Biomedical subjects

E Freed

Publications and source records attributed to E Freed.

At least 19 recordsLinked to original sources

PubMed2001

Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex.

We have discovered an early mitotic inhibitor, Emi1, which regulates mitosis by inhibiting the anaphase promoting complex/cyclosome (APC). Emi1 is a conserved F box protein containing a zinc binding region essential for APC inhibition. Emi1 accumulates before mitosis and is ubiquitylated and destroyed in mitosis, independent of the APC. Emi1 immunodepletion from cycling Xenopus extracts strongly delays cyclin B accumulation and mitotic entry, whereas nondestructible Emi1 stabilizes APC substrates and causes a mitotic block. Emi1 binds the APC activator Cdc20, and Cdc20 can rescue an Emi1-induced block to cyclin B destruction. Our results suggest that Emi1 regulates progression through early mitosis by preventing premature APC activation, and may help explain the well-known delay between cyclin B/Cdc2 activation and cyclin B destruction.

Adenosine Triphosphatases↗

PubMed2000

The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases.

Recently, many new examples of E3 ubiquitin ligases or E3 enzymes have been found to regulate a host of cellular processes. These E3 enzymes direct the formation of multiubiquitin chains on specific protein substrates, and - typically - the subsequent destruction of those proteins. We discuss how the modular architecture of E3 enzymes connects one of two distinct classes of catalytic domains to a wide range of substrate-binding domains. In one catalytic class, a HECT domain transfers ubiquitin directly to substrate bound to a non-catalytic domain. Members of the other catalytic class, found in the SCF, VBC and APC complexes, use a RING finger domain to facilitate ubiquitylation. The separable substrate-recognition domains of E3 enzymes provides a flexible means of linking a conserved ubiquitylation function to potentially thousands of ubiquitylated substrates in eukaryotic cells.

Animals↗

PubMed1999

Components of an SCF ubiquitin ligase localize to the centrosome and regulate the centrosome duplication cycle.

Centrosomes organize the mitotic spindle to ensure accurate segregation of the chromosomes in mitosis. The mechanism that ensures accurate duplication and separation of the centrosomes underlies the fidelity of chromosome segregation, but remains unknown. In Saccharomyces cerevisiae, entry into S phase and separation of spindle pole bodies each require CDC4 and CDC34, which encode components of an SCF (Skp1-cullin-F-box) ubiquitin ligase, but a direct (SCF) connection to the spindle pole body is unknown. Using immunofluorescence microscopy, we show that in mammalian cells the Skp1 protein and the cullin Cul1 are localized to interphase and mitotic centrosomes and to the cytoplasm and nucleus. Deconvolution and immunoelectron microscopy suggest that Skp1 forms an extended pericentriolar structure that may function to organize the centrosome. Purified centrosomes also contain Skp1, and Cul1 modified by the ubiquitin-like molecule NEDD8, suggesting a role for NEDD8 in targeting. Using an in vitro assay for centriole separation in Xenopus extracts, antibodies to Skp1 or Cul1 block separation. Proteasome inhibitors block both centriole separation in vitro and centrosome duplication in Xenopus embryos. We identify candidate centrosomal F-box proteins, suggesting that distinct SCF complexes may direct proteolysis of factors mediating multiple steps in the centrosome cycle.

3T3 Cells↗

PubMed1999

A double-blind, multicentre study to assess the tolerability and efficacy of paroxetine compared with amitriptyline in the treatment of depressed patients in Australian general practice.

OBJECTIVE: This study compared the tolerability and efficacy of paroxetine and amitriptyline in the treatment of depression in general practice. METHODS: In this double-blind, multicentre study conducted in the general practice, patients with depression (Montgomery Asberg Depression Rating Scale [MADRS] score > or = 20) who were regarded as requiring antidepressant therapy were randomly assigned to receive paroxetine (20 mg, n = 184) or amitriptyline (50-100 mg, n = 191) once daily for 9 weeks. RESULTS: More patients completed treatment with paroxetine than with amitriptyline (71.1% vs 56.1%, p = 0.009). Depression rating scores (MADRS and Clinical Global Impression [CGI]) were improved with both agents, but at week 9, paroxetine achieved more favourable scores compared with amitriptyline on MADRS (p=0.019), CGI severity of depression (p=0.044), and CGI efficacy index (p = 0.038). CONCLUSIONS: Depressed patients treated in general practice respond more quickly and are more likely to complete the treatment regimen with paroxetine than with amitriptyline.

Adult↗

PubMed1994

Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation.

To identify proteins that may participate in the activation of the protein kinase Raf, proteins that interact with Raf were selected in a two-hybrid screen. Two members of the 14-3-3 protein family were isolated that interacted with both the amino terminal regulatory regions of Raf and the kinase domain of Raf, but did not compete with the guanine nucleotide-binding protein Ras for binding to Raf. 14-3-3 proteins associated with Raf in mammalian cells and accompanied Raf to the membrane in the presence of activated Ras. In yeast cells expressing Raf and MEK, mammalian 14-3-3 beta or 14-3-3 zeta activated Raf to a similar extent as did expression of Ras. Therefore, 14-3-3 proteins may participate in or be required for the regulation of Raf function. These findings suggest a role for 14-3-3 proteins in Raf-mediated signal transduction.

14-3-3 Proteins↗

PubMed1994

Proteins of the 14-3-3 family associate with Raf and contribute to its activation.

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14-3-3 Proteins↗

PubMed1994

Platelet-derived growth factor-induced p21ras-mediated signaling is independent of platelet-derived growth factor receptor interaction with GTPase-activating protein or phosphatidylinositol-3-kinase.

Stimulation with platelet-derived growth factor (PDGF) results in the association of several SH2 domain-containing proteins with the activated PDGF receptor, including GAP, a GTPase-activating protein of p21ras, and phosphatidylinositol-3-kinase (PI-3K). To investigate the role of GAP-PI-3K receptor interaction in p21ras signaling, we have used cell lines expressing mutant PDGF receptors that either are impaired in GAP binding or fail to bind both GAP and PI-3K. In these cell lines, PDGF treatment resulted in activation of extracellular signal-regulated kinase 2 (ERK2), which could be blocked by the expression of a dominant-negative mutant of p21ras (p21ras(asn17)), indicating that these mutations in the PDGF receptor do not abolish p21ras-mediated activation of ERK2. In addition, the PDGF-induced increase in levels of p21rasGTP, as measured either in intact cells or in permeabilized cells, appears to be normal in the cell lines expressing the mutant PDGF receptors. These results indicate that binding of GAP and/or PI-3K to the PDGF receptor is not necessary for PDGF-induced p21ras activation and p21ras-mediated signaling to ERK2. We also show that, in contrast to the activation of ERK2, PDGF-induced GAP and PI-3K interaction with the PDGF receptor are not inhibited by p21ras(asn17) expression, indicating that these interactions do not require p21ras activation.

Animals↗

PubMed1992

A 41-kilodalton protein is a potential substrate for the p210bcr-abl protein-tyrosine kinase in chronic myelogenous leukemia cells.

Chronic myelogenous leukemia (CML) is characterized by a translocation involving the c-abl protein-tyrosine kinase gene. A chimeric mRNA is formed containing sequences from a chromosome 22 gene (bcr) at its 5' end and all but the variable exon 1 of c-abl sequence. The product of this mRNA, p210bcr-abl, has constitutively high protein-tyrosine kinase activity. We examined K562 cells and other lines established from CML patients for the presence of phosphotyrosine (P-Tyr)-containing proteins which might be p210bcr-abl substrates. Two-dimensional gel separation of 32P-labeled proteins followed by phosphoamino acid analysis of 25 phosphoproteins, which comprised the major alkali-stable phosphoproteins, indicated that three related proteins of 41 kDa are the most prominent P-Tyr-containing proteins detected by this method. The 41-kDa phosphoproteins are found in two other CML lines that we examined but not in lines of similar lineage isolated from patients with distinct leukemic disease. A protein that comigrates with the major form of pp41 (pp41A) and contains P-Tyr is also found in murine fibroblasts and B-lymphoid cells transformed by Abelson murine leukemia virus, which encodes the v-abl protein, and in platelet-derived growth factor-treated fibroblasts, in which it has been described previously. We analyzed three pairs of Epstein-Barr virus-immortalized B-cell lines from individual CML patients and found that only the lines in which active p210bcr-abl was present contained detectable pp41. We also performed immunoblotting with anti-P-Tyr antibodies on the same CML cell lines and detected at least four other putative substrates of p210bcr-abl, which were undetected with use of the two-dimensional gel technique.

Animals↗

PubMed1989

A novel integrin beta subunit is associated with the vitronectin receptor alpha subunit (alpha v) in a human osteosarcoma cell line and is a substrate for protein kinase C.

We demonstrate that a novel integrin beta subunit is present in association with the vitronectin receptor (VNR) alpha subunit on the surface of MG-63 human osteosarcoma cells. This beta subunit and the glycoprotein IIIa beta subunit (beta 3) were both found complexed with VNR alpha on MG-63 cells and in at least two other human cell types we examined. Tryptic peptide mapping indicated that the two beta subunits are related but distinct. The novel beta chain, referred to here as beta s, was not recognized by the monoclonal antibody AP3, which recognizes GPIIIa, nor by an antiserum raised against a peptide from the COOH-terminal cytoplasmic domain of beta 3. Both receptor complexes bound to and were specifically eluted from a column containing the cell adhesion peptide GRGDSP. The unique beta subunit became phosphorylated at high stoichiometry when MG-63 cells or AG1523 human fibroblasts were treated with the phorbol-ester tumor promoter phorbol 12-myristate 13-acetate. This phosphorylation occurred mainly on serine and probably at one major site, as determined by phosphotryptic peptide mapping. Protein kinase C phosphorylated the beta s subunit of intact receptor in vitro, at the same site phosphorylated in treated cells, indicating that protein kinase C is likely to be responsible for this phosphorylation in vivo.

Electrophoresis, Polyacrylamide Gel↗

PubMed1988

Targets for signal-transducing protein kinases.

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Amino Acid Sequence↗

PubMed1988

HIV infection associated with symptoms indistinguishable from functional psychosis.

Patients with acquired immunodeficiency syndrome (AIDS), who present with or develop psychotic symptoms, almost invariably have evidence of cognitive impairment. Three psychotic patients, one with AIDS, and two with AIDS-related complex (ARC), who showed no evidence of cognitive impairment, are reported. It is suggested that the human immunodeficiency virus (HIV) may produce symptoms indistinguishable from those seen in the functional psychoses.

AIDS-Related Complex↗

PubMed1987

Central receptor binding and cardiovascular effects of GABA analogues in the cat.

Several structural analogues of GABA were shown to be inhibitors of GABAA receptor binding in membranes from cat cerebral cortex. These compounds were 3-aminopropanesulfonic acid (APS; IC50 = 0.04 microM), imidazoleacetic acid (IMA; IC50 = 0.4 microM), morpholinopropanesulfonic acid (MOPS; IC50 = 1.6 microM), 5-phenylpyrrolepropionic acid (PPP; IC50 = 15 microM), aminoethanethiosulfonic acid (AETS; IC50 = 22 microM), 3-amino-3-phenylpropionic acid (APP; IC50 = 35 microM), meta-aminobenzoic acid (MABA; IC50 = 58 microM) and urocanic acid (UCA; IC50 = 354 microM). The IC50 value for GABA was 0.03 microM. GABA, PPP, AETS, MABA and UCA were previously shown to reduce arterial pressure in the cat after intracerebroventricular infusion. In the present study MOPS (ED50 = 0.26 nmol/kg), APS (ED50 = 4.7 nmol/kg), APP (ED50 = 49 nmol/kg), and IMA (ED50 = 350 nmol/kg) were also found to be able to decrease blood pressure when infused into the fourth ventricle. All nine compounds reduced blood pressure to the same extent, but in some cases their relative potencies (ED50 values) exhibited significant differences. When the IC50 values for receptor binding were plotted against the ED50 values for the cardiovascular effects, no significant correlation emerged. This lack of a correlation does not necessarily imply that the reductions in blood pressure elicited by the drugs are not related to an activation of central GABAA receptors. Instead, it highlights the difficulties that are sometimes encountered in attempting to obtain quantitative measurements after intracerebroventricular infusion.

Animals↗

PubMed1986

Novel GABA analogues as hypotensive agents.

The actions of four analogues of gamma-aminobutyric acid (GABA) on blood pressure and heart rate were measured in the cat after intracerebroventricular administration. These compounds were previously found to inhibit binding to GABAA receptors of neuronal membranes from the CNS of the rat. Each of the drugs, together with GABA, produced an average maximum reduction in blood pressure of 27.63% +/- 12.5. However, aminoethanethiosulfonic acid (AETS) was the most potent (ED50 = 2.24 X 10(-10) mol/kg) of the drugs, followed by 5-phenyl-2-pyrrole propionic acid (PPP), urocanic acid (UCA), m-aminobenzoic acid (MABA) and GABA. None of the compounds produced a significant effect on heart rate. The fact that these analogues mimicked the action of GABA on the cardiovascular system of the cat and that they were able to inhibit binding to GABAA receptors, indicates that they may be GABA agonists.

Aminobenzoates↗

PubMed1985