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B Zimmermann

Publications and source records attributed to B Zimmermann.

At least 73 records · Page 4Linked to original sources
PubMed1995

Caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release from the endoplasmic reticulum in honeybee photoreceptors.

Light stimulation of invertebrate microvillar photoreceptors causes a large rapid elevation in Cai, shown previously to modulate the adaptational state of the cells. Cai rises, at least in part, as a result of Ins(1,4,5)P3-induced Ca2+ release from the submicrovillar endoplasmic reticulum (ER). Here, we provide evidence for Ca(2+)-induced Ca2+ release (CICR) in an insect photoreceptor. In situ microphotometric measurements of Ca2+ fluxes across the ER membrane in permeabilized slices of drone bee retina show that (a) caffeine induces Ca2+ release from the ER; (b) caffeine and Ins(1,4,5)P3 open distinct Ca2+ release pathways because only caffeine-induced Ca2+ release is ryanodine sensitive and heparin insensitive, and because caffeine and Ins(1,4,5)P3 have additive effects on the rate of Ca2+ release; (c) Ca2+ itself stimulates release of Ca2+ via a ryanodine-sensitive pathway; and (d) cADPR is ineffective in releasing Ca2+. Microfluorometric intracellular Ca2+ measurements with fluo-3 indicate that caffeine induces a persistent elevation in Cai. Electrophysiological recordings demonstrate that caffeine mimics all aspects of Ca(2+)-mediated facilitation and adaptation in drone photoreceptors. We conclude that the ER in drone photoreceptors contains, in addition to the Ins(1,4,5)P3-sensitive release pathway, a CICR pathway that meets key pharmacological criteria for a ryanodine receptor. Coexpression of both release mechanisms could be required for the production of rapid light-induced Ca2+ elevations, because Ca2+ amplifies its own release through both pathways by a positive feedback. CICR may also mediate the spatial spread of Ca2+ release from the submicrovillar ER toward more remote ER subregions, thereby activating Ca(2+)-sensitive cell processes that are not directly involved in phototransduction.

Adenosine Diphosphate Ribose↗
PubMed1995

Basement membrane formation of fetal mouse intestinal epithelial cells in organoid cultures.

Basement membrane formation of fetal mouse intestinal epithelial cells was investigated in organoid cultures. Intestinal cells were dissociated with a commercial collagenase/dispase preparation, and the cells were grown at high density on a membrane filter at the interface between the medium and air. This type of culture allows the histotypical reorganization of cells. After 2 days in culture, epithelial cells began to accumulate on the surface, in particular the periphery of the culture. These cells were usually cuboid, and small vesicles were formed in the center of the culture. Laminin-positive material was observed at peripheral sites. However, no basement membrane could be identified beneath the epithelial cells at the electron-microscopic level. After 3 days, epithelial cells that had gathered at the periphery became columnar in shape. Laminin-positive material extended across the surface of the culture. However, the vesicles formed in the center of the culture were not associated with laminin-positive material. Basement membrane was observed by electron microscopy at some sites beneath groups of epithelial cells, but did not extend continuously beneath these cells. Some epithelial cells made contact with the underlying mesenchymal cells through the discontinuous basement membrane via intercellular contacts. After 5-6 days, the surface of the culture was almost completely covered with epithelial cells and, at some sites, villus-like structures were visible. Laminin-positive material was clearly detectable under epithelial cells, as well as around epithelial vesicles located in the center of the culture. By electron microscopy, basement membrane was clearly visible between the epithelial and mesenchymal cells. After 9 days, villus-like structures were rarely observed. After 3 weeks, the cell mass had become smaller and villi had disappeared. Basement membrane was extensively folded and no basement membrane was visible at some sites. Formation of basement membrane by epithelial cells in monolayer culture occurs in an incomplete and irregular manner. It occurs rapidly in organoid cultures that include mesenchyme and epithelium. The organoid culture used here should be a useful tool for studies of the formation and degeneration of the basement membrane as well as interactions between the epithelium and mesenchyme.

Animals↗
PubMed1995

Changes in integrin expression during chondrogenesis in vitro: an immunomorphological study.

Integrins are receptors composed of ligand-specific alpha-chains and cell type-specific beta-chains which are involved in cell-cell and cell-matrix interactions. The distribution of alpha 1- and alpha 3-integrins as well as collagen Types I and II, was investigated by immunofluorescence and immunoelectron microscopy during chondrogenesis in organ culture after various culture periods. Mesenchymal cells from limb buds of Day 12 mouse embryos were grown at high density. Within the first 2 days of the culture period, only alpha 1-integrin could be detected. Formation of cartilage-specific matrix on Day 3 was accompanied by the occurrence of alpha 3-integrin. On Day 7, alpha 3 was present only in cartilage nodules, whereas alpha 1 was strongly expressed in the perichondrium and was more or less homogeneously distributed in the surrounding mesenchyme. On Day 14, alpha 1-integrin was again detectable in cartilage. We suggest that the change in collagen formation from Type I to Type II during chondrogenesis is accompanied by a change in integrin expression from alpha 1 to alpha 3. Conversely, dedifferentiation of chondrocytes in aging cartilage is accompanied by the occurrence of collagen Type I and alpha 1-integrin. Therefore, a strict correlation between the collagen type synthesized by the cells and the appropriate receptor presented by the cells is suggested.

Animals↗
PubMed1994

Structure/activity characterization of glucagon-like peptide-1.

Glucagon-like peptide-1 is a gastrointestinal hormone that strongly stimulates insulin release via specific receptors on the pancreatic beta-cell. To characterize the side-chain groups required for interaction of glucagon-like peptide-1 with its receptor, we performed binding studies with alanine-substituted glucagon-like peptide-1 analogues on RINm5F insulinoma cells. The binding affinity and biological activity of glucagon-like peptide-1 have been found to be sensitive to alanine exchanges in the N-terminal positions 1, 4, 6 and the C-terminal positions 22 and 23. Alanine substitutions at positions 5, 8, 10-12, 14, 16-21 and 25-30 do not change receptor affinity. These findings could be exploited to design glucagon-like peptide-1 agonists and probably antagonists for further physiological studies.

Amino Acid Sequence↗
PubMed1994

Studies on human porin. XII. Eight monoclonal mouse anti-"porin 31HL" antibodies discriminate type 1 and type 2 mammalian porin channels/VDACs in western blotting and enzyme-linked immunosorbent assays.

Eight mouse monoclonal antibodies directed against the acetylated N-terminal part of the type 1 human VDAC Porin 31HL clearly discriminate type 1 and type 2 mammalian porin channels. This is shown by comparing synthetic N-terminal peptides of either channel type in Western dot blots or by ELISA. The data support the specificity of the anti-Porin 31HL antibodies and thus give further support to our recent observations on extramitochondrial expression of VDAC. In the plasmalemma of different mammalian cells VDAC forms part of an ubiquitous chloride channel complex, which in patch clamp measurements may figure as the outwardly rectifying depolarization-induced chloride channel that is affected in cystic fibrosis.

Amino Acid Sequence↗
PubMed1994

Occurrence of osteoblast necroses during ossification of long bone cortices in mouse fetuses.

Previous investigations concerned with in vitro osteogenesis and mineralization have revealed some indication of a participation of cell necroses in the course of calcification. These observations were confirmed by in vivo investigations on desmoid ossification in fetal mouse calvariae, where abundant necrotic osteoblasts were found at the mineralization border and in the osteoid. In the present study, ossification of long bone cortices from fetal mice was investigated by use of electron microscopy. Specimens obtained from the collection of the Institute of Anatomy, Free University of Berlin (mouse fetuses, forearm; rat fetuses, forearm) were reinvestigated for control purposes. In all cases, mineralization of osteoid was accompanied by cell necroses. Cell degeneration was characterized by swelling of the endoplasmic reticulum and loss of the plasma membrane resulting in freely distributed vesicular structures. Cell debris was incorporated within the mineral. Initially, cell necroses in the perichondrium occurred in the region surrounding the hypertrophic cartilage and the matrix of which showed spots of endochondral mineralization. Necrotic osteoblasts occurred simultaneously with mineralization of the osteoid. During further ossification of the long bone cortices, the number of necrotic cells increased markedly. In addition to necrotic cells, healthy osteoblasts, osteocytes and perichondral tissue were present, indicating that an artifact can be excluded. The importance of cell necroses in the process of mineralization is as yet unclear. Possibly, the cells act as calcium and/or phosphate stores, which are liberated by cell death to increase the amount of mineral constituents at sites of mineralization.

Animals↗
PubMed1994

In vitro platelet function during storage in three different additive solutions.

Three different synthetic media without glucose were studied for platelet storage. The first medium contained acetate and gluconate. The second contained acetate, gluconate and citrate. Finally the third contained phosphate and mannitol. The purpose of the study was to investigate whether there were differences among the various media in terms of preservation of platelet quality. Pools of platelet concentrates were prepared from buffy coats. In vitro function and metabolic parameters were measured during 5 days of storage in these additive solutions as well as in plasma. Platelet aggregation, hypotonic shock response and release of beta-thromboglobulin, platelet factor 4 and lactate dehydrogenase of the cytosol were equivalent in the media containing acetate compared to plasma storage. In vitro platelet functions and pH in these two media were better preserved compared to the medium with phosphate and mannitol. In addition bacteriological studies using platelets suspended in additive solutions or in plasma were carried out. Carryover of 20% of plasma to the synthetic media necessary for successful platelet storage in these additive solutions allows bacteriological growth. As shown, inoculation of 1 colony/ml Staphylococcus epidermidis leads to 10(6)-10(7) organisms/ml after 5 days of storage.

Blood Donors↗
PubMed1994

Purification and characterization of two putative HLA class II associated proteins: PHAPI and PHAPII.

In addition to their well defined role in presentation of processed antigen on the cell surface, class II molecules are able to transduce signals into the cell after binding of ligands. The cytoplasmic regions of class II molecules might function as docking sites for as yet unidentified proteins that are components of this signalling pathway. Here we report on two putative HLA class II associated proteins (PHAPI and PHAPII) which have been purified from the cytosolic fraction of the human lymphoblastoid B-cell line H2LCL using an affinity matrix composed of the synthetic biotinylated cytoplasmic region of the DR2 alpha chain immobilized on avidin agarose. The sequence obtained for PHAPI revealed a novel primary structure with a leucine/isoleucine rich N-terminal region. Protein data and the cDNA sequence obtained for PHAPII agree with the cDNA sequence of SET that has been described recently. Both PHAPI and PHAPII have an extended highly acidic C-terminal region. Based on their primary structure we speculate that PHAPI and PHAPII are involved in the generation of intracellular signalling events that lead to regulation of transcriptional activity after binding of a ligand to HLA class II molecules.

Amino Acid Sequence↗
PubMed1994

Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes and bovine skeletal muscle, reversibly binds adenosine triphosphate (ATP).

A new aspect of mammalian porin (mammalian VDAC = mammalian voltage-dependent anion channel) is presented: channel active VDAC binds adenosine triphosphate (ATP) in the absence of Ca2+. Channel active "Porin 31HL" or "Porin 31BM", enriched from crude membranes of human B lymphocytes or whole cell lysates of bovine skeletal muscle, respectively, was bound to a nine atoms spacer ATP-agarose at pH 7.4 or 5.0 and reeluted from the resin by 10 mM ATP disodium salt. Furthermore, channel active "Porin 31BM" was labelled by [32P]ATP in a 1:1 stoichiometric relation. Binding of ATP to human porin was confirmed by studying the interaction of the synthetic porin fragment Type-1/Ac-35, comprising the putative nucleotide binding site G Y G F G, with trinitrophenyl-ATP (TNT-ATP) by scanning fluorometry. Peptide/TNP-ATP complexes clearly show enhancement of fluorescence intensity and a spectral shift of the fluorescence maximum. In a control experiment, using a porin fragment lacking the putative nucleotide binding site, no change of fluorescence emission was observed. Further confirmation for ATP binding by human VDAC arose from an autoradiographic experimental approach: the porin fragment Type-1/Ac-35 could be labelled by [32P]ATP, while a second porin fragment ending immediately before the putative nucleotide binding site could not; nor could a synthetic non porin peptide.

Adenosine Triphosphate↗
PubMed1994

Inhibition of chondrogenesis and endochondral mineralization in vitro by different calcium channel blockers.

Limb bud mesenchymal cells from mouse embryos grown at high density at the medium/air interphase undergo chondrogenesis and form numerous nodules of mature cartilage. Addition of beta-glycerophosphate (5 mM) induced endochondral mineralization within these nodules. Ca2+ accumulation, matrix formation and alkaline phosphatase activity were recorded for each culture. Treatment with the L-type channel-specific blockers nifedipine and verapamil during the entire culture period caused an inhibition of mineralization. Sequential treatment reduced mineralization only when added during the early part of the culture period in the course of chondrogenesis. In all cases, matrix formation, estimated by alcian blue binding, was concomitantly diminished. Lanthanum acetate, which blocks Ca2+ channels non-specifically, also reduced Ca2+ accumulation in the cultures when added continuously. After sequential treatment, Ca2+ content was only diminished when the cultures were treated in the last part of the culture period during mineralization. Matrix formation was not altered by lanthanum acetate, but alkaline phosphatase was decreased. These results show that chondrogenic differentiation is under control of L-type Ca2+ channels, whereas matrix calcification depends on intracellular Ca2+ accumulation mediated by lanthanum-sensitive Ca2+ channels. This points to the importance of intracellular Ca2+ accumulation in the process of endochondral mineralization.

Animals↗
PubMed1994

[Exceptional production of leukocyte-free erythrocyte concentrates using filtration with the BPF 4 BBS leukocyte filter].

Leukocyte depletion prevents some undesirable effects of red cell transfusions. We evaluated the blood filter system BPF 4 BBS (PALL) by filtering 26 units of buffy coat-free red cell concentrates, prepared from 500 ml of whole blood and suspended in SAG-M-additive solution, over 8 min within 48 h after donation at room temperature. Filtration reduced the leukocyte content by more than 99.99% in 25 of the 26 units examined. In 20 instances, no leukocyte was found in the Nageotte counting chamber (2 x 50 microliters) corresponding to a total leukocyte count below 2.38 x 10(4). In 5 instances a single leukocyte was seen, corresponding to a total leukocyte count of (2.35 +/- 0.01) x 10(4). One unit contained 2.93 x 10(5) leukocytes after filtration, corresponding to a 99.98% leukocyte reduction. Red cell recovery was 92.5 +/- 3.8%. This system is well suited for routine use.

Cytapheresis↗
PubMed1993

Probing the human receptor for C5a anaphylatoxin with site-directed antibodies. Identification of a potential ligand binding site on the NH2-terminal domain.

Molecular cloning has revealed the DNA sequence of the human receptor for the C5a anaphylatoxin (C5aR). In this study, mAb and polyclonal antibodies with specificities for deduced hydrophilic sequences of the receptor protein were employed to determine the expression and the topography of C5aR on PBL. Evidence was obtained that an antigenically homogenous receptor exists on human neutrophils, eosinophils, and monocytes that binds C5a and C5a(desArg). The assignment of epitopes to extra- or intracellular receptor domains as determined by FACS analysis of intact or permeabilized cells confirmed the general topography of the C5aR that had been predicted by hydropathy analysis. Competitive binding studies were performed to examine whether extracellular receptor domains participate in ligand binding. The occupation of the receptor by C5a inhibited only the binding of antibodies that were specific for the receptor's aminoterminal domain (C5aR-EX1). The anti-EX1 mAb S5/1 effectively antagonized C5a/C5a(desArg) biological activity. A heptameric peptide (D15DKDTLD21) was identified as the smallest receptor fragment that was recognized by the mAb S5/1 or by polyclonal rabbit anti-EX1 lg. These results imply that an amino acid sequence rich in aspartate within the receptor aminoterminus represents both an immunodominant epitope and a ligand binding site on the C5aR.

Amino Acid Sequence↗
PubMed1993

Dexamethasone induces chondrogenesis in organoid culture of cell mixtures from mouse embryos.

The effect of dexamethasone on morphogenesis and differentiation of cells obtained from mouse embryos grown at high density in vitro was investigated. Cells from decapitated mouse embryos of day 10 to day 13 were isolated by enzymatic treatment and grown at high density at the medium/air interface in organoid culture. After 28 days in culture, organoid-like structures such as vesicles, gland-like structures and cell aggregates had developed, dependent on the stage of the embryos. After the addition of 10(-7) M dexamethasone, cartilage had formed in cultures of cells from day-10, -11 and -12 embryos. It was maximal in cultures of day-11 cells and rare in day-10 cells. No cartilage was found in cultures of day-13 cells. Cartilage induction was similar in cultures treated with 10(-6) and 10(-7) M dexamethasone, but clearly less in cultures treated with 10(-8) M. Only minute amounts of cartilage were detectable after dexamethasone treatment of cells obtained from decapitated embryos (day-11 and -12) whose limb buds had been cut off. In organoid cultures of pure limb bud cells, 10(-7) M dexamethasone had no influence on chondrogenesis. The results indicate that the cells inducible to form cartilage by dexamethasone originate from the limb buds. Glucocorticoid induction of chondrogenesis has not been described in vivo. The dependency of both chondrogenesis and expression of glucocorticoid receptor on cell density in vitro may be the cause for this effect.

Animals↗
PubMed1993

The covalent linkage of secretory component to IgA. Structure of sIgA.

Immunoglobulin A which is secreted into external fluids is synthesized in plasma cells as an (IgA)2-J-chain complex. This complex docks on to the polyimmunoglobulin receptor which is located at the basolateral surface of epithelial cells. After docking the (IgA)2-J-receptor complex is internalized and processed. The polyimmunoglobulin receptor loses its C-terminal tail and thus becomes the secretory component. This secretory component is then covalently linked to the (IgA)2-J-chain complex by a disulfide bond, and protects the so formed sIgA from denaturation and proteolysis in external fluids. In order to establish this disulfide bond between IgA and the secretory component, sIgA, purified from human colostrum, was subjected to several enzymatic and chemical fragmentation reactions. One of the resulting polypeptides allowed us to characterize the covalent linkage of the secretory component to IgA in sIgA. IgA was found to be covalently linked to the secretory piece by a single disulfide bond between Cys 311 of one alpha-chain and Cys 467 of the secretory component. Cys 501 of the secretory component and Cys 311 of the other alpha-chain are blocked by cysteines. With this last paper of a series the structure of an entire sIgA molecule has been elucidated.

Amino Acid Sequence↗
PubMed1993

Primary structure of the murine monoclonal IgG2a antibody mAb735 against alpha (2-8) polysialic acid. 2. Amino acid sequence of the heavy (H-) chain Fd' region.

The complete amino acid sequence of the Fd' region including the VH part, the CH1 domain, and the hinge segment of the biologically relevant monoclonal mouse anti-alpha (2-8) polysialic acid antibody mAb735 is presented. The reduced and carboxymethylated H-chain was digested with trypsin and cyanogen bromide. For subfragmentation selected peptides were cleaved with thermolysin and endoproteinase Asp-N. The generated peptides were isolated by RP-HPLC and characterized by sequence analysis, plasma desorption mass spectrometry (PDMS), and amino acid analysis. The N-terminal sequence was determined after enzymatic deprotection with pyroglutamate aminopeptidase. According to Kabat et al. the variable region of the H-chain belongs to the subgroup II. Sequence data from the constant region indicate that mAb735 represents the gamma 2a isotype.

Amino Acid Sequence↗
PubMed1992

Kinetics of beta-glycerophosphate-induced endochondral mineralization in vitro. Calcium accumulation, alkaline phosphatase activity, and effects of levamisole.

Isolated mesenchymal limb bud cells from day-12 mouse embryos grown at high density in organoid culture at the medium/air interphase differentiate into chondrocytes and form cartilage nodules. Upon addition of beta-glycerophosphate (beta-GP), cartilage undergoes endochondral mineralization. This beta-GP-induced mineralization was investigated by measuring the calcium content in the cultures and the activity of alkaline phosphatase (AP) in the cell mass and the medium. Calcium incorporation depended on the amount of beta-GP added. After continuous treatment, mineralization began on day 8 of the culture period and increased linearly until day 15. In long-term cultures, periodical treatment for 6 days caused an increase in mineralization the older the cultures were, but the slope of increase was proportionately less steep. Treatment at the latest period on days 19-24 resulted in a markedly reduced mineralization. After short-term treatment (48 hours), mineralization increased also the older the cultures were and proceeded during further cultivation in beta-GP-free medium. This kinetic behavior indicates a dependency of mineralization on cartilage maturation in this in vitro system. AP activity increased enormously and nearly logarithmically in the cell mass in beta-GP-free medium, whereas beta-GP treatment inhibited this drastic increase. In the medium, considerable activities of AP were also measurable from day 10 onward. It increased in beta-GP-free medium up to day 14, but was diminished after mineralization had been induced. Levamisole inhibited AP activity dose dependently when added directly to the enzyme-containing medium (100% inhibition at 10(-3) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1992

Degeneration of osteoblasts involved in intramembranous ossification of fetal rat calvaria.

Ossification of calvariae from day-21 rat fetuses was reinvestigated by electron microscopy using different fixation techniques (glutaraldehyde/OsO4, tannic acid, ruthenium red, K-pyroantimonate). An osteoid layer with scattered mineral deposits was found at the mineralization front. Directly beyond this layer, a sheet of one to two layers of necrotic and degenerating osteoblasts was present. Above this sheet, normal and healthy cells were seen, formed by six to eight layers of flattened cells, embedded in a collagenous matrix. The osteoblasts on the less mineralizing opposite side of the calcified calvariae and the osteocytes embedded in the calcified calvariae appeared healthy. Closer inspection of the necrotic zone revealed apatite crystals in vesicles which most probably originated from mitochondria of the degenerated cells. Large K-pyroantimonate deposits were found throughout the osteoid and the necrotic zone, whereas only small granules were scattered in the cytoplasm and at the plasma membrane of the healthy cells directly adjacent to the necrotic zone. A concept of intramembranous mineralization is outlined, according to which osteoblasts store enormous amounts of calcium, which are liberated by physiological cell death in the vicinity of the mineralizing front.

Animals↗