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Biomedical subjects

M Mann

Publications and source records attributed to M Mann.

At least 73 records · Page 4Linked to original sources
PubMed2000

Association of yeast RNA polymerase I with a nucleolar substructure active in rRNA synthesis and processing.

A novel ribonucleoprotein complex enriched in nucleolar proteins was purified from yeast extracts and constituents were identified by mass spectrometry. When isolated from rapidly growing cells, the assembly contained ribonucleic acid (RNA) polymerase (pol) I, and some of its transcription factors like TATA-binding protein (TBP), Rrn3p, Rrn5p, Rrn7p, and Reb1p along with rRNA processing factors, like Nop1p, Cbf5p, Nhp2p, and Rrp5p. The small nucleolar RNAs (snoRNAs) U3, U14, and MRP were also found to be associated with the complex, which supports accurate transcription, termination, and pseudouridylation of rRNA. Formation of the complex did not depend on pol I, and the complex could efficiently recruit exogenous pol I into active ribosomal DNA (rDNA) transcription units. Visualization of the complex by electron microscopy and immunogold labeling revealed a characteristic cluster-forming network of nonuniform size containing nucleolar proteins like Nop1p and Fpr3p and attached pol I. Our results support the idea that a functional nucleolar subdomain formed independently of the state of rDNA transcription may serve as a scaffold for coordinated rRNA synthesis and processing.

Cell Nucleolus↗
PubMed2000

Attention deficit/hyperactivity disorder children with a 7-repeat allele of the dopamine receptor D4 gene have extreme behavior but normal performance on critical neuropsychological tests of attention.

An association of the dopamine receptor D4 (DRD4) gene located on chromosome 11p15.5 and attention deficit/hyperactivity disorder (ADHD) has been demonstrated and replicated by multiple investigators. A specific allele [the 7-repeat of a 48-bp variable number of tandem repeats (VNTR) in exon 3] has been proposed as an etiological factor in attentional deficits manifested in some children diagnosed with this disorder. In the current study, we evaluated ADHD subgroups defined by the presence or absence of the 7-repeat allele of the DRD4 gene, using neuropsychological tests with reaction time measures designed to probe attentional networks with neuroanatomical foci in D4-rich brain regions. Despite the same severity of symptoms on parent and teacher ratings for the ADHD subgroups, the average reaction times of the 7-present subgroup showed normal speed and variability of response whereas the average reaction times of the 7-absent subgroup showed the expected abnormalities (slow and variable responses). This was opposite the primary prediction of the study. The 7-present subgroup seemed to be free of some of the neuropsychological abnormalities thought to characterize ADHD.

Alleles↗
PubMed2000

Detailed analysis of the phosphorylation of the human La (SS-B) autoantigen. (De)phosphorylation does not affect its subcellular distribution.

The La (SS-B) autoantigen is an evolutionarily conserved phosphoprotein which plays an important role, most likely as an RNA chaperone, in various processes, such as the biosynthesis and maturation of RNA polymerase III transcripts in the cell nucleus and (internal) initiation of translation in the cytoplasm. In this study, the phosphorylation state of this protein from human HeLa and HEp-2 cells was characterized by high-resolution two-dimensional IEF/SDS-PAGE analysis, and phosphorylation sites were mapped by nanoelectrospray mass spectrometry. Furthermore, the effect of phosphorylation at the sites identified on the subcellular distribution of the protein was studied by site-directed mutagenesis. At least 14 isoelectric isoforms were discerned on 2-D gels with La protein from both types of cells. Metabolic labeling in combination with alkaline phosphatase treatment revealed that only a limited number of these isoforms could be attributed to phosphorylation. Four phosphorylation sites, Thr-302, Ser-325, Thr-362, and Ser-366, were mapped by mass spectrometric analysis of the isolated La protein from HeLa cells or the carboxy-terminal half of this protein. The analysis of mutants of La, in which the respective phosphorylated residues were replaced by either a neutral (alanine) or an acidic (aspartate) residue, by microinjection into Xenopus laevis oocytes on the one hand and transfection of HEp-2 cells on the other hand revealed that the subcellular distribution of this protein was not affected by these amino acid substitutions. These results strongly suggest that the signals that determine the subcellular distribution of this protein are not regulated by (de)phosphorylation of the target residues examined.

Amino Acid Sequence↗
PubMed2000

Gemin4. A novel component of the SMN complex that is found in both gems and nucleoli.

The survival of motor neurons (SMN) protein, the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, is localized both in the cytoplasm and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins including Gemin2 (formerly SIP1) and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with snRNP Sm core proteins and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing by serving in the regeneration of spliceosomes. These functions are likely impaired in cells of SMA patients because they have reduced levels of functional SMN. Here, we report the identification by nanoelectrospray mass spectrometry of a novel component of the SMN complex that we name Gemin4. Gemin4 is associated in vivo with the SMN complex through a direct interaction with Gemin3. The tight interaction of Gemin4 with Gemin3 suggests that it could serve as a cofactor of this DEAD box protein. Gemin4 also interacts directly with several of the Sm core proteins. Monoclonal antibodies against Gemin4 efficiently immunoprecipitate the spliceosomal U snRNAs U1 and U5 from Xenopus oocytes cytoplasm. Immunolocalization experiments show that Gemin4 is colocalized with SMN in the cytoplasm and in gems. Interestingly, Gemin4 is also detected in the nucleoli, suggesting that the SMN complex may also function in preribosomal RNA processing or ribosome assembly.

Animals↗
PubMed2000

A urokinase receptor-associated protein with specific collagen binding properties.

The plasminogen activation cascade system, directed by urokinase and the urokinase receptor, plays a key role in extracellular proteolysis during tissue remodeling. To identify molecular interaction partners of these trigger proteins on the cell, we combined covalent protein cross-linking with mass spectrometry based methods for peptide mapping and primary structure analysis of electrophoretically isolated protein conjugates. A specific tri-molecular complex was observed upon addition of pro-urokinase to human U937 cells. This complex included the urokinase receptor, pro-urokinase, and an unknown, high molecular weight urokinase receptor-associated protein. The tryptic peptide mixture derived from a cross-linked complex of pro-urokinase and the latter protein was analyzed by nanoelectrospray tandem mass spectrometric sequencing. This analysis identified the novel protein as the human homologue of a murine membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices.

Amino Acid Sequence↗
PubMed2000

Model for stathmin/OP18 binding to tubulin.

Stathmin/OP18 is a regulatory phosphoprotein that controls microtubule (MT) dynamics. The protein does not have a defined three-dimensional structure, although it contains three distinct regions (an unstructured N-terminus, N: 1-44; a region with high helix propensity, H 1: 44-89; and a region with low helix propensity, H 2: 90-142). The full protein and a combination of H 1 and H 2 inhibits tubulin polymerization, while the combination of H 1 and the N-terminus is less efficient. None of the individual three regions alone are functional in this respect. However, all of them cross-link to alpha-tubulin, but only full-length stathmin produces high-molecular-weight products. Mass spectrometry analysis of alpha-tubulin-stathmin/OP18 and its truncation products shows that full-length stathmin/OP18 binds to the region around helix 10 of alpha-tubulin, a region that is involved in longitudinal interactions in the MT, sequestering the dimer and possibly linking two tubulin heterodimers. In the absence of the N-terminus, stathmin/OP18 binds to only one molecule of alpha-tubulin, at the top of the free tubulin heterodimer, preventing polymerization.

Cloning, Molecular↗
PubMed2000

A generic strategy to analyze the spatial organization of multi-protein complexes by cross-linking and mass spectrometry.

Most cellular functions are performed by multi-protein complexes. The identity of the members of such complexes can now be determined by mass spectrometry. Here we show that mass spectrometry can also be used in order to define the spatial organization of these complexes. In this approach, components of a protein complex are purified via molecular interactions using an affinity tagged member and the purified complex is then partially cross-linked. The products are separated by gel electrophoresis and their constituent components identified by mass spectrometry yielding nearest-neighbor relationships. In this study, a member of the yeast nuclear pore complex (Nup85p) was tagged and a six-member sub-complex of the pore was cross-linked and analyzed by 1D SDS-PAGE. Cross-linking reactions were optimized for yield and number of products. Analysis by MALDI mass spectrometry resulted in the identification of protein constituents in the cross-linked bands even at a level of a few hundred femtomoles. Based on these results, a model of the spatial organization of the complex was derived that was later supported by biological experiments. This work demonstrates, that the use of mass spectrometry is the method of choice for analyzing cross-linking experiments aiming on nearest neighbor relationships.

Cross-Linking Reagents↗
PubMed2000

Mlp2p, a component of nuclear pore attached intranuclear filaments, associates with nic96p.

A fraction of the yeast nucleoporin Nic96p is localized at the terminal ring of the nuclear basket. When Nic96p was affinity purified from glutaraldehyde-treated spheroplasts, it was found to be associated with Mlp2p. Mlp2p, together with Mlp1p, are the yeast Tpr homologues, which form the nuclear pore-attached intranuclear filaments (Strambio-de-Castillia, C., Blobel, G., and Rout, M. P. (1999) J. Cell Biol. 144, 839-855). Double disruption mutants of MLP1 and MLP2 are viable and apparently not impaired in nucleocytoplasmic transport. However, overproduction of MLP1 causes nuclear accumulation of poly(A)(+) RNA in a chromatin-free area of the nucleus.

Amino Acid Sequence↗
PubMed2000

Purification of protein A-tagged yeast ran reveals association with a novel karyopherin beta family member, Pdr6p.

The small GTPase Ran (encoded by GSP1 and GSP2 in yeast) plays a central role in nucleocytoplasmic transport. GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1 null mutant. After affinity purification of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, known karyopherin beta transport receptors (e.g. Kap121p and Kap123p) and a novel member of this protein family, Pdr6p, were found to be associated with yeast Ran. Subsequent tagging of Pdr6p with green fluorescent protein revealed association with the nuclear pore complexes in vivo. Thus, functional tagging of yeast Ran allowed the study of its in vivo distribution and interaction with known and novel Ran-binding proteins.

Cell Compartmentation↗
PubMed2000

Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors.

Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2) or phosphotyrosine interaction domains (PID). Additionally, several cytoplasmic proteins that may or may not associate with the receptor undergo tyrosine phosphorylation. To identify several components of the EGFR signaling pathway in a single step, we have immunoprecipitated molecules that are tyrosine phosphorylated in response to EGF and analyzed them by one-dimensional gel electrophoresis followed by mass spectrometry. Combining matrix-assisted laser desorption/ionization (MALDI) and nanoelectrospray tandem mass spectrometry (MS/MS) led to the identification of nine signaling molecules, seven of which had previously been implicated in EGFR signaling. Several of these molecules were identified from low femtomole levels of protein loaded onto the gel. We identified Vav-2, a recently discovered guanosine nucleotide exchange factor that is expressed ubiquitously, as a substrate of the EGFR. We demonstrate that Vav-2 is phosphorylated on tyrosine residues in response to EGF and associates with the EGFR in vivo. Binding of Vav-2 to the EGFR is mediated by the SH2 domain of Vav-2. In keeping with its ubiquitous expression, Vav-2 seems to be a general signaling molecule, since it also associates with the platelet-derived growth factor (PDGF) receptor and undergoes tyrosine phosphorylation in fibroblasts upon PDGF stimulation. The strategy suggested here can be used for routine identification of downstream components of cell surface receptors in mammalian cells.

ErbB Receptors↗
PubMed2000

Identification and characterization of PaMTH1, a putative O-methyltransferase accumulating during senescence of Podospora anserina cultures.

A differential protein display screen resulted in the identification of a 27-kDa protein which strongly accumulates during the senescence of Podospora anserina cultures grown under standard conditions. After partial determination of the amino-acid sequence by mass-spectrometry analysis of trypsin-generated fragments, pairs of degenerated primers were deduced and used to amplify parts of the sequence coding for the protein. These PCR products were utilized to select specific cDNA and genomic clones from DNA libraries of P. anserina. A subsequent DNA-sequence analysis revealed that the 27-kDa protein is encoded by a discontinuous gene, PaMth1, capable of coding for 240 amino acids. The first three amino-terminal residues appear to be removed post-translationally. The deduced amino-acid sequence shows significant homology to S-adenosylmethionine (SAM)-dependent methyltransferases. We hypothesize that the 27-kDa protein, PaMTH1, is involved in age-related methylation reactions protecting aging cultures against increasing oxidative stress.

Amino Acid Sequence↗
PubMed2000

Trait anxiety and prior exposure to non-stressful stimuli: effects on psychophysiological arousal and anxiety.

Arousal and anxiety responses to stressful stimulation are products of multiple factors that may include the physiological, behavioral, cognitive, affective, trait, and state components of our six-system model, as well as mediational and non-mediational perspectives. Within the context of this model, the present experiment examined the effects of prior exposure to neutral stimuli on arousal and anxiety responses to stress. High and low trait-anxious participants were randomly assigned to one of three prior exposure conditions in which they were exposed to stressful stimuli following exposure to: (a) neutral, non-stressful stimulation in the same modality (the Intramodality Prior Exposure or IPE experimental condition); (b) neutral stimuli in a different modality (the Cross-modality Prior Exposure or CPE control condition); or (c) a rest period (the Stress Only or SO control condition). Results showed that low trait anxious subjects had consistently larger arousal and anxiety responses to stress than did highs and that prior exposure to certain same-modality neutral stimuli reduced responses to subsequent stressors. In addition, arousal was greater in response to cognitive than to affective and in response to non-mediational than mediational conditions. Results are discussed in terms of the inverted-U arousal function and of the six-system model and its implications for understanding anxiety and arousal.

Acoustic Stimulation↗
PubMed2000

p95-APP1 links membrane transport to Rac-mediated reorganization of actin.

Motility requires protrusive activity at the cellular edge, where Rho family members regulate actin dynamics. Here we show that p95-APP1 (ArfGAP-putative, Pix-interacting, paxillin-interacting protein 1), a member of the GIT1/PKL family, is part of a complex that interacts with Rac. Wild-type and truncated p95-APP1 induce actin-rich protrusions mediated by Rac and ADP-ribosylation factor 6 (Arf6). Distinct p95-APP1-derived polypeptides have different distributions, indicating that p95-APP1 cycles between the cell surface and endosomes. Our results show that p95-APP1 functionally interacts with Rac and localizes to endosomal compartments, thus identifying p95-APP1 as a molecular link between actin organization, adhesion, and membrane transport during cell motility.

ADP-Ribosylation Factor 6↗
PubMed2000

Cell-cycle-regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres.

Telomeres allow cells to distinguish natural chromosome ends from damaged DNA and protect the ends from degradation and fusion. In human cells, telomere protection depends on the TTAGGG repeat binding factor, TRF2 (refs 1-4), which has been proposed to remodel telomeres into large duplex loops (t-loops). Here we show by nanoelectrospray tandem mass spectrometry that RAD50 protein is present in TRF2 immunocomplexes. Protein blotting showed that a small fraction of RAD50, MRE11 and the third component of the MRE11 double-strand break (DSB) repair complex, the Nijmegen breakage syndrome protein (NBS1), is associated with TRF2. Indirect immunofluorescence demonstrated the presence of RAD50 and MRE11 at interphase telomeres. NBS1 was associated with TRF2 and telomeres in S phase, but not in G1 or G2. Although the MRE11 complex accumulated in irradiation-induced foci (IRIFs) in response to gamma-irradiation, TRF2 did not relocate to IRIFs and irradiation did not affect the association of TRF2 with the MRE11 complex, arguing against a role for TRF2 in DSB repair. Instead, we propose that the MRE11 complex functions at telomeres, possibly by modulating t-loop formation.

Acid Anhydride Hydrolases↗
PubMed2000

Aseptic necrosis in HIV seropositive patients: a possible etiologic role for megestrol acetate.

The association between pharmacologic doses of corticosteroids and the development of aseptic bone necrosis has been well documented. Recent reports have described the corticosteroid activity of megestrol acetate. A retrospective review of adverse events reported to the U.S. Food and Drug Administration identified three human immunodeficiency virus (HIV) seropositive patients who developed avascular necrosis of the femoral head during treatment with megestrol acetate. All were males, ages 34, 36, and 55 years, and were on therapy for 6, 1.5, and 18 months, respectively, when symptoms of aseptic necrosis occurred in the absence of antecedent trauma. Megestrol acetate doses were 640, 320, and 600-1200 mg/d, respectively. Two patients had no history of corticosteroid use whereas the third had taken an undisclosed dose and duration of corticosteroids concurrent with pentamidine administration. Notably, despite the predominant use of megestrol in women for hormone sensitive malignancies, none of the reports of aseptic necrosis occurred in this population. Megestrol acetate may be associated with the development of avascular necrosis via its glucocorticoid-like effects. Cachectic acquired immunodeficiency syndrome (AIDS) patients may have additional risk factors that predispose them to aseptic necrosis when receiving megestrol acetate.

Adult↗
PubMed2000

Mitotic regulation of the APC activator proteins CDC20 and CDH1.

The ordered activation of the ubiquitin protein ligase anaphase-promoting complex (APC) or cyclosome by CDC20 in metaphase and by CDH1 in telophase is essential for anaphase and for exit from mitosis, respectively. Here, we show that CDC20 can only bind to and activate the mitotically phosphorylated form of the Xenopus and the human APC in vitro. In contrast, the analysis of phosphorylated and nonphosphorylated forms of CDC20 suggests that CDC20 phosphorylation is neither sufficient nor required for APC activation. On the basis of these results and the observation that APC phosphorylation correlates with APC activation in vivo, we propose that mitotic APC phosphorylation is an important mechanism that controls the proper timing of APC(CDC20) activation. We further show that CDH1 is phosphorylated in vivo during S, G2, and M phase and that CDH1 levels fluctuate during the cell cycle. In vitro, phosphorylated CDH1 neither binds to nor activates the APC as efficiently as does nonphosphorylated CDH1. Nonphosphorylatable CDH1 mutants constitutively activate APC in vitro and in vivo, whereas mutants mimicking the phosphorylated form of CDH1 are constitutively inactive. These results suggest that mitotic kinases have antagonistic roles in regulating APC(CDC20) and APC(CDH1); the phosphorylation of APC subunits is required to allow APC activation by CDC20, whereas the phosphorylation of CDH1 prevents activation of the APC by CDH1. These mechanisms can explain the temporal order of APC activation by CDC20 and CDH1 and may help to ensure that exit from mitosis is not initiated before anaphase has occurred.

Anaphase-Promoting Complex-Cyclosome↗