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1.
Exp Cell Res ; 313(12): 2521-30, 2007 Jul 15.
Article in English | MEDLINE | ID: mdl-17574238

ABSTRACT

Alternative spliced variants of the human discs large (hDlg) tumour suppressor are characterized by combinations of insertions. Here, using insertions I2- and I3-specific antibodies, we show that I2 and I3 variants have distinct distributions in epidermal and cervical epithelia. In skin and cervix, I3 variants are found in the cytoplasm. Cytoplasmic localization of I3 variants decreases as cervical keratinocytes differentiate, concomitant with relocalization to the cell periphery. I2 variants are found at the cell periphery of differentiated epidermal and cervical keratinocytes. Nuclear localization of I2 variants was evident in both tissues, with concentration of nuclear I2 variants in basal and parabasal cervical keratinocytes. A prominent nuclear localization of hDlg in cells of hyperproliferative layers of psoriatic lesions, but not in mature differentiated keratinocytes, together with I2 redistribution in differentiating keratinocytes, suggests that nuclear hDlg functions may be pertinent to growth of undifferentiated cells. Supporting our findings in squamous tissues, a decrease of nuclear hDlg and an increase of membrane-bound and cytoplasmic hDlg upon calcium-induced keratinocyte differentiation were not concomitant processes. Furthermore, we confirm that the exit of I2 variants from the nucleus is linked to stimulation of epithelial differentiation. The dynamic redistribution of hDlg also correlated with a marked increase in the expression of I3 variants while the level of I2 variants showed only a moderate decrease. Because changes in the intracellular distribution of hDlg splice variants, and in their expression levels, correlate with changes in differentiation state we hypothesize that the different hDlg isoforms play distinct roles at various stages of epithelial differentiation.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Alternative Splicing/genetics , Cell Differentiation , Keratinocytes/cytology , Keratinocytes/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Alternative Splicing/drug effects , Antibodies/immunology , Calcium/pharmacology , Cell Differentiation/drug effects , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cells, Cultured , Cervix Uteri/cytology , Cervix Uteri/drug effects , Cervix Uteri/metabolism , Discs Large Homolog 1 Protein , Epidermis/drug effects , Epidermis/pathology , Female , Humans , Keratinocytes/drug effects , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Transport/drug effects , Time Factors
2.
Mol Cell Biol ; 21(7): 2608-16, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11259607

ABSTRACT

An abridged prion protein (PrP) molecule of 106 amino acids, designated PrP106, is capable of forming infectious miniprions in transgenic mice (S. Supattapone, P. Bosque, T. Muramoto, H. Wille, C. Aagaard, D. Peretz, H.-O. B. Nguyen, C. Heinrich, M. Torchia, J. Safar, F. E. Cohen, S. J. DeArmond, S. B. Prusiner, and M. Scott, Cell 96:869-878, 1999). We removed additional sequences from PrP106 and identified a 61-residue peptide, designated PrP61, that spontaneously adopted a protease-resistant conformation in neuroblastoma cells. Synthetic PrP61 bearing a carboxy-terminal lipid moiety polymerized into protease-resistant, beta-sheet-enriched amyloid fibrils at a physiological salt concentration. Transgenic mice expressing low levels of PrP61 died spontaneously with ataxia. Neuropathological examination revealed accumulation of protease-resistant PrP61 within neuronal dendrites and cell bodies, apparently causing apoptosis. PrP61 may be a useful model for deciphering the mechanism by which PrP molecules acquire protease resistance and become neurotoxic.


Subject(s)
Mice, Transgenic , Neurodegenerative Diseases/genetics , Prions/genetics , Animals , Mice , Neurodegenerative Diseases/etiology , Peptides/genetics
3.
J Virol ; 74(24): 11928-34, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11090193

ABSTRACT

An abridged PrP molecule of 106 amino acids designated PrP106 can form infectious miniprions in transgenic (Tg) mice (29). Addition of six-histidine (His(6)) affinity tags to selective sites within PrP106 resulted unexpectedly in new PrP proteins that spontaneously adopted protease-resistant conformations when expressed in neuroblastoma cells and Tg mice. Acquisition of protease resistance depended on the length, charge, and placement of the affinity tag. Introduction of the disease-linked mutation E200K into the sequence of PrP106(140/6His) increased the recovery of protease-resistant PrP fivefold, whereas introduction of the mutations C213A and Delta214-220 did not affect the recovery of protease-resistant PrP. Treatment of cultured cells expressing affinity-tagged PrP106 mutants with polypropyleneimine dendrimer rendered these proteins sensitive to protease digestion in a manner similar to wild-type PrP(Sc). We conclude that certain affinity-tagged PrP106 proteins spontaneously fold into conformations partially resembling, yet distinct from, wild-type PrP(Sc). These proteins might be useful tools in the identification of new disease-causing mutations as well as for screening compounds for therapeutic efficacy.


Subject(s)
Endopeptidases/metabolism , Prions/chemistry , Prions/metabolism , Protein Conformation , Animals , Mice , Mice, Transgenic , Structure-Activity Relationship
4.
Anesthesiology ; 93(3): 765-73, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10969310

ABSTRACT

BACKGROUND: Moxonidine, a novel imidazoline-alpha2-adrenergic receptor-selective analgesic, was recently identified as antinociceptive but has yet to be evaluated in neuropathic pain models. alpha2-adrenergic receptor-selective analgesics, and high-efficacy opioids, effectively inhibit neuropathic pain behaviors in rodents. In contrast, morphine potency and efficacy decreases in states of neuropathic pain, both in rodents and in humans, but may be restored or enhanced by coadministration of morphine with alpha2-adrenergic receptor-selective analgesics. The current experiments extend the evaluation of opioid-coadjuvant interactions in neuropathic subjects by testing the respective antihyperalgesic interactions of moxonidine and clonidine with morphine in a test of mechanical hyperalgesia. METHODS: Nerve-injured mice (Chung model) were spinally administered moxonidine, clonidine, morphine, and the combinations moxonidine-morphine and clonidine-morphine. Hyperalgesia was detected by von Frey monofilament stimulation (3.3 mN) to the hind paws (plantar surface). The ED50 values were calculated and the interactions tested by isobolographic analysis. RESULTS: In nerve-injured mice, moxonidine, clonidine, and morphine all dose-dependently inhibited mechanical hyperalgesia. Furthermore, the combinations of moxonidine-morphine and clonidine-morphine resulted in substantial leftward shifts in the dose-response curves compared with those of each agonist administered separately. The calculated ED50 values of the dose-response curves of these combinations were significantly lower than their corresponding theoretical additive ED50 values. These results confirmed that both interactions were synergistic. CONCLUSIONS: Moxonidine and clonidine both synergize with morphine to inhibit paw withdrawal from nociceptive mechanical stimuli in nerve-injured mice.


Subject(s)
Adrenergic alpha-2 Receptor Agonists , Adrenergic alpha-Agonists/therapeutic use , Analgesics/therapeutic use , Hyperalgesia/drug therapy , Imidazoles/therapeutic use , Morphine/therapeutic use , Animals , Clonidine/therapeutic use , Drug Synergism , Imidazoles/adverse effects , Male , Mice , Morphine/adverse effects , Peripheral Nerve Injuries
5.
Proc Natl Acad Sci U S A ; 97(19): 10584-9, 2000 Sep 12.
Article in English | MEDLINE | ID: mdl-10984543

ABSTRACT

Antagonists of glutamate receptors of the N-methyl-d-aspartate subclass (NMDAR) or inhibitors of nitric oxide synthase (NOS) prevent nervous system plasticity. Inflammatory and neuropathic pain rely on plasticity, presenting a clinical opportunity for the use of NMDAR antagonists and NOS inhibitors in chronic pain. Agmatine (AG), an endogenous neuromodulator present in brain and spinal cord, has both NMDAR antagonist and NOS inhibitor activities. We report here that AG, exogenously administered to rodents, decreased hyperalgesia accompanying inflammation, normalized the mechanical hypersensitivity (allodynia/hyperalgesia) produced by chemical or mechanical nerve injury, and reduced autotomy-like behavior and lesion size after excitotoxic spinal cord injury. AG produced these effects in the absence of antinociceptive effects in acute pain tests. Endogenous AG also was detected in rodent lumbosacral spinal cord in concentrations similar to those previously detected in brain. The evidence suggests a unique antiplasticity and neuroprotective role for AG in processes underlying persistent pain and neuronal injury.


Subject(s)
Agmatine/therapeutic use , Analgesics/therapeutic use , Inflammation/complications , Pain/drug therapy , Peripheral Nervous System Diseases/complications , Spinal Cord Injuries/complications , Animals , Immunohistochemistry , Male , Mice , N-Methylaspartate/physiology , Pain/etiology , Rats , Rats, Sprague-Dawley
6.
Arch Virol Suppl ; (16): 113-24, 2000.
Article in English | MEDLINE | ID: mdl-11214913

ABSTRACT

There is growing concern that bovine spongiform encephalopathy (BSE) may have passed from cattle to humans, resulting in approximately 70 cases of an atypical, variant CJD (vCJD) in teenagers and young adults. We report here that transgenic (Tg) mice expressing full-length bovine (Bo) PrP serially propagate BSE prions and that there is no species barrier for transmission from cattle to Tg(BoPrP) mice. Surprisingly, these same mice were also highly susceptible to vCJD and natural sheep scrapie. The incubation times (approximately 250 d), neuropathology, and PrP(Sc) isoforms in Tg(BoPrP) mice inoculated with vCJD and BSE brain extracts were indistinguishable and differed dramatically from those seen in these mice injected with natural scrapie. In efforts to identify PrP sequences required for prion formation, we found that a redacted prion protein of only 106 amino acids (PrP106) containing two large deletions supported prion propagation. In Tg(PrP106) mice, an artificial transmission barrier for the passage of full-length mouse prions was diminished by the coexpression of full-length wt MoPrP(C), suggesting that wt MoPrP acts in trans to accelerate the replication of "miniprions" containing PrP(Sc)106. Following a single passage (approximately 300 d) in Tg(PrP106) mice, the miniprions efficiently transmitted disease to Tg(PrP106) mice after only approximately 66 days. Our findings with Tg(BoPrP) mice provide compelling evidence that prions from cattle with BSE have infected humans and caused fatal neurodegeneration, the unique features of miniprions offer new insights into the mechanism of prion replication, and the trans-acting effects of full-length PrP coexpression suggest a new approach to the development of even more efficient animal models for prion diseases.


Subject(s)
Disease Models, Animal , Mice, Transgenic , Prion Diseases , Prions , Animals , Brain/pathology , Cattle , Creutzfeldt-Jakob Syndrome/etiology , Creutzfeldt-Jakob Syndrome/pathology , Creutzfeldt-Jakob Syndrome/physiopathology , Creutzfeldt-Jakob Syndrome/transmission , Encephalopathy, Bovine Spongiform/etiology , Encephalopathy, Bovine Spongiform/pathology , Encephalopathy, Bovine Spongiform/physiopathology , Encephalopathy, Bovine Spongiform/transmission , Humans , Mice , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/metabolism , PrPSc Proteins/chemistry , PrPSc Proteins/metabolism , Prion Diseases/etiology , Prion Diseases/pathology , Prion Diseases/physiopathology , Prion Diseases/transmission , Prions/chemistry , Prions/genetics , Prions/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Species Specificity , Transgenes
7.
Proc Natl Acad Sci U S A ; 96(26): 15137-42, 1999 Dec 21.
Article in English | MEDLINE | ID: mdl-10611351

ABSTRACT

There is growing concern that bovine spongiform encephalopathy (BSE) may have passed from cattle to humans. We report here that transgenic (Tg) mice expressing bovine (Bo) prion protein (PrP) serially propagate BSE prions and that there is no species barrier for transmission from cattle to Tg(BoPrP) mice. These same mice were also highly susceptible to a new variant of Creutzfeldt-Jakob disease (nvCJD) and natural sheep scrapie. The incubation times (approximately 250 days), neuropathology, and disease-causing PrP isoforms in Tg(BoPrP)Prnp(0/0) mice inoculated with nvCJD and BSE brain extracts were indistinguishable and differed dramatically from those seen in these mice injected with natural scrapie prions. Our findings provide the most compelling evidence to date that prions from cattle with BSE have infected humans and caused fatal neurodegeneration.


Subject(s)
Encephalopathy, Bovine Spongiform/transmission , PrPSc Proteins/pathogenicity , Prions/pathogenicity , Animals , Brain/pathology , Cattle , Creutzfeldt-Jakob Syndrome/genetics , Creutzfeldt-Jakob Syndrome/pathology , Creutzfeldt-Jakob Syndrome/transmission , Humans , Mice , Mice, Transgenic , PrPSc Proteins/genetics , Prions/genetics , Risk , Scrapie/genetics , Scrapie/pathology , Scrapie/transmission , Species Specificity , Tissue Distribution
8.
Proc Natl Acad Sci U S A ; 96(25): 14529-34, 1999 Dec 07.
Article in English | MEDLINE | ID: mdl-10588739

ABSTRACT

We report that branched polyamines, including polyamidoamide dendimers, polypropyleneimine, and polyethyleneimine, are able to purge PrP(Sc), the protease-resistant isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture. The removal of PrP(Sc) by these compounds depends on both the concentration of branched polymer and the duration of exposure. Chronic exposure of ScN2a cells to low noncytotoxic concentrations of branched polyamines for 1 wk reduced PrP(Sc) to an undetectable level, a condition that persisted at least 3 wk after removal of the compound. Structure-activity analysis revealed that a high surface density of primary amino groups is required for polyamines to eliminate PrP(Sc) effectively from cells. The removal of PrP(Sc) by branched polyamines is attenuated by chloroquine in living cells, and exposure of scrapie-infected brain extracts with branched polyamines at acidic pH rendered the PrP(Sc) susceptible to protease in vitro, suggesting that endosomes or lysozomes may be the site of action. Our studies suggest that branched polyamines might be useful therapeutic agents for treatment of prion diseases and perhaps a variety of other degenerative disorders.


Subject(s)
Polyamines/therapeutic use , Prions/metabolism , Scrapie/drug therapy , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Humans , Hydrogen-Ion Concentration , Mice , Neuroblastoma/drug therapy , Structure-Activity Relationship , Time Factors
9.
Cell ; 96(6): 869-78, 1999 Mar 19.
Article in English | MEDLINE | ID: mdl-10102274

ABSTRACT

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.


Subject(s)
PrPSc Proteins/metabolism , Amino Acid Sequence , Animals , Brain/metabolism , Mice , Mice, Transgenic , PrPSc Proteins/chemistry , PrPSc Proteins/genetics , PrPSc Proteins/pathogenicity , Protein Conformation , Sequence Deletion
10.
Neuroreport ; 8(14): 3131-5, 1997 Sep 29.
Article in English | MEDLINE | ID: mdl-9331928

ABSTRACT

Two highly-selective mu-opioid receptor agonists, endomorphin-1 and -2, were recently purified from bovine brain and are postulated to be endogenous mu-opioid receptor ligands. We sought to determine the effects of these ligands at the spinal level in mice. Endomorphin-1 and -2 produced short acting, naloxone-sensitive antinociception in the tail flick test and inhibited the behavior elicited by intrathecally injected substance P. Both endomorphin-1 and -2 were anti-allodynic in the dynorphin-induced allodynia model. Although acute tolerance against both endomorphins developed rapidly, endomorphin-1 required a longer pretreatment time before tolerance was observed. We conclude that the endomorphins are potent spinal antinociceptive and anti-allodynic agents and that they or related compounds may prove therapeutically useful as spinal analgesics.


Subject(s)
Analgesics, Opioid/pharmacology , Oligopeptides/pharmacology , Receptors, Opioid, mu/agonists , Animals , Cattle , Drug Tolerance , Hot Temperature , Injections, Spinal , Male , Mice , Mice, Inbred ICR , Substance P/antagonists & inhibitors
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