Biomedical subjects

M Mann

Publications and source records attributed to M Mann.

At least 163 records · Page 9Linked to original sources

PubMed1997

Nup93, a vertebrate homologue of yeast Nic96p, forms a complex with a novel 205-kDa protein and is required for correct nuclear pore assembly.

Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human, rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAAO225, present in the human database. The putative human nucleoporin of 205 kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. The analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex-depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates.

Amino Acid Sequence↗

PubMed1997

Lack of clinical utility of bronchoalveolar lavage cultures for cytomegalovirus in HIV infection.

This study assessed the presence of cytomegalovirus (CMV) in bronchoalveolar lavage (BAL) in three subpopulations of HIV-infected patients and correlated its presence with clinical status during 3 mo of follow-up. Nineteen asymptomatic volunteers, six patients with CMV retinitis, and 46 patients with acute pulmonary symptoms underwent BAL and were assessed for CMV by cytopathology, conventional shell vial cultures, and antigen detection. Transbronchial biopsies were also obtained when possible and evaluated for histopathologic changes of CMV. All patients were followed for approximately 3 mo. Cytomegalovirus was detected in BAL in nine of 19 (47%) asymptomatic volunteers, in all six patients with CMV retinitis, and in 33 of 46 (72%) patients with pulmonary symptoms. Only one symptomatic patient with a positive CMV BAL culture developed clinically significant CMV pulmonary disease; this patient developed disseminated CMV and died. The only other death occurred in a patient with CMV retinitis who developed staphylococcal bacteremia. None of the asymptomatic volunteers or patients with CMV retinitis developed evidence of CMV pneumonia or any other organ disease with CMV. Cytomegalovirus is frequently detected in BAL from HIV-infected patients regardless of their pulmonary symptoms and its presence does not clinically predict significant pulmonary morbidity or mortality in 3 mo of follow-up.

AIDS-Related Opportunistic Infections↗

PubMed1997

Canadian beef quality audit.

A study was conducted in 4 Canadian processing plants in 1995-96 to determine the prevalence of quality defects in Canadian cattle. One percent of the annual number of cattle processed in Canada were evaluated on the processing floor and 0.1% were graded in the cooler. Brands were observed on 37% and multiple brands on 6% of the cattle. Forty percent of the cattle had horns, 20% of which were scurs, 33% were stubs, 10% were tipped, and 37% were full length. Tag (mud and manure on the hide) was observed on 34% of the cattle. Bruises were found on 78% of the carcasses, 81% of which were minor in severity. Fifteen percent of the bruises were located on the round, 29% on the loin, 40% on the rib, 16% on the chuck, and 0.02% on the brisket. Grubs were observed in 0.02% of the steers, and injection sites were observed in 1.3% of whole hanging carcasses. Seventy percent of the livers were passed for human food and 14% for pet food; 16% were condemned. Approximately 71% of the liver condemnations were due to liver abscesses. Four percent of the heads, 6% of the tongues, and 0.2% of whole carcasses were condemned. The pregnancy rate in female cattle was approximately 6.7%. The average hot carcass weight was 357 kg (s = 40) in steers, 325 kg (s = 41) in heifers, 305 kg (s = 53) in cows, 388 kg (s = 62) in virgin bulls and 340 kg (s = 39) in mature bulls. The average ribeye area in all cattle was 84 cm2 (s = 12); range 29 cm2 to 128 cm2. Grade fat was highly variable and averaged 9 mm (s = 4) for steers and heifers, 6 mm (s = 6) for cows, 5 mm (s = 1) for virgin bulls, and 4 mm (s = 0.5) for mature bulls. The average lean meat yield was 59.7% in cattle (s = 3.4); range 39% to 67%. One percent of the carcasses were devoid of marbling, 1% were dark cutters, and 0.05% of the steer carcasses were staggy. Six percent of the carcasses had poor conformation, 3.7% were underfinished, and 0.7% were overfinished. Yellow fat was observed in 4% of the carcasses; 10% of carcasses were aged. Based on January 1996 prices, the economic analysis showed that the Canadian beef industry lost $70.52 per head or $189.6 million annually from quality nonconformities. Methods identified to reduce these nonconformities included improvements in management, animal identification, handling, genetic selection, marketing, grading, and information transfer.

Abattoirs↗

PubMed1997

[Surgical management of dysfunctions of dialysis fistulas].

Due to the superficial position of shunt vessels we do not use complicated equipment or diagnostic procedures in the morphological assessment of shunt insufficiency or shunt occlusion. Preoperatively, we merely conduct a clinical examination including inspection, pulse, palpation of the shunt veins and arteries with and without venous congestion, and shunt auscultation. Subsequently, we reoperate the shunt under local anesthesia, at which time the anastomosis is usually checked and repositioned. From January 1995 to May 1996, 539 shunt operations were performed in 371 patients, whereby 263 of these were reoperations. The reoperations were performed due to shunt occlusion (n = 144), shunt stenoses (n = 60), shunt aneurysms (n = 17), steal syndrome (n = 3), and rare complications such as hematoma, shunt infection, seroma, and other disturbances (n = 6) (32 patients were treated in other clinics after reoperation or the functional disturbance of the shunt was not recorded). Angiography was only conducted if the clinical examination did not provide enough information about the shunt problems, and so, preoperatively, only six angiographic examinations were conducted (stenosis, n = 3; aneurysm, n = 1; steal syndrome, n = 2). All reoperations, with only few exceptions (PTFE shunt), were conducted under local anesthesia. At reoperation, 184 new proximal shunts were made, 14 thrombectomies conducted, seven PTFE fistulas made, 13 shunts positioned on the opposite side, five shunts ligated, and eight various other operations performed (32 patients were given further treatment elsewhere or no treatment records were available). If during reoperation flow disturbances were suspected (arterial stenosis) or the blood was flowing towards center (proximal venous stenosis) angiography was performed intraoperatively to assess the condition of the vessels. The 4% rate of early occlusion using this procedure was very low. Only 21 patients had to have more than two reoperations. After 2 years 65% of the reoperated AV fistulas were still functional. Without further diagnostic procedures, we performed immediate, outpatient reoperation under local anesthesia, preferably positioning new proximal shunts so that dialysis could be conducted immediately using the existing dialysis shunt. Only if there were particularly complex functional shunt disturbances (steal syndrome, proximal venous flow disturbance, or arterial stenosis) did we employ other diagnostic procedures (angiography, DSA). With this approach the functional shunt disturbances could be eliminated quickly and effectively, which also minimized the cost and stress for the patient.

Adult↗

PubMed1996

Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels.

The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials, the method led to unambiguous identification in all cases. In the largest individual protein identification project to date, a total of 150 gel spots-many of them at subpicomole amounts-were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base. More than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome. This study establishes that mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of choice to connect genome and proteome.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1996

Isoprenylation of the G protein gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.

We have previously shown that isoprenylation and/or additional post-translational processing of the G protein gamma 1 subunit carboxyl terminus is required for beta 1 gamma 1 subunit stimulation of phospholipase C-beta 2 (PLC beta 2) [Dietrich, A., Meister, M., Brazil, D., Camps, M., & Gierschik, P. (1994) Eur. J. Biochem. 219, 171-178]. To examine whether isoprenylation of the gamma 1 subunit alone is sufficient for beta 1 gamma 1-mediated PLC beta 2 stimulation or whether any of the two subsequent modifications, proteolytic removal of the carboxyl-terminal tripeptide and/or carboxylmethylation, is required for this effect, nonisoprenylated recombinant beta 1 gamma 1 dimers were produced in baculovirus-infected insect cells, purified to near homogeneity, and then isoprenylated in vitro using purified recombinant protein farnesyltransferase. Analysis of the beta 1 gamma 1 dimer after in vitro farnesylation by reversed phase high-performance liquid chromatography followed by delayed extraction matrix-assisted laser desorption/ionization mass spectrometry confirmed that the gamma 1 subunit was carboxyl-terminally farnesylated but not proteolyzed and carboxylmethylated. Functional reconstitution of in vitro-farnesylated beta 1 gamma 1 dimers with a recombinant PLC beta 2 isozyme revealed that farnesylation rendered recombinant nonisoprenylated beta 1 gamma 1 dimers capable of stimulating PLC beta 2 and that the degree of this stimulation was only approximately 45% lower for in vitro-farnesylated beta 1 gamma 1 dimers than for fully modified native beta 1 gamma 1 purified from bovine retinal rod outer segments. Taken together, these results suggest that isoprenylation of the gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.

Alkyl and Aryl Transferases↗

PubMed1996

Clostridium novyi alpha-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins.

The lethal and edema-inducing alpha-toxin from Clostridium novyi causes rounding up of cultured cell lines by redistribution of the actin cytoskeleton. alpha-Toxin belongs to the family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A and B and the lethal toxin from Clostridium sordellii. Toxin A and toxin B have been recently identified as monoglucosyltransferases to modify the low molecular mass GTPases of the Rho subfamily (Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503 and Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) J. Biol. Chem. 270, 13932-13936). We report here the identification of the alpha-toxin-catalyzed modification of Rho. Using electrospray mass spectrometry, the mass of the modification was determined as 203 Da, consistent with a N-acetyl-hexosamine moiety. UDP-N-acetyl-glucosamine selectively served as cosubstrate for alpha-toxin-catalyzed modification into the Rho subfamily proteins Rho, Rac, Cdc42, and RhoG. The acceptor amino acid of N-acetyl-glucosaminylation was identified by mutagenesis as Thr-37 in Rho (equivalent to Thr-35 in Rac/Cdc42), which is located in the effector domain of the GTPases. C. novyi alpha-toxin seems to mediate its cytotoxic effects on cells by mimicking endogenous post-translational modification of cellular proteins.

3T3 Cells↗

PubMed1996

The yeast protein Arc1p binds to tRNA and functions as a cofactor for the methionyl- and glutamyl-tRNA synthetases.

Arc1p was found in a screen for components that interact genetically with Los1p, a nuclear pore-associated yeast protein involved in tRNA biogenesis. Arc1p is associated with two proteins which were identified as methionyl-tRNA and glutamyl-tRNA synthetase (MetRS and GluRS) by a new mass spectrometry method. ARC1 gene disruption leads to slow growth and reduced MetRS activity, and synthetically lethal arc1- mutants are complemented by the genes for MetRS and GluRS. Recombinant Arc1p binds in vitro to purified monomeric yeast MetRS, but not to an N-terminal truncated form, and strongly increases its apparent affinity for tRNAMet. Furthermore, Arc1p, which is allelic to the quadruplex nucleic acid binding protein G4p1, exhibits specific binding to tRNA as determined by gel retardation and UV-cross-linking. Arc1p is, therefore, a yeast protein with dual specificity: it associates with tRNA and aminoacyl-tRNA synthetases. This functional interaction may be required for efficient aminoacylation in vivo.

Acylation↗

PubMed1996

The purification and characterization of the catalytic domain of Src expressed in Schizosaccharomyces pombe. Comparison of unphosphorylated and tyrosine phosphorylated species.

The catalytic domain of chicken Src including the C-terminal tail (Src-CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono-phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono-phosphorylated form is phosphorylated either at Tyr416 or at Tyr436. The stability of Src-CD is comparable to the wild-type protein. Src-CD auto-phosphorylates and efficiently phosphorylates substrate peptides and proteins. Auto-phosphorylation occurs by an intermolecular mechanism and is completely inhibited by an excess of substrate peptide. Kinetic measurements for two exogenous substrates, the Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showed that the overall activity (kcat) of the Src-CD molecule is about 10 times higher than that of wild-type Src. The kcat values for phosphorylation of the Src substrate peptide are similar for the unphosphorylated and monophosphorylated Src-CD (50 min-1), but the apparent K(m) values differ significantly (approximately 3 microM and 10 microM, respectively). Therefore, at low substrate concentrations in vitro the mono-phosphorylated form is more active, in agreement with the importance of Tyr416 for in vivo activity. The apparent K(m) values of the mono-phosphorylated Src-CD and wild-type Src for the Src substrate peptide and enolase are similar, indicating that, under these conditions, the kinase domain is mainly responsible for substrate binding.

Amino Acid Sequence↗

PubMed1996

Sequence tag identification of intact proteins by matching tanden mass spectral data against sequence data bases.

Molecular and fragment ion data of intact 8- to 43-kDa proteins from electrospray Fourier-transform tandem mass spectrometry are matched against the corresponding data in sequence data bases. Extending the sequence tag concept of Mann and Wilm for matching peptides, a partial amino acid sequence in the unknown is first identified from the mass differences of a series of fragment ions, and the mass position of this sequence is defined from molecular weight and the fragment ion masses. For three studied proteins, a single sequence tag retrieved only the correct protein from the data base; a fourth protein required the input of two sequence tags. However, three of the data base proteins differed by having an extra methionine or by missing an acetyl or heme substitution. The positions of these modifications in the protein examined were greatly restricted by the mass differences of its molecular and fragment ions versus those of the data base. To characterize the primary structure of an unknown represented in the data base, this method is fast and specific and does not require prior enzymatic or chemical degradation.

Amino Acid Sequence↗

PubMed1996

FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death--inducing signaling complex.

To identify CAP3 and CAP4, components of the CD95 (Fas/APO-1) death-inducing signaling complex, we utilized nano-electrospray tandem mass spectrometry, a recently developed technique to sequence femtomole quantities of polyacrylamide gel-separated proteins. Interestingly, CAP4 encodes a novel 55 kDa protein, designated FLICE, which has homology to both FADD and the ICE/CED-3 family of cysteine proteases. FLICE binds to the death effector domain of FADD and upon overexpression induces apoptosis that is blocked by the ICE family inhibitors, CrmA and z-VAD-fmk. CAP3 was identified as the FLICE prodomain which likely remains bound to the receptor after proteolytic activation. Taken together, this is unique biochemical evidence to link a death receptor physically to the proapoptotic proteases of the ICE/CED-3 family.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.

Proteins from silver-stained gels can be digested enzymatically and the resulting peptide analyzed and sequenced by mass spectrometry. Standard proteins yield the same peptide maps when extracted from Coomassie- and silver-stained gels, as judged by electrospray and MALDI mass spectrometry. The low nanogram range can be reached by the protocols described here, and the method is robust. A silver-stained one-dimensional gel of a fraction from yeast proteins was analyzed by nano-electrospray tandem mass spectrometry. In the sequencing, more than 1000 amino acids were covered, resulting in no evidence of chemical modifications due to the silver staining procedure. Silver staining allows a substantial shortening of sample preparation time and may, therefore, be preferable over Coomassie staining. This work removes a major obstacle to the low-level sequence analysis of proteins separated on polyacrylamide gels.

Amino Acid Sequence↗

PubMed1996

Parent ion scans of unseparated peptide mixtures.

The nanoelectrospray ion source (NanoES) developed recently has proven to be an excellent tool for the sequencing of peptides out of unseparated mixtures. At lower levels of analyte, however, it is increasingly difficult to distinguish the peptide ions in the spectrum, limiting the overall sensitivity of the procedure. Scans for the parents of common fragmentation products of peptides such as immonium ions allow determination of peptide ion masses even when these ions have signal-to-noise ratios of < 1 in the mass spectrum. With this technique, concentration limits for sequencing peptides could be improved to < 5 fmol/microL in favorable cases. Parent ions scan were used to identify posttranslational modifications of peptides in mixtures, with phosphorylation and glycosylation as examples. Detecting phosphorylation by parent scans increased sensitivity by 1-2 orders of magnitude. Modified peptides can be subjected to tandem mass spectrometry in the same experiment to localize the modification. The present approach expands the range of application of nanoelectrospray and complements established liquid chromatography/mass spectrometry methods.

Amino Acid Sequence↗

PubMed1996

Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.

Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.

Amino Acid Sequence↗

PubMed1996

Analytical properties of the nanoelectrospray ion source.

The nanoelectrospray ion source (nanoES) has recently been developed and described theoretically. It is different from conventional electrospray sources and from other miniaturized electrospray sources by (i) its 1-2 microns spraying orifice achieved by pulling the spraying capillary to a fine tip, (ii) its very low flow rate of approximately 20 nL/min and the small size of droplets it generates, and (iii) the absence of solvent pumps and inlet valves. The fabrication and operation of nanoES needles is described in detail. Solutions with up to 0.1 M salt contents could be sprayed without sheath flow or pneumatic assist. Improved desolvation in nanoES led to instrument-limited resolution of the signals of a glycoprotein and the ability to signal average extensively allowed the C-terminal sequencing of a 40 kDa protein. Extensive mass spectrometric and tandem mass spectrometric investigation of the components of an unseparated peptide mixture was demonstrated by verification of 93% of the sequence of carbonic anhydrase. A rapid and robust desalting/concentration step coupled to the nanoES procedure allows the direct analysis of impure samples such as peptide mixtures extracted after in-gel digestion.

Amino Acid Sequence↗

PubMed1996

Aspects of the sequencing of carbohydrates and oligonucleotides by matrix-assisted laser desorption/ionization post-source decay.

The possibility of structural elucidation of carbohydrates and oligonucleotides by matrix-assisted laser desorption/ionization followed by post-source decay (MALDI-PSD) is investigated. Spectra of both classes of compounds exhibit the better signal-to-noise ratios for the intact species in the negative-ion mode, but the most informative spectra for structural elucidation by PSD are obtained in the positive-ion mode. A novel fragmentation mode is demonstrated on a model sialylated complex type N-linked oligosaccharide. Prompt fragmentation in the positive-ion mode can remove the sialic acids at high laser irradiance if a suitable matrix is used. The remaining sugar backbone is then characterized by post-source decay. A modified oligonucleotide is analyzed with 3-hydroxypicolinic acid as the matrix and ammonium tartrate to displace sodium. Enough structural information is obtained to verify the modification.

Gas Chromatography-Mass Spectrometry↗

PubMed1996

Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionization peptide maps.

Peptide mass maps obtained by matrix-assisted laser desorption ionization (MALDI) are an attractive means to identify proteins by searches in sequence databases. Here we demonstrate that the recently introduced delayed ion-extraction technique, when coupled to reflectron MALDI time-of-flight mass spectrometry, leads to dramatically improved search specificity. Routine resolution in the range of 6,000 to 12,000 allows assignment of monoisotopic masses throughout the peptide mass range. Database searches can be performed with high precision by use of a mass accuracy which is currently better than 30 ppm over a wide mass range and better than 5 ppm for a narrow mass range. This high performance makes it possible to identify proteins with fewer peptide masses than before. Additional low intensity peaks can be assigned after a search because of the improved signal-to-noise ratio of delayed-extraction peptide mass spectra, increasing sequence coverage of matched proteins. The improvements in database search specificity can be used to identify the components of simple protein mixtures. In combination with advanced sample preparation and automation techniques, delayed-extraction MALDI time-of-flight mass spectrometry is now an extremely powerful tool for the database identification of proteins.

Amino Acid Sequence↗

PubMed1996

Sequence patterns produced by incomplete enzymatic digestion or one-step Edman degradation of peptide mixtures as probes for protein database searches.

Mass spectrometric peptide mapping of proteins isolated by polyacrylamide gel electrophoresis is a rapid method for identifying proteins in sequence databases. A majority of tryptic peptide maps were found to contain pairs of peptide ion peaks separated by the molecular weight of the lysyl or arginyl residue. These peaks originate from amino acid sequence patterns such as Lys-Lys where trypsin has cleaved C-terminals to either one of the lysines. The peptide mass and the pattern define an N- or C-terminal sequence tag. Searching sequence databases by such a sequence tag results in only a moderate number of matches and significantly reduces the number of database matches when used in combination with a peptide mass map. Two N- or C-terminal sequence tags alone unambiguously identify a protein in most cases. The technique discussed here is simple, does not require additional measurements, and increases the percentage of protein samples that can be identified by their mass maps alone. N-Terminal peptide sequence tags for database searching can also be generated by manual one-step Edman degradation of the unseparated peptide mixture.

Databases, Factual↗