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M Mann

Publications and source records attributed to M Mann.

At least 37 records · Page 2Linked to original sources
PubMed2001

Ephrin B1 is expressed on neuroepithelial cells in correlation with neocortical neurogenesis.

To identify molecules involved in neurogenesis, we have raised monoclonal antibodies against embryonic day 12.5 mouse telencephalon. One antibody, monoclonal antibody 25H11, stains predominantly the ventricular zone of the anterior and lateral telencephalon. Purification of the 25H11 antigen, a 47 kDa integral membrane protein, from approximately 2500 mouse telencephali reveals its identity with ephrin B1. Ephrin B1 appears at the onset of neocortical neurogenesis, being first expressed in neuron-generating neuroepithelial cells and rapidly thereafter in virtually all neuroepithelial cells. Expression of ephrin B1 persists through the period of neocortical neurogenesis and is downregulated thereafter. Ephrin B1 is present on the ventricular as well as basolateral plasma membrane of neuroepithelial cells and exhibits an ventricular-high to pial-low gradient across the ventricular zone. Expression of ephrin B1 is also detected on radial glial cells, extending all the way to their pial endfeet, and on neurons in the mantle/intermediate zone but not in the cortical plate. Our results suggest that ephrin B1, presumably via ephrin-Eph receptor signaling, has a role in neurogenesis. Given the ventricular-to-pial gradient of ephrin B1 on the neuroepithelial cell surface and its known role in cell migration in other systems mediated by its repulsive properties, we propose that ephrin B1 may be involved in the migration of newborn neurons out from the ventricular zone toward the neocortex.

Animals↗
PubMed2001

Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion mode.

Phosphorylation is a common form of protein modification. To understand its biological role, the site of phosphorylation has to be determined. Generally, only limited amounts of phosphorylated proteins are present in a cell, thus demanding highly sensitive procedures for phosphorylation site determination. Here, a novel method is introduced which enables the localization of tyrosine phosphorylation in gel-separated proteins in the femtomol range. The method utilizes the immonium ion of phosphotyrosine at m/z 216.043 for positive ion mode precursor ion scanning combined with the recently introduced Q2-pulsing function on quadrupole TOF mass spectrometers. The high resolving power of the quadrupole TOF instrument enables the selective detection of phosphotyrosine immonium ions without interference from other peptide fragments of the same nominal mass. Performing precursor ion scans in the positive ion mode facilitates sequencing, because there is a no need for polarity switching or changing pH of the spraying solvent. Similar limits of detection were obtained in this approach when compared to triple-quadrupole mass spectrometers but with significantly better selectivity, owing to the high accuracy of the fragment ion selection. Synthetic phosphopeptides could be detected at 1 fmol/microL, and 100 fmol of a tyrosine phosphorylated protein in gel was sufficient for the detection of the phosphorylated peptide in the unseparated digestion mixture and for unambiguous phosphorylation site determination. The new method can be applied to unknown protein samples, because the identification and localization of the modification is performed on the same sample.

Amino Acid Sequence↗
PubMed2001

Molecular patterning of the oikoplastic epithelium of the larvacean tunicate Oikopleura dioica.

Appendicularia are protochordates that rely on a complex mucous secretion, the house, to filter food particles from seawater. A monolayer of cells covering the trunk of the animal, the oikoplastic epithelium, secretes the house. This epithelium contains a fixed number of cells arranged in characteristic patterns with distinct sizes and nuclear morphologies. Certain house structures appear to be spatially related to defined, underlying groups of cells in the epithelium. We show that the house is composed of at least 20 polypeptides, a number of which are highly glycosylated, with glycosidase treatments resulting in molecular mass shifts exceeding 100 kDa. Nanoelectrospray tandem mass spectrometric microsequencing of house polypeptides was used to design oligonucleotides to screen an adult Oikopleura dioica cDNA library. This resulted in the isolation of cDNAs coding for three different proteins, oikosin 1, oikosin 2, and oikosin 3. The latter two are novel proteins unrelated to any known data base entries. Oikosin 1 has 13 repeats of a Cys domain, previously identified as a subunit of repeating sequences in some vertebrate mucins. We also find one repeat of this Cys domain in human cartilage intermediate layer protein but find no evidence of this domain in any invertebrate species, including those for which entire genomes have been sequenced. The three oikosins show distinct and complementary expression patterns restricted to the oikoplastic epithelium. This easily accessible epithelium, with differential gene expression patterns in readily identifiable groups of cells with distinctive nuclear morphologies, is a highly attractive model system for molecular studies of pattern formation.

Amino Acid Sequence↗
PubMed2001

Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion.

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear. Ca2+/calmodulin controls this terminal process in many intracellular fusion events. Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles. Between docking and bilayer fusion, V0 sectors from opposing membranes form complexes. V0 trans-complex formation occurs downstream from trans-SNARE pairing, and depends on both the Rab-GTPase Ypt7 and calmodulin. The maintenance of existing complexes and completion of fusion are independent of trans-SNARE pairs. Reconstituted proteolipids form sealed channels, which can expand to form aqueous pores in a Ca2+/calmodulin-dependent fashion. V0 trans-complexes may therefore form a continuous, proteolipid-lined channel at the fusion site. We propose that radial expansion of such a protein pore may be a mechanism for intracellular membrane fusion.

Calmodulin↗
PubMed2001

A functional interaction between the survival motor neuron complex and RNA polymerase II.

The survival motor neuron (SMN) protein, the protein product of the spinal muscular atrophy (SMA) disease gene, plays a role in the assembly and regeneration of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. By nanoelectrospray mass spectrometry, we identified RNA helicase A (RHA) as an SMN complex-associated protein. RHA is a DEAH box RNA helicase which binds RNA polymerase II (pol II) and reportedly functions in transcription. SMN interacts with RHA in vitro, and this interaction is impaired in mutant SMNs found in SMA patients. Coimmunoprecipitation demonstrated that the SMN complex is associated with pol II, snRNPs, and RHA in vivo. In vitro experiments suggest that RHA mediates the association of SMN with the COOH-terminal domain of pol II. Moreover, transfection of cells with a dominant negative mutant of SMN, SMNDeltaN27, causes accumulation of pol II, snRNPs, and RHA in nuclear structures that contain the known markers of gems and coiled bodies, and inhibits RNA pol I and pol II transcription in vivo. These findings indicate a functional as well as physical association of the SMN complex with pol II and suggest a role for the SMN complex in the assembly of the pol II transcription/processing machinery.

Animals↗
PubMed2001

Mass spectrometry allows direct identification of proteins in large genomes.

Proteome projects seek to provide systematic functional analysis of the genes uncovered by genome sequencing initiatives. Mass spectrometric protein identification is a key requirement in these studies but to date, database searching tools rely on the availability of protein sequences derived from full length cDNA, expressed sequence tags or predicted open reading frames (ORFs) from genomic sequences. We demonstrate here that proteins can be identified directly in large genomic databases using peptide sequence tags obtained by tandem mass spectrometry. On the background of vast amounts of noncoding DNA sequence, identified peptides localize coding sequences (exons) in a confined region of the genome, which contains the cognate gene. The approach does not require prior information about putative ORFs as predicted by computerized gene finding algorithms. The method scales to the complete human genome and allows identification, mapping, cloning and assistance in gene prediction of any protein for which minimal mass spectrometric information can be obtained. Several novel proteins from Arabidopsis thaliana and human have been discovered in this way.

Amino Acid Sequence↗
PubMed2001

Quadrupole time-of-flight versus triple-quadrupole mass spectrometry for the determination of phosphopeptides by precursor ion scanning.

An API 3000 triple-quadrupole instrument and a QSTAR Pulsar quadrupole time-of-flight (TOF) mass spectrometer were compared for the determination of phosphopeptides by precursor ion scanning in both the positive and negative nanoelectrospray ionization modes. The limits of detection for synthetic phosphopeptides were similar (500 amol microl(-1)) for both types of instruments when monitoring precursors of -79 Da (PO(3)(-)). However, the quadrupole TOF system was approximately fivefold more sensitive (1 fmol microl(-1)) than the triple-quadrupole instrument (5 fmol microl(-1)) when monitoring precursors of 216 Da (immonium ion of phosphotyrosine). The recently introduced Q(2)-pulsing function, which enhances the transmission of fragment ions of a selected m/z window from the collision cell into the TOF part, improved the sensitivity of precursor ion scans on a quadrupole TOF instrument. The selectivity of precursor ion scans is much better on quadrupole TOF systems than on triple quadrupoles because the high resolving power of the reflectron-TOF mass analyzer permits high-accuracy fragment ion selection at no expense of sensitivity. This minimizes interferences from other peptide fragment ions (a-, b-, and y- type) of the same nominal mass but with sufficient differences in their exact masses. As a result, the characteristic immonium ion of phosphotyrosine at m/z 216.043 can be utilized for the selective detection of tyrosine phosphorylated peptides. Our data suggest that, in addition to their superior performance for peptide sequencing, quadrupole TOF instruments also offer a very viable alternative to triple quadrupoles for precursor ion scanning, thus combining high sensitivity and selectivity for both MS and MS/MS experiments in one instrument.

Amino Acid Sequence↗
PubMed2001

Similarity between condensed phase and gas phase chemistry: fragmentation of peptides containing oxidized cysteine residues and its implications for proteomics.

Amino acid residues containing thioethers are easily oxidized during protein purification, derivatization, and/or digestion. For instance, oxidation of methionine residues in proteins during SDS-PAGE is commonly observed. Under low energy collision induced dissociation this gives rise to a second series of fragment ion of lower abundance that are shifted by -64 Da when compared to the oxidized methionine-containing fragments. We report here that alkylated cysteine residues can be found in their oxidized form too, indicating that the oxidation of thioethers can occur during and following protein digestion and not only during SDS-PAGE or reduction and alkylation. Collision induced dissociation experiments on the singly- and multiply-charged species reveals that these peptides preferentially undergo elimination reactions that forms a dehydroalanine from the oxidized, alkylated cysteine residue. This contrasts to the less abundant elimination reaction of peptides containing oxidized methionines which cannot form an alpha,beta-unsaturated compound, but parallels the condensed phased chemistry of sulfoxides. The masses of both precursor and product ions are shifted such that these peptides cannot be identified in database searches with current algorithms. Incorporation of this fragmentation pattern is important for the isotope-coded affinity tag approach since this method is based on peptides containing cysteine residues.

Cysteine↗
PubMed2001

A novel proliferation-associated variant of CFR-1 defined by a human monoclonal antibody.

The germline coded human monoclonal IgM antibody 103/51 was isolated from a gastric carcinoma patient. This antibody binds to a 130-kd membrane molecule and has a mitotic effect on tumor cells in vitro. To characterize the target, we sequenced the protein and showed that the antibody binds to the cysteine-rich fibroblast growth factor receptor (CFR)-1, which is highly homologous to MG-160 and the E-selectin-ligand (ESL)-1. The epitope was determined by glycosidase-digestion experiments to be an N-linked carbohydrate side chain. Immunohistochemistry was used to investigate the tissue distribution of CFR-1. Different healthy tissues were tested and only the collecting tubes of the kidney, the Golgi apparatus, and the glomerular and fascicular zones of the adrenal gland stained positive. However, on malignant tissue the receptor is overexpressed in nearly all tested stomach cancers (12 of 15) and other tested carcinomas (13 of 15). Most interestingly, the receptor is also present in Helicobacter pylori gastritis and gastric dysplasia, but absent on uninflamed stomach mucosa. This restricted tissue pattern indicates that antibody 103/51 reacts with a membrane-bound variant of CFR-1, which is mainly expressed on transformed cells and precursor lesions and is essential for proliferation processes. The possible activity of antibody 103/51 as an activating ligand in these proliferative changes of gastric epithelial mucosa is discussed.

Adenocarcinoma↗
PubMed2001

Targeting cyclooxygenase 2 and HER-2/neu pathways inhibits colorectal carcinoma growth.

BACKGROUND & AIMS: The cyclooxygenase 2 (COX-2) and ErbB/HER pathways are important modulators of cancer cell growth. We sought to determine the effects of treatment with a specific COX-2 inhibitor and/or a monoclonal antibody against the ErbB receptor subtype HER-2/neu on carcinoma cell growth. METHODS: A cell-proliferation assay was used to determine the response of HCA-7 cells to the HER-3/HER-4 ligand heregulin beta-1 (HRGbeta-1). Both in vitro and in vivo assays were used to determine the effects of the selective COX-2 inhibitor, celecoxib, and/or an anti-HER-2/neu monoclonal antibody (either Herceptin [Genetech Inc., S. San Francisco, CA] or 2C4) on cell growth. RESULTS: HCA-7 cells express HER-2/neu messenger RNA and protein, and exposure of these cells to HRGbeta-1 results in a significant stimulation of cell growth. Celecoxib or Herceptin inhibits HCA-7 cell growth in vitro and in vivo. Combination therapy with celecoxib plus Herceptin or celecoxib plus 2C4 resulted in additive effects that resulted in almost complete inhibition of tumor growth. CONCLUSIONS: Combined treatment with COX-2 and HER-2/neu inhibitors more effectively reduces colorectal carcinoma growth than either agent alone. Therefore, targeting of both the COX-2 and ErbB signaling pathways may represent a novel approach for the treatment and/or prevention of colorectal cancer in humans.

Animals↗
PubMed2001

Detection of caustic oesophageal injury with technetium 99m-labelled sucralfate.

BACKGROUND/PURPOSE: Radiolabelled sucralfate has been used to show the extent and severity of reflux oesophagitis, peptic ulceration, and inflammatory bowel disease. Endoscopy under general anaesthetic has been the preferred method to assess the injury after caustic ingestion. The aim of this study was to assess whether sucralfate has an affinity for the chemically injured oesophageal mucosa and, if so, to assess the accuracy of radiolabeled sucralfate as an indicator of presence and extent of oesophageal injury. METHODS: A prospective study was conducted of 22 patients with mean age of 30 months (range, 13 to 90) admitted consecutively with a history of caustic ingestion between January 1998 and January 2000. A sucralfate-labelled scan followed by endoscopic assessment of upper gastrointestinal tract with documentation of extent and grade of injury was performed in all patients within 24 hours of admission except the first 6 who underwent scan after the endoscopy. The sucralfate was labelled by the direct stannous reduction method. Oesophageal transit was studied by recording 120 images (64 x 64 matrix size) at 1 image per second while the child swallowed 5 mL of labelled sucralfate containing 2 to 3 MBq Technetium 99m. Retention of radiolabelled oesophageal activity was considered abnormal. RESULTS: The caustic substances ingested were household cleaners in 18, potassium permanganate in 3, and pool chlorine in 1. There were 11 scans that showed residual activity in the oesophagus, which correlated exactly with endoscopic findings. The other 11 patients had normal oesophageal mucosa, but 2 were found to be falsely positive on scanning. In 2 cases repeat sucralfate scan results correlated well with the healing process assessed endoscopically. CONCLUSIONS: The results indicate that technetium 99m sucralfate swallow is an accurate technique for assessing oesophageal injury after ingestion of caustic substances. In addition, it may be used to document healing.

Burns, Chemical↗
PubMed2001

The nuclear export receptor Xpo1p forms distinct complexes with NES transport substrates and the yeast Ran binding protein 1 (Yrb1p).

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin beta-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.

Active Transport, Cell Nucleus↗
PubMed2001

Mass spectrometric analysis of a UV-cross-linked protein-DNA complex: tryptophans 54 and 88 of E. coli SSB cross-link to DNA.

Protein-nucleic acid complexes are commonly studied by photochemical cross-linking. UV-induced cross-linking of protein to nucleic acid may be followed by structural analysis of the conjugated protein to localize the cross-linked amino acids and thereby identify the nucleic acid binding site. Mass spectrometry is becoming increasingly popular for characterization of purified peptide-nucleic acid heteroconjugates derived from UV cross-linked protein-nucleic acid complexes. The efficiency of mass spectrometry-based methods is, however, hampered by the contrasting physico-chemical properties of nucleic acid and peptide entities present in such heteroconjugates. Sample preparation of the peptide-nucleic acid heteroconjugates is, therefore, a crucial step in any mass spectrometry-based analytical procedure. This study demonstrates the performance of four different MS-based strategies to characterize E. coli single-stranded DNA binding protein (SSB) that was UV-cross-linked to a 5-iodouracil containing DNA oligomer. Two methods were optimized to circumvent the need for standard liquid chromatography and gel electrophoresis, thereby dramatically increasing the overall sensitivity of the analysis. Enzymatic degradation of protein and oligonucleotide was combined with miniaturized sample preparation methods for enrichment and desalting of cross-linked peptide-nucleic acid heteroconjugates from complex mixtures prior to mass spectrometric analysis. Detailed characterization of the peptidic component of two different peptide-DNA heteroconjugates was accomplished by matrix-assisted laser desorption/ionization mass spectrometry and allowed assignment of tryptophan-54 and tryptophan-88 as candidate cross-linked residues. Sequencing of those peptide-DNA heteroconjugates by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry identified tryptophan-54 and tryptophan-88 as the sites of cross-linking. Although the UV-cross-linking yield of the protein-DNA complex did not exceed 15%, less than 100 pmole of SSB protein was required for detailed structural analysis by mass spectrometry.

Amino Acids↗
PubMed2001

The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins.

snRNPs, integral components of the pre-mRNA splicing machinery, consist of seven Sm proteins which assemble in the cytoplasm as a ring structure on the snRNAs U1, U2, U4, and U5. The survival motor neuron (SMN) protein, the spinal muscular atrophy disease gene product, is crucial for snRNP core particle assembly in vivo. SMN binds preferentially and directly to the symmetrical dimethylarginine (sDMA)-modified arginine- and glycine-rich (RG-rich) domains of SmD1 and SmD3. We found that the unmodified, but not the sDMA-modified, RG domains of SmD1 and SmD3 associate with a 20S methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 and a JBP1-interacting protein, pICln. JBP1 binds SmD1 and SmD3 via their RG domains, while pICln binds the Sm domains. JBP1 produces sDMAs in the RG domain-containing Sm proteins. We further demonstrate the existence of a 6S complex that contains pICln, SmD1, and SmD3 but not JBP1. SmD3 from the methylosome, but not that from the 6S complex, can be transferred to the SMN complex in vitro. Together with previous results, these data indicate that methylation of Sm proteins by the methylosome directs Sm proteins to the SMN complex for assembly into snRNP core particles and suggest that the methylosome can regulate snRNP assembly.

Arginine↗