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Biomedical subjects

M Mann

Publications and source records attributed to M Mann.

At least 181 records · Page 10Linked to original sources
PubMed1996

New carbamate supports for the preparation of 3'-amino-modified oligonucleotides.

A novel approach for the preparation of oligonucleotides carrying amino groups at the 3'-end is described. Several CPG supports having aminoalkyl groups and 3'-amino-2',3'-dideoxynucleosides linked through base-labile carbamate linkages such as 2-(2-nitrophenyl)ethoxycarbonyl and fluorenylmethoxycarbonyl were prepared using two different strategies. These supports are compatible to the standard solid phase phosphite-triester methodology and yield oligonucleotides containing amino groups at the 3'-end. Several properties of the 3'-amino oligonucleotides, such as nuclease resistance, hybridization, and preparation of oligonucleotide conjugates are discussed.

Carbamates↗
PubMed1996

Developments in matrix-assisted laser desorption/ionization peptide mass spectrometry.

Most characteristics of matrix-assisted laser desorption/ionization (MALDI) are ideal for the analysis of biomolecules. New preparation techniques have dramatically increased mass accuracy and resolution, making MALDI a high-performance mass spectrometric technique for peptide mass analysis. Attempts to obtain amino acid sequence information by MALDI have been partially successful. The technique has been put to novel uses in protein primary structure characterization.

Amino Acid Sequence↗
PubMed1996

Identification of the major membrane and core proteins of vaccinia virus by two-dimensional electrophoresis.

Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known about the molecular events that occur during that process. Towards this goal, we have identified the major membrane and core proteins of the intracellular mature virus (IMV). Pure IMV preparations were subjected to Nonidet P-40 (NP-40) and dithiothreitol (DTT) treatment to separate the core proteins from the membrane proteins. These proteins were subsequently separated by two-dimensional (2D) gel electrophoresis, and the major polypeptide spots, as detected by silver staining and 35S labeling, were identified by either matrix-assisted laser desorption/ionization mass spectrometry, N-terminal amino acid sequencing, or immunoprecipitation with defined antibodies. Sixteen major spots that partitioned into the NP-40-DTT-soluble fraction were identified; 11 of these were previously described virally encoded proteins and 5 were cellular proteins, mostly of mitochondrial origin. The core fraction revealed four major spots of previously described core proteins, two of which were also detected in the membrane fraction. Subsequently, the NP-40-DTT-soluble and -insoluble fractions from purified virus preparations, separated by 2D gels, were compared with postnuclear supernatants of infected cells that had been metabolically labeled at late times (6 to 8 h) postinfection. This relatively short labeling period as well as the apparent shutoff of host protein synthesis allowed the selective detection in such postnuclear supernatants of virus-encoded proteins. These postnuclear supernatants were subsequently treated with Triton X-114 or with sodium carbonate to distinguish the membrane proteins from the soluble proteins. We have identified the major late membrane and nonmembrane proteins of the IMV as they occur in the virus as well as in infected cells. This 2D gel map should provide an important reference for future molecular studies of vaccinia virus morphogenesis.

Amino Acid Sequence↗
PubMed1996

Sexual behaviour in travellers abroad attending an inner-city genitourinary medicine clinic.

OBJECTIVE: To investigate frequency of sexual encounters with new partners abroad in patients attending a genitourinary clinic (GUM). METHODS: In a case series 464 attenders at a genitourinary medicine clinic completed an anonymous self-administered questionnaire, if they had been abroad recently, enquiring about sexual behaviour abroad. RESULTS: 28.4% of subjects admitted to sex with a new partner abroad with only 41.7 per cent consistently using condoms. There were no significant differences in condom use for gender, ethnicity or type of visit and relationship. Twenty-nine per cent of those admitting to sex abroad had more than one partner. The risk of multiple partners was not associated with gender, ethnicity, type of visit (holiday or business) or type of relationship (heterosexual or homosexual). The first partner abroad for 63% of men and 62.5% of women was of a nationality other than that of United Kingdom residents. Non-Caucasians and homosexuals were significantly more likely to have first partners abroad from outside the UK than Caucasians and heterosexuals respectively. CONCLUSION: The occurrence of casual sex abroad in GUM attenders suggests that further research is needed to establish targetable risk factors for this type of behaviour amendable to change through health promotion.

Adolescent↗
PubMed1995

Automation of micro-preparation and enzymatic cleavage of gel electrophoretically separated proteins.

To achieve high throughput, protein microcharacterization sample preparation must be automated. We describe a cartesian robot capable of processing 32 protein samples in parallel. The system is based on specially designed flow-through reactors for contamination-free reagent delivery and removal. Washing of excised gel pieces, reduction and alkylation, proteolytic cleavage and peptide extraction are performed in these reactors. Compatibility of the system with HPLC peptide separation and Edman degradation as well as with laser desorption mass spectrometry of the unseparated mixture is demonstrated. This is the first report describing automated preparation and processing of multiple protein samples.

Amino Acid Sequence↗
PubMed1995

The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins.

The enterotoxin from Clostridium difficile (ToxA) is one of the causative agents of the antibiotic-associated pseudomembranous colitis. In cultured monolayer cells ToxA exhibits cytotoxic activity to induce disassembly of the actin cytoskeleton, which is accompanied by morphological changes. ToxA-induced depolymerization of actin filaments is correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, K. (1995) J. Clin. Invest. 95, 1026-1031). Here we report on the identification of the ToxA-induced modification of Rho. Applying electrospray mass spectrometry, the mass of the modification was determined as 162 Da, which is consistent with the incorporation of a hexose into Rho. From several hexoses tested UDP-glucose selectively served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated peptide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (RhoA, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, and Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [14C]glucose, indicating previous glucosylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA mediates its cytotoxic effects on cells.

Amino Acids↗
PubMed1995

Glucosylation of Rho proteins by Clostridium difficile toxin B.

Toxin A and B, the major virulence factors of Clostridium difficile, are the causative agents of antibiotic-associated pseudomembranous colitis. In cultured cell lines their potent cytotoxicity results from their ability to induce disaggregation of the microfilament cytoskeleton. Toxin B acts on the low-molecular-mass GTPase RhoA, which is involved in the regulation of the actin cytoskeleton. We report here that toxin B catalyses the incorporation of up to one mole of glucose per mole of RhoA at the amino acid threonine at position 37. The modification was identified and localized by tandem electrospray mass spectrometry. UDP-glucose selectively serves as cosubstrate for the monoglucosylation reaction catalysed by toxin B. Microinjection of RhoA previously glucosylated by toxin B into monolayer cells caused disaggregation of actin filaments, indicating a dominant-negative activity of glucosylated RhoA.

Actins↗
PubMed1995

Life-span, T-cell responses, and incidence of lymphomas in congenic mice.

Survival, T-cell functions, and postmortem histopathology were studied in H-2 congenic strains of mice bearing H-2b, H-2k, and H-2d haplotypes. Males lived longer than females in all homozygous and heterozygous combinations except for H-2d homozygotes, which showed no differences between males and females. Association of heterozygosity with longer survival was observed only with H-2b/H-2b and H-2b/H-2d mice. Analysis using classification and regression trees (CART) showed that both males and females of H-2b homozygous and H-2k/H-2b mice had the shortest life-span of the strains studied. In histopathological analyses, lymphomas were noted to be more frequent in females, while hemangiosarcomas and hepatomas were more frequent in males. Lymphomas appeared earlier than hepatomas or hemangiosarcomas. The incidence of lymphomas was associated with the H-2 haplotype--e.g., H-2b homozygous mice had more lymphomas than did mice of the H-2d haplotype. More vigorous T-cell function was maintained with age (27 months) in H-2d, H-2b/H-2d, and H-2d/H-2k mice as compared with H-2b, H-2k, and H-2b/H-2k mice, which showed a decline of T-cell responses with age.

Animals↗
PubMed1995

Identification of genes showing altered expression in preimplantation and early postimplantation parthenogenetic embryos.

Uniparental embryos have been instrumental in studying imprinting because contributions from the parental genomes can be determined unambiguously. In this study, we set out to identify imprinted genes showing differential expression between parthenogenetic and fertilized embryos during preimplantation and early postimplantation stages of development. We identified three genes--apolipoprotein E, pyruvate kinase-3, and protein phosphatase 1 gamma--that represent excellent candidates for imprinted genes, based on the results of the differential screen, their function in differentiation and the cell cycle, and their location within imprinted chromosomal regions. In addition, two novel genes expressed in trophoblast were identified, 1661 and RA81. These genes, together with four known imprinted genes, H19, Igf2r, Igf2, and Snrpn, showed evidence of expression from both parental alleles in early stage embryos, indicating a role for postfertilization processes in regulating imprinted gene function.

Animals↗
PubMed1995

Electrospray mass spectrometry for protein characterization.

Mass spectrometry is a venerable analytical tool that has been used for some time in biochemistry for the analysis of small molecules, such as steroids. More recently, physicists have solved the problems associated with vaporizing and ionizing proteins and peptides, thereby allowing mass spectrometry to take on new roles in investigating protein sequences, structures and modifications.

Mass Spectrometry↗
PubMed1995

Identification of hnRNP P2 as TLS/FUS using electrospray mass spectrometry.

Protein complexes assembled on mRNA precursors can be separated by gel filtration chromatography to yield spliceosomal and H complex fractions (Reed R, Griffith J, Maniatis T, 1988, Cell 53:949-961; Reed R, 1990, Proc Natl Acad Sci USA 87:8031-8035.). Here we use Nano electrospray mass spectrometry (Wilm M, Mann M, 1994, Int J Mass Spectrometry Ion Processes 136:167-180) to identify proteins complexed with Adeno-pre-mRNA in the H complex peak. Four of the major hnRNP proteins, A1, B1, C1, and G, were identified by database analysis based on peptide mass and sequence information. A fifth protein in the H complex peak, corresponding to hnRNP P2, is shown to be the product of the TLS/FUS gene. This was originally identified as a chimeric oncogene formed by the chromosome translocation t(12;16) that is responsible for myxoid liposarcoma. The involvement of hnRNP P2 in oncogenesis provides a clear example of the importance of hnRNP proteins in molecular disease.

Amino Acid Sequence↗
PubMed1994

Error-tolerant identification of peptides in sequence databases by peptide sequence tags.

We demonstrate a new approach to the identification of mass spectrometrically fragmented peptides. A fragmentation spectrum usually contains a short, easily identifiable series of sequence ions, which yields a partial sequence. This partial sequence divides the peptide into three parts-regions 1, 2, and 3-characterized by the added mass m1 of region 1, the partial sequence of region 2, and the added mass m3 of region 3. We call the construct, m1 partial sequence m3, a "peptide sequence tag" and show that it is a highly specific identifier of the peptide. An algorithm developed here that uses the sequence tag to find the peptide in a sequence database is up to 1 million-fold more discriminating than the partial sequence information alone. Peptides can be identified even in the presence of an unknown posttranslational modification or an amino acid substitution between an entry in the sequence database and the measured peptide. These concepts are demonstrated with model and practical examples of electrospray mass spectrometry/mass spectrometry of tryptic peptides. Just two to three amino acid residues derived by fragmentation are enough to identify these peptides. In peptide mapping applications, even less information is necessary.

Algorithms↗
PubMed1994

Identification of proteins in polyacrylamide gels by mass spectrometric peptide mapping combined with database search.

Mass spectrometric peptide mapping of proteins separated by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis has been investigated. The best results are obtained after blotting of the proteins onto polyvinylidene difluoride membranes followed by enzymatic digestion of the protein on the membrane. The peptide maps were investigated in terms of completeness and applicability for protein identification using a previously developed database search program as well as for the possibility for full characterization of covalent modifications in the proteins. The most complete peptide maps were obtained when the proteins were reduced and alkylated on the membrane prior to enzymatic digestion followed by separation of the resulting mixture by high performance liquid chromatography prior to mass spectrometric analysis. Such peptide maps cover up to 98% of the sequence and consequently may allow complete characterization of post-translational modifications in proteins for which the amino acid sequence is known. The fastest and most sensitive procedure to obtain peptide maps sufficient for protein identification was direct analysis of the extracted peptide mixture by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The use of external and internal calibration of MALDI spectra for database searches is evaluated as well as the possibility of including a post-calibration routine within the search program.

Alkylation↗
PubMed1994

Metamorphosin A: a novel peptide controlling development of the lower metazoan Hydractinia echinata (Coelenterata, Hydrozoa).

Animal development depends on cell communication by signals. We have investigated the role of signals and of signal transduction in the development of the marine hydroid Hydractinia echinata. The larvae undergo metamorphosis in response to a chemical signal provided by environmental bacteria. Metamorphosis can be induced by a variety of different compounds interfering with biochemical signal transduction pathways. Sectioned posterior parts cannot be induced by most compounds known to induce whole larvae to metamorphose. We identified a novel peptide, pGlu-Gln-Pro-Gly-Leu-TrpNH2 ("metamorphosin A"), which induces isolated posterior parts to undergo metamorphosis and hence reactivates pattern formation, cell proliferation, cell differentiation, and morphogenesis. We suggest this peptide to be part of an internal signaling system involved in control of metamorphosis.

Amino Acid Sequence↗
PubMed1994

Genomic imprinting--defusing the ovarian time bomb.

Why do mammals imprint their parental genomes? Imprinting is seen in many phyla, but that in mammals is by far the most dramatic. Is there something peculiar to mammals that calls for such a striking phenomenon? We propose that imprinting is a device that protects female mammals from the potential ravages of ovarian trophoblast disease. Without imprinting, the ovarian teratomas that frequently arise from parthenogenetically activated oocytes in situ might be capable of forming malignant trophoblast. An allele that favored imprinting would spread rapidly because of the great increase in fitness associated with suppressing a lethal cancer of females.

Alleles↗