by a procedure involving salt/detergent extraction, concanavalin-A precipitation, S-Sepharose, MonoP and concanavalin-A-Sepharose chromatographies. A highly purified OST preparation exerting catalytic activity, contained two protein subunits of 48 kDa and 66 kDa, from which the 66-kDa species was identified by immunoblotting as ribophorin I. The function of ribophorin I in this dimeric protein complex is unknown. The high degree of similarity between its transmembrane region and a putative dolichol-recognition consensus sequence suggests that ribophorin I could be involved in glycolipid binding and delivery. Several lines of evidence indicate that the catalytically active 48-kDa/66-kDa polypeptides are associated in the endoplasmic reticulum membrane with other proteins, including ribophorin II and a 40-kDa glycoprotein. The implication of ribophorins I and II in the translocation machinery and their apparent association with the OST activity point to a close relationship between polypeptide synthesis, translocation and N-glycosylation, both spacially and temporally. Kinetic studies with the MonoP-purified oligosaccharyl transferase showed that the enzyme transfers dolichyl-diphosphate-linked GlcNAc2 to synthetic tripeptides and hexapeptides, containing the Asn-Xaa-Thr motif, at a comparable rate. The glycosylation reaction was found to have a pH optimum close to 7 and to require divalent metal ions, with Mn2+ being most effective. Substitution of threonine in the N-glycosylation motif by serine impairs its function as an acceptor, measured by Vmax/Km, by approximately 17-fold, consisting of a 7.3-fold increase in Km and a 2.3-fold decrease in Vmax. This indicates that the side chain structure of the hydroxyamino acid influences both binding and catalysis, consistent with previous studies highlighting its participation in the catalytic mechanism of transglycosylation. The Km values of peptide acceptors improved significantly when dolichyl-phosphate-bound oligosaccharides were used instead of lipid-linked GlcNAc2 as the glycosyl donor. We conclude from this observation that the sugar residues on the outer branches of the glycolipid donor induce conformational changes in the active site of the oligosaccharyl transferase, thus influencing the association constant of the peptide substrate.

Amino Acid Sequence↗

PubMed1995

Helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats.

Gastric biopsies from the fundic gland region of 122 dogs and 127 cats were subjected to histopathological examination. The aim of the study was to determine infection rates and degrees of colonization by Helicobacter-like organisms (HLOs), and to ascertain their possible relationship to histopathological changes. In all, 82% of the dogs and 76% of the cats had an HLO infection. The most striking histopathological changes were glandular degeneration with accumulation of lymphocytes and neutrophilic granulocytes (dogs, 21%; cats, 39%), fibrosis of the lamina propria mucosae (dogs, 41%; cats, 58%), oedema in the lamina propria mucosae (dogs, 54%; cats, 23%), lymphoid follicles (dogs, 17%; cats, 19%) and lymphoplasmacytic infiltrates. A relation between the degree of colonization by HLOs and the extent of histopathological changes could only be discovered in the cats. It was not possible to ascertain whether these bacteria, irrespective of the degree of colonization, were responsible for the histopathological changes in the dogs.

Animals↗

PubMed1995

Transport of iron and other transition metals into cells as revealed by a fluorescent probe.

Transport of nontransferrin-bound iron into cells is thought to be mediated by a facilitated mechanism involving either the trivalent form Fe(III) or the divalent form Fe(II) following reduction of Fe(III) at the cell surface. We have made use of the probe calcein, whose fluorescence is rapidly and stoichiometrically quenched by divalent metals such as Fe(II), Cu(II), Co(II), and Ni(II) and is minimally affected by variations in ionic strength, Ca(II) and Mg(II). Addition of Fe(II) salts to calcein-loaded human erythroleukemia K-562 cells elicited a slow quenching response that was markedly accelerated by the ionophore A-23187 and was reversed by membrane-permeant but not by impermeant chelators. These observations were confirmed by fluorescence imaging of cells. Other divalent metals such as Co(II), Ni(II), and Mn(II) permeated into cells at roughly similar rates, and their uptake, like that of Fe(II), was blocked by trifluoperazine, bepridil, and impermeant sulfhydryl-reactive organomercurials, indicating the operation of a common transport mechanism. This method could provide a versatile tool for studying the transport of iron and other transition metals into cells.

Biological Transport↗

PubMed1995

[Acute myeloid leukemia (promyelocytic leukemia) with detection of virus particles in a boar. Histologic, histochemical and ultrastructural findings].

Acute myeloid leukemia is a rare finding in swine. A case of acute promyelocytic leukemia is described by light microscopy, enzyme- and immunohistochemistry as well as electron microscopy. Tumour cells can be demonstrated in bone marrow, lymph nodes, liver, spleen, kidneys, small intestine and myocardium. The tumorous neutrophil precursor cells are positive for naphthol-AS-D-chloroacetate esterase and lysozyme. They show ultrastructurally numerous granules as typical for promyelocytes. Virus particles of the oncorna-c-type can be demonstrated within the tumour cells.

Animals↗

PubMed1995

[Toxoplasmosis causing death in a mole (Talpa europaea)].

Pathologic lesions caused by Toxoplasma gondii in a juvenile male common mole are described. Main degenerative lesions are localized in liver, heart and lung combined with excessive proliferation of the typical microparasitic cysts. Besides of this a moderate coccidiosis in the mucosa of bile ducts was diagnosed.

Animals↗

PubMed1994

Light microscopic, ultrastructural, and immunohistochemical examinations of two calcifying epithelial odontogenic tumors (CEOT) in a dog and a cat.

Two calcifying epithelial odontogenic tumors from a 10-year-old cross-breed dog and a 9-year-old cat were characterized histologically and electron microscopically. Important characteristics were plexiform to squamous epithelial structure, amyloid in the tumor tissue with partial calcification (Liesegang's rings), and a low rate of mitosis. The amyloid found in the tumors was subjected to immunohistochemical examination, using a series of antibodies (anti-AA, -AL, -AF, -ASc1, -AB, -cytokeratin, -vimentin, -desmin, -laminin). The exclusive reaction to anti-cytokeratin and anti-laminin suggests that the amyloid is of epithelial origin.

Amyloid↗

PubMed1994

Familial nephropathy in Bernese mountain dogs.

Between January 1988 and March 1992 nephropathies were frequently diagnosed in Bernese Mountain Dogs. During this period, 20 animals (16 females, four males), ages 2-5 years (average age at time of diagnosis = 3.3 years) presented with clinically renal insufficiency. Morphologic diagnosis of the renal lesions was identical in all cases, i.e., membranoproliferative glomerulonephritis (MPGN) with concomitant interstitial nephritis. Deposits of immunoglobulin-M (IgM) and of the third complement component were regularly demonstrated immunohistochemically in the glomeruli; deposits of immunoglobulin-A (IgA) and immunoglobulin-G (IgG) were found only in isolated cases. Reduplication of glomerular basement membranes, mesangial interposition, and subendothelial deposits of the immunocomplex type were also detected by electron microscopy. A pedigree analysis indicated that the MPGN in these 20 Bernese Mountain Dogs of approximately the same age was of hereditary genesis. Thus, MPGN should be allocated to the group of familial nephropathies. Serologically, high IgG titers against Borrelia burgdorferi were found in 17 dogs. These findings are discussed in relation to familial nephropathies in humans.

Animals↗

PubMed1993

Protein kinase C mediates down-regulation of cystic fibrosis transmembrane conductance regulator levels in epithelial cells.

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) in epithelial cells is known to be down-regulated by the action of phorbol myristate acetate (PMA). We show here that in addition to suppressing the rate of transcription of the CFTR gene, PMA treatment stimulates degradation of the CFTR protein. HT-29 colon epithelial cells and the CFTR-transfected pancreatic cells PLJ-4.7 lost 55-80% of their CFTR protein after 3-6 h of treatment with 100 nM PMA, as analyzed by quantitative Western blotting. In contrast to PMA, actinomycin D and cycloheximide reduced the CFTR protein content by 19 and 9% in HT-29 cells and by 22 and 40% in PLJ-4.7 cells, respectively, while inhibiting total cellular RNA and protein synthesis by over 80%. The PMA-induced loss of CFTR was partially reversed by the protein kinase C inhibitor GF109203X. The PMA-induced degradation of CFTR may represent a regulatory pathway for terminating CFTR-mediated chloride and mucin secretion.

Adenocarcinoma↗

PubMed1993

Immunoglobulin-producing tumours in dogs and cats.

Tumours with a plasmacytoid pattern taken from 32 dogs and four cats were examined for the presence of immunoglobulins, which would allow them to be designated as B-cell lymphomas. Within a total of 19 immunoglobulin-positive tumours, three types could be distinguished: extramedullary plasmacytoma (15), multiple myeloma (two) and immunocytoma (two). These tumours occurred in 18 of the dogs, and in one cat (extramedullary plasmacytoma). The characteristics of the immunoglobulin-producing tumours were investigated by light and electron-microscopy as well as by immunohistochemical methods. Seventeen of the 19 tumours expressed lambda-type light chains and one tumour kappa-type light chains. Heavy chains were also synthesized by five tumours.

Animals↗

PubMed1993

Papovavirus-induced trichogenous tumours in Syrian hamsters (Mesocricetus auratus).

Tumorous, virus-induced skin lesions in two golden hamsters (Mesocricetus auratus) were characterized macroscopically and by means of light- and electron-microscopy. Evidence of a virus was demonstrated in the ultra-thin sections and by the negative staining method. The morphological findings confirm the assumption that infections with papoviruses--probably of the polyomavirus genus--were involved.

Animals↗

PubMed1993

Induction of multidrug resistance downregulates the expression of CFTR in colon epithelial cells.

The epithelial cell line HT-29, which constitutively expresses the cystic fibrosis transmembrane conductance regulator (CFTR), was induced to become drug resistant by cultivation in the presence of colchicine. The gradual acquisition of drug resistance was associated with a corresponding increase in the expression of the multidrug resistance P-glycoprotein (P-gp) and a marked (> 80%) decrease in the constitutive levels of CFTR protein, as determined by immunoblotting. The reduction in CFTR content occurred at the onset of acquisition of drug resistance when P-gp expression was still relatively low. Reversal of drug resistance by removal of colchicine from the culture medium led to a 70% decrease in P-gp levels and a concomitant 40% increase in CFTR. The levels of other membrane proteins such as Na(+)-K(+)-ATPase and alkaline phosphatase remained relatively constant (< 26% variation). We propose that a selective downregulation of CFTR is elicited by acquisition of the multidrug resistance (MDR) phenotype and that induction of P-gp expression leads to a reversible repression of CFTR biosynthesis. These findings provide an experimental foundation for the complementary patterns of expression of the CFTR and MDR1 genes observed in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

[Chronic gastritis caused by Ollulanus tricuspis (Leuckart, 1865) in a tiger].

A chronic gastritis caused by an infestation with O. tricuspis is described. The differential-diagnostic importance of this parasitosis in chronic maldigestive diseases is emphasized. Conventional coproscopic diagnostic procedures fail as the life cycle of the parasite is confined to the gastrum of the host.

Animals↗

PubMed1992

Effect of substrate structure on the activity of Man9-mannosidase from pig liver involved in N-linked oligosaccharide processing.

Man9-mannosidase, an alpha 1,2-specific enzyme located in the endoplasmic reticulum and involved in N-linked-oligosaccharide processing, has been isolated from crude pig-liver microsomes and its substrate specificity studied using a variety of free and peptide-bound high-mannose oligosaccharide derivatives. The purified enzyme displays no activity towards synthetic alpha-mannosides, but removes three alpha 1,2-mannose residues from the natural Man9-(GlcNAc)2 substrate (M9). The alpha 1,2-mannosidic linkage remaining in the M6 intermediate is cleaved about 40-fold more slowly. Similar kinetics of hydrolysis were determined with Man9-(GlcNAc)2 N-glycosidically attached to the hexapeptide Tyr-Asn-Lys-Thr-Ser-Val (GP-M9), indicating that the specificity of the enzyme is not influenced by the peptide moiety of the substrate. The alpha 1,2-mannose residue which is largely resistant to hydrolysis, was found to be attached in both the M6 and GP-M6 intermediate to the alpha 1,3-mannose of the peripheral alpha 1,3/alpha 1,6-branch of the glycan chain. Studies with glycopeptides varying in the size and branching pattern of the sugar chains, revealed that the relative rates at which the various alpha 1,2-mannosidic linkages were cleaved, differed depending on their structural complexity. This suggests that distinct sugar residues in the aglycon moiety may be functional in substrate recognition and binding. Reduction or removal of the terminal GlcNAc residue of the chitobiose unit in M9 increased the hydrolytic susceptibility of the fourth (previously resistant) alpha 1,2-mannosidic linkage significantly. We conclude from this observation that, in addition to peripheral mannose residues, the intact chitobiose core represents a structural element affecting Man9-mannosidase specificity. A possible biological role of the enzyme during N-linked-oligosaccharide processing is discussed.

Amino Acid Sequence↗

PubMed1992

Induction of expression of the cystic fibrosis transmembrane conductance regulator.

The cystic fibrosis transmembrane conductance regulator (CFTR) was studied in HT-29 human colonic carcinoma cells with the aim of assessing possible mechanisms of up-regulation of its expression. CFTR was identified and quantified in total cell extracts by Western immunoblots using a monoclonal anti-CFTR antibody and was functionally assessed by tracer Cl-efflux from intact cells. It was found that various stimuli that lead to a sustained (greater than or equal to 8 h) elevation of intracellular cyclic AMP elicited a marked and specific increase in CFTR expression in cell membranes and concomitant activation of Cl- secretion. Further activation of Cl- secretion was obtained by additional short term activation by cyclic AMP analogues or cyclic AMP-inducing agents. Blockers of transcription or translation largely depressed the cAMP-mediated induction of CFTR levels and associated function, indicating that the inductive phenomenon was at the transcriptional level. The results imply the involvement of putative cyclic AMP responsive (and related) elements that are present in the CFTR gene promoter and that are known to modulate eukaryotic gene expression. Activation of these elements by various stimuli might provide pharmacological tools for up-regulation of CFTR expression at both biochemical and physiological levels.

Adenocarcinoma↗