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B Riederer

Publications and source records attributed to B Riederer.

14 recordsLinked to original sources
PubMed2005

Superficial and deep changes of cellular mechanical properties following cytoskeleton disassembly.

The cytoskeleton, composed of actin filaments, intermediate filaments, and microtubules, is a highly dynamic supramolecular network actively involved in many essential biological mechanisms such as cellular structure, transport, movements, differentiation, and signaling. As a first step to characterize the biophysical changes associated with cytoskeleton functions, we have developed finite elements models of the organization of the cell that has allowed us to interpret atomic force microscopy (AFM) data at a higher resolution than that in previous work. Thus, by assuming that living cells behave mechanically as multilayered structures, we have been able to identify superficial and deep effects that could be related to actin and microtubule disassembly, respectively. In Cos-7 cells, actin destabilization with Cytochalasin D induced a decrease of the visco-elasticity close to the membrane surface, while destabilizing microtubules with Nocodazole produced a stiffness decrease only in deeper parts of the cell. In both cases, these effects were reversible. Cell softening was measurable with AFM at concentrations of the destabilizing agents that did not induce detectable effects on the cytoskeleton network when viewing the cells with fluorescent confocal microscopy. All experimental results could be simulated by our models. This technology opens the door to the study of the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.

Actins↗
PubMed2004

The Na+/H+ exchanger isoform 2 is the predominant NHE isoform in murine colonic crypts and its lack causes NHE3 upregulation.

The Na(+)/H(+) exchanger isoform NHE2 is highly expressed in the intestinal tract, but its physiological role has remained obscure. The aim of this study was to define its expression, location, and regulatory properties in murine colon and to look for the compensatory changes in NHE2 (-/-) colon that allow normal histology and absorptive function. To this end, we measured murine proximal colonic surface and crypt cell NHE1, NHE2, and NHE3 expression levels, transport rates in response to acid, hyperosmolarity and cAMP in murine proximal colonic crypts, as well as changes in transcript levels and acid-activated NHE activity in NHE2 (-/-) crypts. We found that NHE2 was expressed most abundantly in crypts, NHE1 equally in crypts and surface cells, and NHE3 much stronger in surface cells. NHE2, like NHE1, was activated by low intracellular pH (pH(i)), hyperosmolarity, and cAMP, whereas NHE3 was activated only by low pH(i). Crypts isolated from NHE2 (-/-) mice displayed increased acid-activated NHE1- and NHE3-attributable Na(+)/H(+) exchange activity, no change in NHE1 expression, and NHE3 expression levels twice as high as in normal littermates. No change in cellular ultrastructure was found in NHE2 (-/-) colon. Our results demonstrate high NHE2 expression in the crypts and suggest a role for NHE2 in cryptal pH(i) and volume homeostasis.

Acids↗
PubMed2003

Independence of apical Cl-/HCO3- exchange and anion conductance in duodenal HCO3- secretion.

Reduced gastrointestinal HCO3- secretion contributes to malabsorption and obstructive syndromes in cystic fibrosis. The apical HCO3- transport pathways in these organs have not been defined. We therefore assessed the involvement of apical Cl-/HCO3- exchangers and anion conductances in basal and cAMP-stimulated duodenal HCO3- secretion. Muscle-stripped rat and rabbit proximal duodena were mounted in Ussing chambers, and electrical parameters, HCO3- secretion rates, and 36Cl-, 22Na+, and 3H+ mannitol fluxes were assessed. mRNA expression levels were measured by a quantitative PCR technique. Removal of Cl- from or addition of 1 mM DIDS to the luminal perfusate markedly decreased basal HCO3- secretion but did not influence the HCO3- secretory response to 8-bromo-cAMP, which was inhibited by luminal 5-nitro-2-(3-phenylpropylamino)-benzoate. Bidirectional 22Na+ and 36Cl- flux measurements demonstrated an inhibition rather than a stimulation of apical anion exchange during cAMP-stimulated HCO3- secretion. The ratio of Cl- to HCO3- in the anion secretory response was compatible with both Cl- and HCO3- being secreted via the CFTR anion channel. CFTR expression was very high in the duodenal mucosa of both species. We conclude that in rat and rabbit duodena, an apical Cl-/HCO3- exchanger mediates a significant part of basal HCO3- secretion but is not involved in the HCO3- secretory response to cAMP analogs. The inhibitor profile, the strong predominance of Cl- over HCO3- in the anion secretory response, and the high duodenal CFTR expression levels suggest that a major portion of cAMP-stimulated duodenal HCO3- secretion is directly mediated by CFTR.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2003

Islet-brain1/C-Jun N-terminal kinase interacting protein-1 (IB1/JIP-1) promoter variant is associated with Alzheimer's disease.

Islet-brain1 (IB1) or c-Jun NH2 terminal kinase interacting protein-1 (JIP-1), the product of the MAPK8IP1 gene, functions as a neuronal scaffold protein to allow signalling specificity. IB1/JIP-1 interacts with many cellular components including the reelin receptor ApoER2, the low-density lipoprotein receptor-related protein (LRP), kinesin and the Alzheimer's amyloid precursor protein. Coexpression of IB1/JIP-1 with other components of the c-Jun NH2 terminal-kinase (JNK) pathway activates the JNK activity; conversely, selective disruption of IB1/JIP-1 in mice reduces the stress-induced apoptosis of neuronal cells. We therefore hypothesized that IB1/JIP-1 is a risk factor for Alzheimer's disease (AD). By immunocytochemistry, we first colocalized the presence of IB1/JIP-1 with JNK and phosphorylated tau in neurofibrillary tangles. We next identified a -499A>G polymorphism in the 5' regulatory region of the MAPK8IP1 gene. In two separate French populations the -499A>G polymorphism of MAPK8IP1 was not associated with an increased risk to AD. However, when stratified on the +766C>T polymorphism of exon 3 of the LRP gene, the IB1/JIP-1 polymorphism was strongly associated with AD in subjects bearing the CC genotype in the LRP gene. The functional consequences of the -499A>G polymorphism of MAPK8IP1 was investigated in vitro. In neuronal cells, the G allele increased transcriptional activity and was associated with an enhanced binding activity. Taken together, these data indicate that the increased transcriptional activity in the presence of the G allele of MAPK8IP1 is a risk factor to the onset of in patients bearing the CC genotype of the LRP gene.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo.

The present study describes the postnatal expression of calbindin, calretinin and parvalbumin and glutamic acid decarboxylase (GAD) and microtubule-associated protein 2 (MAP2) in organotypic monocultures of rat dorsal thalamus compared to the thalamus in vivo. Cultures were maintained for up to 7 weeks. Cortex-conditioned medium improved the survival of thalamic cultures. MAP2-immunoreactive material was present in somata and dendrites of small and large-sized neurons throughout the cultures. Parvalbumin immunoreactivity was present in larger multipolar or bitufted neurons along the edge of a culture. These neurons also displayed strong parvalbumin mRNA and GAD mRNA expression, and GABA immunoreactivity. They likely corresponded to cells of the nucleus reticularis thalami. Parvalbumin mRNA, but neither parvalbumin protein nor GAD mRNA, was expressed in neurons with large somata within the explant. They likely represented relay cells. GAD mRNA, but not parvalbumin mRNA, was expressed in small neurons within the explants. Small neurons also displayed calbindin- and calretinin-immunoreactivity. The small neurons likely represented local circuit neurons. The time course of expression of the calcium-binding proteins revealed that all were present at birth with the predicted molecular weights. A low, but constant parvalbumin expression was observed in vitro without the developmental increase seen in vivo, which most likely represented parvalbumin from afferent sources. In contrast, the explantation transiently downregulated the calretinin and calbindin expression, but the neurons recovered the expression after 14 and 21 days, respectively. In conclusion, thalamic monocultures older than three weeks represent a stable neuronal network containing well differentiated neurons of the nucleus reticularis thalami, relay cells and local circuit neurons.

Aging↗
PubMed1997

Mechanism of alkaloid cyclopeptide synthesis in the ergot fungus Claviceps purpurea.

BACKGROUND: Previous analyses of the biosynthesis of the alkaloid cyclopeptides from the ergot fungus Claviceps purpurea were hampered by a lack of suitable systems for study in vitro, and this led to conflicting results concerning the mechanism of alkaloid cyclopeptide formation. Recently, D-lysergyl peptide synthetase (LPS) of the ergot fungus Claviceps purpurea, which assembles the non-cyclol precursors of the ergopeptines, has been partially purified and shown to consist of two polypeptide chains of 370 kDa (LPS 1) and 140 kDa (LPS 2); these contain all the sites necessary for the assembly of the D-lysergyl peptide backbone. The mechanism of D-lysergyl peptide synthesis remained unclear, however. RESULTS: We have identified the obligatory peptidic intermediates in D-lysergyl peptide synthesis and the sequential order of their formation. The two LPS subunits catalyze the formation of D-lysergyl mono-, di-, and tripeptides as enzyme-thioester intermediates, the formation of which appears to be irreversible. Peptide synthesis starts when D-lysergic acid binds to the LPS 2 subunit, which most probably occurs after the previous round of synthesis has been completed by the release of the end product from the LPS enzyme. CONCLUSIONS: We have shown that the mechanism of D-lysergyl peptide synthesis is an ordered process of successive acyl transfers on a multienzyme complex. This knowledge opens the way for enzymatic and genetic investigations into the formation of novel alkaloid cyclopeptides.

Alkaloids↗
PubMed1996

D-Lysergyl peptide synthetase from the ergot fungus Claviceps purpurea.

The ergot fungus Claviceps purpurea produces the medically important ergopeptines, which consist of a cyclol-structured tripeptide and D-lysergic acid linked by an amide bond. An enzyme activity capable of non-ribosomal synthesis of D-lysergyl-L-alanyl-L-phenylalanyl-L-proline lactam, the non-cyclol precursor of the ergopeptine ergotamine, has been purified about 18-fold from the ergotamine-producing C. purpurea strain D1. Analysis of radioactively labeled enzyme-substrate complexes revealed a 370-kDa lysergyl peptide synthetase 1 (LPS 1) carrying the amino acid activation domains for alanine, phenylalanine, and proline. The activation of D-lysergic acid is catalyzed by a 140-kDa peptide synthetase (LPS 2) copurifying with LPS 1. LPS 1 and LPS 2 contain 4'-phosphopantetheine and bind their substrates covalently by thioester linkage. Kinetic analysis of the synthesis reaction revealed a Km of approximately 1.4 microM for both D-lysergic acid and its structural homolog dihydrolysergic acid, which is one to two orders of magnitude lower than the Km values for the other amino acids involved. The Km values for the amino acids reflect their relative concentrations in the cellular pool of C. purpurea. This may indicate that in in vivo conditions D-lysergyl peptide formation is limited by the D-lysergic acid concentration in the cell. In vitro, the multienzyme preparation catalyzes the formation of several different D-lysergyl peptide lactams according to the amino acids supplied. Specific antiserum was used to detect LPS 1 in various C. purpurea strains. In C. purpurea wild type, the enzyme was expressed at all stages of cultivation and in different media, suggesting that it is produced constitutively.

Amino Acids↗
PubMed1990

Self-splicing of the mobile group II intron of the filamentous fungus Podospora anserina (COI I1) in vitro.

The first intron of the mitochondrial gene coding for cytochrome oxidase subunit I (COI I1) of Podospora anserina can undergo self-splicing in vitro at high concentrations of NH4Cl or KCl. Under these conditions cleavage at the 5' splice junction takes place without branch formation probably via hydrolysis by water or OH- and the intron is released in a linear form. In vitro transcripts that contain mutated introns with large deletions in nonconserved domain IV comprising greater than 50% of the intronic sequence display a more efficient splicing reaction and, surprisingly, 5' cleavage via transesterification and lariat formation is re-established to a low degree under NH4Cl. In contrast to the self-splicing group II introns aI5 gamma and bI1 from yeast mitochondria cleavage at the 3' splice site of the Podospora intron is reduced and cleavage by hydrolysis in trans (i.e. exon reopening) is almost completely suppressed. Both observations could be interpreted as a result of unfavourable spatial conformations of the intron that (i) lead to a steric hindrance of the 5' exon to attack the 3' splice site in cis and (ii) block intron-dependent cleavage reaction of the ligated exons in trans. Alternatively, the possibility that a weak overall interaction of the postulated exon- with the corresponding intron-binding sites (EBS-IBS pairings) is responsible for the remarkable differences to the self-splicing reaction of other group II introns is discussed.

Ascomycota↗
PubMed1987

Influence of monoclonal antibodies on microtubule assembly.

The influence on microtubule assembly in vitro of monoclonal antibodies against microtubule-associated proteins (MAPs) was studied. Light scattering was used for measuring net polymer formation and electron microscopy for determining the influence of antibodies on microtubule morphology. Control experiments showed that nonimmune mouse IgG had no effect on either the assembly or appearance of microtubules. The same was true for monoclonal antibodies against MAP1. At low levels, antibodies against MAP2 caused the aggregation of microtubules into bundles, an effect that did not occur with antibodies against any other MAP type studied. At increasing concentrations, anti-MAP2 progressively inhibited tubulin polymerization, producing irregular, shortened filaments. Anti-MAP5 produced a striking fragmentation of microtubules into very short pieces that were otherwise morphologically identical to control microtubules. The different effects of these antibodies show the potential of monoclonal antibodies for investigating MAP function and form an important adjunct to cellular microinjection experiments.

Animals↗
PubMed1986

MAP5: a novel brain microtubule-associated protein under strong developmental regulation.

A novel microtubule-associated protein, MAP5, is described, whose chemical properties and cytological distribution distinguish it from other known microtubule-associated proteins (MAPs). Its status as a MAP is indicated by the observations that (i) it co-assembles efficiently with microtubules in vitro, (ii) it is localized on microtubules in brain sections by immunogold staining with monoclonal antibody against MAP5 and (iii) immunoaffinity purified MAP5 stimulates tubulin polymerization. Immunoperoxidase staining of brain sections showed that MAP5 is present in neurons throughout the brain and that in them it is evenly distributed throughout axons, dendrites and cell bodies. In this respect it differs from previously described MAPs (1, 2, 3 and tau) which are differentially compartmentalized in brain neurons. MAP5 is not present in axon terminals, dendritic spines or other synaptic elements. It is present at substantially higher levels in neonatal brain than adult and it is more abundant than either MAP1 or MAP2a up to postnatal day 10. The fall in amount of MAP5, from juvenile to adult levels, is completed between postnatal days 10 and 20. This suggests that MAP5 is particularly important in modulating microtubule function during the formation of neuronal processes.

Animals↗
PubMed1985

Differential expression of distinct microtubule-associated proteins during brain development.

The levels of three different microtubule-associated proteins (MAP1, -2, and -3) in brain were found to undergo large changes during postnatal development. MAP1 was barely detectable at birth but thereafter steadily increased, reaching adult levels by postnatal day 20 (P20). Both MAP2 and MAP3 showed differential expression patterns of their component peptides. At birth, MAP2 was represented by the smaller of two Mr 280,000 peptides (MAP2b) and three antigenically related Mr 70,000 peptides. The larger of the Mr 280,000 peptides (MAP2a) first appeared between P10 and P20, and the Mr 70,000 components disappeared at the same time. Of the two MAP3 peptides, the larger (MAP3a) was present in the late embryo, several days before MAP3b appeared. Between P10 and P20, both MAP3 components underwent a striking decrease in abundance (a factor of 10), which correlated with their disappearance from all neuronal compartments except neurofilament-containing axons. These developmental changes in expression are different and characteristic for each of the three MAPs, yet in each case they are detectable in brain homogenates, indicating that they occur concurrently throughout the brain.

Animals↗
PubMed1984

Sodium ions regulate a specific population of acidic amino acid receptors in synaptic membranes.

The regulatory effects of Na+ on C1-/Ca2+-dependent and C1-/Ca2+-independent L-glutamate binding sites were examined. In Tris-C1-/Ca2+ buffer, the binding of L-[3H]-glutamate to rat brain synaptic membranes was 5-fold higher than in Tris-acetate buffer. Low concentrations of Na+ (less than 5 mM) markedly depressed L-glutamate binding when assayed in Tris-C1/Ca2+ buffer, and this effect was attenuated by the selective blocker of C1-/Ca2+-dependent binding sites, DL-2-amino-4-phosphonobutyrate (APB). Scatchard analyses indicated that the effect of Na+ was due to a decrease in the number of C1-/Ca2+-dependent binding sites with no change in affinity. In Tris-acetate buffer, low concentrations of Na+ had little effect on L-glutamate binding. Dose-response curves for the inhibition of L-glutamate binding by DL-APB indicated a predominant high-affinity (Ki 5-10 microM) inhibitory component in Tris-C1-/Ca2+ buffer, but mainly a low-affinity component (Ki 1-2 mM) in Tris-acetate buffer and in Tris-C1-/Ca2+ buffer containing Na+. These data indicate that low concentrations of Na+ regulate specifically the C1-/Ca2+-dependent, APB-sensitive class of L-glutamate binding sites.

Aminobutyrates↗
PubMed1984

The effect of age on the microheterogeneous pattern of human myelin basic protein.

Dry weight, total basic protein and myelin basic protein (MBP) in human corpus callosum tissue were found to be significantly reduced with age, confirming other reports. The heterogeneous patterns of the MBP samples were investigated and could be classified into three groups: a normal one with peak 1 larger than peak 3; an old-age pattern with peak 1 smaller than peak 3; and a pathological pattern with peak 1 greatly reduced. Because peak 1 is the only one which is significantly reduced with age, it is suggested that future investigations could be simplified. The physiological and pathophysiological role of MBP heterogeneity is discussed.

Adult↗