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B Stahl

Publications and source records attributed to B Stahl.

At least 19 recordsLinked to original sources
PubMed1999

Analysis of high-molecular-weight oligosaccharides from human milk by liquid chromatography and MALDI-MS.

Pooled human milk oligosaccharides were fractionated by anion-exchange chromatography on AG 1-X2 and by an improved gel filtration procedure that allowed the separation of large oligosaccharides on Toyopearl HW 40 (S) and Bio-Gel P-6 columns, respectively. The analysis of the resulting nonderivatizated fractions by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) revealed several neutral and acidic high-molecular-weight oligosaccharides. So far unknown acidic oligosaccharides containing up to 20 monomers were detected in a molecular mass range of 2094-3626 Da. Furthermore, neutral structures containing up to 35 monosaccharides were identified after fractionation on Toyopearl HW 40 (S) and subsequent P-6 fractionation, demonstrating the suitability of the applied method for the preparation of oligosaccharides in this high-molecular-mass range. The composition of the detected oligosaccharides was found to be the same as those previously identified in oligosaccharides of lower masses. However, an enormous structural heterogeneity was observed when acidic and neutral fractions were characterized by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). From our analysis we may conclude that each molecular mass identified by MALDI-MS corresponds to a variety of isomeric structures. The total number of oligosaccharides occurring in human milk may consequently be much higher than estimated before.

Carbohydrate Sequence↗
PubMed1999

Direct interaction of Alzheimer's disease-related presenilin 1 with armadillo protein p0071.

Alzheimer's disease-related presenilins are thought to be involved in Notch signaling during embryonic development and/or cellular differentiation. Proteins mediating the cellular functions of the presenilins are still unknown. We utilized the yeast two-hybrid system to identify an interacting armadillo protein, termed p0071, that binds specifically to the hydrophilic loop of presenilin 1. In vivo, the presenilins constitutively undergo proteolytic processing, forming two stable fragments. Here, we show that the C-terminal fragment of presenilin 1 directly binds to p0071. Nine out of 10 armadillo repeats in p0071 are essential for mediating this interaction. Since armadillo proteins, like beta-catenin and APC, are known to participate in cellular signaling, p0071 may function as a mediator of presenilin 1 in signaling events.

Alzheimer Disease↗
PubMed1999

Matrix optimization for matrix-assisted laser desorption/ionization mass spectrometry of oligosaccharides from human milk.

Neutral and acidic oligosaccharides from human milk were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). These experiments require suitable matrices; their selection and particularly their preparation protocols must be optimized. Important criteria are sensitivity, reproducibility, tolerance against impurities and resolution over a wide mass range. For analytical investigations of these oligosaccharides, containing labile fucosylated and sialylated components, another property of a matrix becomes a significant factor, namely the influence on ion stability and the extent of (metastable) fragmentation. The experience gained with the MALDI/MS of neutral and acidic oligosaccharides is summarized taking into account different intentions of measurement and typical problems, such as impurities after enzymatic treatment. For a rapid screening of an oligosaccharide sample, superior results were obtained with a new preparation technique using 5-chloro-2-mercaptobenzothiazole (CMBT) as the first layer for 2,5-dihydroxybenzoic acid. For structural analysis by post-source decay, CMBT as the first layer for 3-aminoquinoline is a favoured preparation protocol, because extensive fragmentation is achieved. For acidic oligosaccharides, a special preparation protocol makes it possible to determine the number of sialic acids by inducing highly effective cationization. Matrix-assisted laser desorption/ionization mass spectrometry; matrices; oligosaccharides; post-source decay.

Gentisates↗
PubMed1999

Two distinct domains in hsc70 are essential for the interaction with the synaptic vesicle cysteine string protein.

The cysteine string protein (csp) is a synaptic vesicle protein found to be essential for normal neurotransmitter release. The precise function of csp in the synaptic vesicle cycle is still enigmatic. By interacting with the heat-shock cognate hsc70, a cochaperone-chaperone complex with an unknown function is formed. We report here that the formation of this complex is mediated by two distinct domains in hsc70. The ATPase domain and the substrate-binding domain must cooperate to create a binding site for csp. The C-terminal domain of hsc70 seems to function as a regulator for the formation of the cochaperone-chaperone complex. We also show that the interaction of csp with heat-shock proteins is confined to hsc70 and hsp70. Other heat-shock proteins, like hsp60 and hsp90, do not interact with csp.

Animals↗
PubMed1999

Chemical analysis and antimicrobial activity of the resin Ladano, of its essential oil and of the isolated compounds.

Fractionation of the resin Ladano from Cistus creticus subsp. creticus and susceptibility testing using the chromatographic fractions showed that its antistaphylococcal activity was mainly due to the diterpene sclareol. The antimicrobial activity of its essential oil, of the chromatographic fractions, and of the isolated compounds was also evaluated against Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus hominis.

Anti-Bacterial Agents↗
PubMed1997

High-sensitivity analysis of neutral underivatized oligosaccharides by nanoelectrospray mass spectrometry.

Nanoscale electrospray ionization (nano-ESI) overcomes the sensitivity problems found and reported for neutral oligosaccharides in conventional microscale forced-flow ESI. For a series of compounds ranging from trisaccharides to larger polymers with molecular masses up to 6 kDa, sample concentrations of 10(-5) M, i.e., 10 pmol total sample load, yielded very intense singly or multiply cationized molecule ions in an ion-trap mass spectrometer. In a dilution series, it is exemplified that, at the 10(-8) M level, molecule ion signals can be clearly registered with a S/N ratio of about 7. Only 100 amol of sample has been consumed in this experiment. Investigation of an oligosaccharide-peptide mixture revealed that the oligosaccharide is suppressed in conventional ESI, whereas in nano-ESI both analytes are detected at comparable and high intensities. Mechanistic implications are discussed, emphasizing the influence of surface activity for the two ESI techniques. The very low flow rates inherent to nano-ESI of about 30 nL/min, together with the high signal intensity, make it possible to fully employ the MSn capabilities of an ion-trap mass spectrometer for structural analysis. From less than 1 microL of sample solution it is possible to make consecutive fragmentation experiments up to MS7 to obtain valuable information about the structure of complex oligosaccharides.

Mass Spectrometry↗
PubMed1997

Analysis of fructans from higher plants by matrix-assisted laser desorption/ionization mass spectrometry.

In this communication both matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and high-performance anion-exchange chromatography (HPAEC) have been applied to analyze fructans from higher plants. Size distribution of a commercially available fructan preparation from Dahlia variabilis L. was determined by MALDI-MS. Molecular masses ranged from 2,000 up to 6,000 Da with a peak value of distribution at 2,635 Da. Essentially the same pattern was obtained using HPAEC. Low-molecular-weight fructans from onion bulbs (Allium cepa L.) were studied in more detail. Tissue extracts were analyzed by MALDI-MS without any analyte purification. Mass-spectra of both proteins and oligosaccharides were obtained. For identification, metastable ion scanning was performed. Neither deproteinization nor deionization of the samples affected the oligosaccharide pattern. Using HPAEC, a more complex oligosaccharide pattern was obtained because isomeric glycans were differentiated. However, the overall size distribution was similar to that obtained by MALDI-MS. In further experiments epidermal or parenchyma cell layers of the onion bulb were placed into matrix solution and were then subjected to MALDI-MS and metastable ion scanning as well. By taking this approach, analyte desorption was achieved immediately from plant tissue. Oligosaccharide mass spectra were essentially the same as those of the extracts. To our knowledge, this is the first time that MALDI-MS has been applied as a microprobe to plant tissue. Finally MALDI-MS analysis was performed using single-cell extracts from onion tissues without any purification of the analyte.

Allium↗
PubMed1996

A peptidoglycan binding domain in the porin-associated protein (PAP) of Rhodospirillum rubrum FR1.

The porin-associated protein of Rhodospirillum rubrum FR1 was found to contain a peptidoglycan binding motif. A partial fragment of 179 amino acids, obtained by cleavage of PAP with trypsin, Asp-N protease, and CNBr, was sequenced. Substantial sequence homology was found of the C-terminal part (residues 126-179) of porin-associated protein with OmpA, the peptidoglycan-associated lipoprotein of several bacteria, protein F of Pseudomonas aeruginosa, and PIII of Neisseria gonorrhoeae, the latter being also a porin-associated protein. The 179 amino acid fragment comprised about 67% of the mass spectrometrically determined total mass of PAP of 27850 Da.

Amino Acid Sequence↗
PubMed1996

Peripheral blood mononuclear cells of a patient with advanced Hodgkin's lymphoma give rise to permanently growing Hodgkin-Reed Sternberg cells.

A novel Hodgkin's disease (HD) derived cell line, L1236, was established from the peripheral blood of a patient with advanced Hodgkin's disease. Analysis of immunoglobulin (Ig) gene rearrangements revealed a biallelic Ig heavy chain and a monoallelic Ig kappa light chain gene rearrangement, pointing to a B-lymphoid origin of these cells. No DNA of Epstein-Barr virus was detected in L1236. The cells expressed the HD-associated surface antigens CD30 and CD15 as well as the transferrin receptor (CD71). Cytogenetic analysis of early passages of L1236 cells revealed a grossly disordered karyotype including cytogenetic aberrations described previously in other HD-derived cell lines. The Hodgkin/Reed-Sternberg (H-RS) cell origin of L1236 cells is further confirmed by Kanzler et al (Blood 87:3429, 1996), who found identical Ig gene rearrangement sequences in L1236 cells and H-RS cells of the same patient's bone marrow. L1236 cells expressed antigens necessary for efficient antigen presentation to T cells including HLA class I and II, B7.1 and B7.2, as well as adhesion molecules ICAM 1 and LFA 3. The cells secreted the interleukins (IL)-6, -8, -10, tumor necrosis factor (TNF) alpha, interferon (IFN) gamma, transforming growth factor (TGF) beta, and the granulocyte-macrophage colony stimulating factor (GM-CSF). After subcutaneous inoculation into SCID mice, a necrotic regression of initially growing tumors at the injection site was followed by disseminated intralymphatic growth. Our findings, together with the results of Kanzler et al, demonstrate that H-RS cells of B-lymphoid origin were present in the peripheral blood of a patient with advanced HD. These cells exerted a malignant phenotype with regard to their in vitro and in vivo characteristics.

Adult↗
PubMed1996

Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras.

GTP activates the interaction between the synaptic vesicle proteins rabphilin and rab3. This raises the question of whether rabphilin is a resident vesicle protein that recruits rab3 in a stage-dependent fashion, or if it is instead an effector protein recruited by rab3. We now show that rabphilin, like rab3, dissociates from synaptic vesicles after exocytosis in a manner requiring both Ca2+ and membrane fusion. Rabphilin interacts with GTP-rab3 via a N-terminal domain comprising a novel Zn2+(-)finger motif, and this interaction is essential for rabphilin binding to synaptic vesicles. Thus, in the same way that ras recruits raf to the plasma membrane, rab3 reversibly recruits rabphilin to synaptic vesicles in a stage-dependent manner. These results reveal an unexpected similarity between the molecular mechanisms by which small G protein function in recruiting effector proteins to membranes during membrane traffic and signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Cell-associated glucans of Burkholderia solanacearum and Xanthomonas campestris pv. citri: a new family of periplasmic glucans.

The cell-associated glucans produced by Burkholderia solanacearum and Xanthomonas campestris pv. citri were isolated by trichloroacetic acid treatment and gel permeation chromatography. The compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry, and high-performance anion-exchange chromatography. B. solanacearum synthesizes only a neutral cyclic glucan containing 13 glucose residues, and X. campestris pv. citri synthesizes a neutral cyclic glucan containing 16 glucose residues. The two glucans were further purified by high-performance anion-exchange chromatography. Methylation analysis revealed that these glucans are linked by 1,2-glycosidic bonds and one 1,6-glycosidic bond. Our 600-MHz homonuclear and 1H-13C heteronuclear nuclear magnetic resonance experiments revealed the presence of a single alpha-1,6-glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The presence of this single alpha-1,6 linkage, however, induces such structural constraints in these cyclic glucans that all individual glucose residues could be distinguished. The different anomeric proton signals allowed complete sequence-specific assignment of both glucans. The structural characteristics of these glucans contrast with those of the previously described osmoregulated periplasmic glucans.

Burkholderia↗
PubMed1996

Hereditary gingival fibromatosis: identification, treatment, control.

Hereditary gingival fibromatosis, or HGF, is characterized by varying degrees of attached gingival hyperplasia. The authors describe a case of generalized severe hereditary gingival fibromatosis involving the maxillary and mandibular arches. Removal of excess gingival tissue by conventional gingivectomy dramatically improved the patient's appearance.

Adult↗
PubMed1995

Localization of the palmitoylation site in the transmembrane protein p12E of Friend murine leukaemia virus.

Friend murine leukaemia virus complex was propagated on murine cells in the presence of [9,10-3H]palmitic acid. Virus particles were harvested from the culture supernatant and lysed with detergents. The viral transmembrane protein, p12E, was isolated from the lysates by size-exclusion chromatography and purified by narrowbore reverse-phase HPLC. Analysis of the purified product by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed that the protein is palmitoylated carrying one fatty acid residue. The radiolabelled fatty acid was released by hydroxylamine treatment at pH 7, indicating that acylation occurred via a thioester linkage. For allocation of the acylation site, p12E was digested with trypsin. The resulting peptides were either directly subjected to MALDI-TOF-MS or fractionated by microbore reverse-phase HPLC prior to mass spectrometry. The results revealed that p12E of Friend murine leukaemia virus is acylated at a cysteine residue situated at the C-terminal side of the putative transmembrane anchor of the polypeptide. Fatty acid analysis of the purified acylpeptide demonstrated that p12E carries almost exclusively palmitic acid.

Acylation↗
PubMed1994

The amino acid sequence of the red kidney bean Fe(III)-Zn(II) purple acid phosphatase. Determination of the amino acid sequence by a combination of matrix-assisted laser desorption/ionization mass spectrometry and automated Edman sequencing.

Purple acid phosphatase of the common bean Phaseolus vulgaris is a homodimeric 110-kDa glycoprotein with a Fe(III)-Zn(II) center in the active site of each monomer. After exchange of Zn(II) for Fe(II), the enzyme spectroscopically and kinetically resembles the mammalian purple acid phosphatases with Fe(III)-Fe(II) centers in monomeric 35-kDa proteins. The kidney bean enzyme consists of 432 amino acids/monomer with five N-glycosylated asparagine residues. The complete amino acid sequence was determined by a combination of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and classical sequencing methods. Our strategy involved mass determination and sequence analysis of all cyanogen-bromide-generated fragments by automated Edman degradation. Limited cleavages with cyanogen bromide were performed to obtain fragments containing still uncleaved Met-Xaa linkages. MALDI mass spectra of these products allowed the characterization of each fragment and the determination of the order of the cyanogen bromide fragments in the intact protein without producing overlapping peptides. For one large 30-kDa methionine-free fragment, the alignment of the Edman-degraded tryptic peptides was obtained by MALDI-MS analysis and enzymic microscale peptide laddering of overlapping Glu-C-generated fragments. The employed strategy shows that the classical method, in combination with modern mass spectrometry, is an attractive approach for primary structure determination in addition to the DNA sequencing method.

Acid Phosphatase↗
PubMed1994

GTP cleavage by the small GTP-binding protein Rab3A is associated with exocytosis of synaptic vesicles induced by alpha-latrotoxin.

Neurotransmitter release from presynaptic nerve terminals is a highly regulated form of exocytosis. Small GTP-binding proteins of the Rab family have been proposed to act as central regulators in this process that cycle between a GTP- and GDP-bound form. Previous work has shown that the synaptic vesicle protein Rab3A undergoes a membrane association-dissociation cycle that is associated with neurotransmitter release. Using isolated nerve terminals as our model system, we have now analyzed the GDP/GTP status of Rab3A. Synaptic vesicle-bound Rab3A was almost exclusively in the GTP form whereas cytosolic Rab3A contained only GDP. Approximately equal amounts of GTP and GDP were found in the pool of Rab3A localized to a membrane fraction containing plasma membrane-synaptic vesicle complexes. In contrast to Rab3A, Rab5 (an endosomal G-protein) was predominantly GDP-bound in all analyzed compartments. To analyze whether Rab3A-bound GTP is cleaved during exocytosis, synaptosomes were stimulated with alpha-latrotoxin, the active component of black widow spider venom. This resulted in massive exocytosis. A significant increase of the GDP/GTP ratio of Rab3A was observed under these conditions that was not due to a nonspecific loss of high energy nucleotides. Our findings suggest that cleavage of Rab3A-bound GTP is a crucial step in regulated exocytosis of synaptic vesicles.

Animals↗
PubMed1994

Rab3C is a synaptic vesicle protein that dissociates from synaptic vesicles after stimulation of exocytosis.

Rab3 proteins are small GTP-binding proteins of the Ras superfamily. Four highly homologous Rab3 proteins termed Rab3A, Rab3B, Rab3C, and Rab3D have been described. Rab3A has previously been shown to be a constituent of synaptic vesicles in neurons that undergoes membrane dissociation-association cycles during synaptic vesicle recycling. Here we report that Rab3C copurifies with Rab3A during the isolation of synaptic vesicles. Organelles immunoisolated with monoclonal antibodies directed against Rab3A led to a coenrichment of Rab3A and Rab3C, demonstrating that both Rab3 proteins are colocalized on the same organelle. In isolated nerve terminals, stimulation of neurotransmitter release resulted in a dissociation of Rab3C from synaptic vesicle and/or recycling membranes. This dissociation parallels that of Rab3A observed under the same conditions. In contrast, no change was observed in the membrane-association of Rab5, a Rab protein localized on early endosomes. We conclude that in the nervous system Rab3C is localized on synaptic vesicles and, like Rab3A, cycles on and off the synaptic vesicle membrane in parallel with exocytotic release of neurotransmitter.

Amino Acid Sequence↗