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1.
J Neurochem ; 78(1): 155-62, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11432982

RESUMO

Tight junctions form the diffusion barrier of brain microcapillary endothelial cells and support cell polarity. Also astrocytes express tight junction components such as occludin, claudin-1, ZO-1 and ZO-2, but do not establish a permeability barrier. However, little is known about the function and regulation of these molecules in astrocytes. We studied the impact of tumour necrosis factor (TNF) on occludin and ZO-1 expression in astrocytes. TNF decreased occludin, but not ZO-1 expression. In brain microcapillary endothelial cells, as well as in epithelial cells, occludin expression was not influenced by TNF. Removal of TNF from astrocytes restored the basal level of occludin. Down-regulation was inhibited by caffeic acid phenethyl ester, a specific inhibitor of nuclear factor-kappaB (NF-kappaB) activation. Exposure of astrocytes isolated from mice deficient in either TNF type-1 receptor (TNFR1), TNF type-2 receptor (TNFR2), or both, respectively, revealed that down-regulation was mediated entirely by TNFR1. ZO-1, which can interact with occludin, was found to co-precipitate connexin43, but not occludin. These findings demonstrate that TNF selectively down-regulates occludin in astrocytes, but not in cells forming established tight junctions, through TNFR1 and suggest that NF-kappaB is involved as a negative regulator.


Assuntos
Antígenos CD/fisiologia , Astrócitos/metabolismo , Proteínas de Membrana/metabolismo , NF-kappa B/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Antígenos CD/genética , Células Cultivadas , Circulação Cerebrovascular , Regulação para Baixo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Microcirculação , Ocludina , Fosfoproteínas/metabolismo , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Proteína da Zônula de Oclusão-1
2.
J Neurosci Res ; 64(1): 70-8, 2001 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-11276053

RESUMO

The extracellular matrix glycoprotein tenascin-R (TN-R) has been implicated in a variety of cell-matrix interactions involved in the molecular control of axon guidance and neural cell migration during development and regeneration of the central nervous system (CNS). Whereas TN-R is amply expressed in the early postnatal and adult mammalian CNS, the protein has so far not been detected in different compartments of the peripheral nervous system (PNS). Here we provide first evidence that TN-R (predominantly TN-R 160 isoform) is transiently expressed in the sciatic nerve of late embryonic (E14-18) and neonatal mice, while at later developmental stages, both protein and mRNA are downregulated. In vitro, TN-R protein was found to be expressed by both undifferentiated and neuronally differentiated PC12 cells and by L1-positive Schwann cells (SC), but not by other neural and non-neural cell types in cell cultures derived from embryonic (E17/18) hindlimbs and neonatal sciatic nerves. In the developing PNS, TN-R expression correlated with axon growth and SC migration during the period of skeletal muscle innervation. Based on different in vitro approaches, we found that the substrate-bound glycoprotein selectively inhibits the fibronectin-dependent: (1) neurite outgrowth from dorsal root ganglion neurons (strongly expressing alpha5beta1 integrin and the disialoganglioside GD3) by a ganglioside-sensitive signaling mechanism; and (2) migration of primary myoblasts and other non-neuronal cells in a ganglioside-independent manner. Our findings suggest the functional role of TN-R in PNS pattern formation during distinct stages of axon pathfinding and skeletal muscle innervation.


Assuntos
Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Tenascina/metabolismo , Envelhecimento/metabolismo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Feto/metabolismo , Fibronectinas/fisiologia , Camundongos , Neuritos/fisiologia , Células PC12/metabolismo , Ratos , Células de Schwann/fisiologia , Nervo Isquiático/citologia , Nervo Isquiático/embriologia , Nervo Isquiático/crescimento & desenvolvimento
3.
Acta Crystallogr D Biol Crystallogr ; 57(Pt 1): 37-43, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11134925

RESUMO

The crystal structure of affinity-purified Thermomonospora fusca beta-mannanase has been solved despite the lack of the major part of the amino-acid sequence. A high-quality electron-density map allowed the identification of a stretch of eight amino acids close to the C-terminus which was used to design a degenerate downstream PCR primer. Together with a specific primer previously derived from the N-terminus, 95.7% of the mannanase gene sequence was obtained from genomic T. fusca DNA by PCR. The structure-derived sequence was then compared with the DNA-derived sequence and corrected when necessary. Applying the presented protocol, there was no need to manually build a model at an early stage of structure determination, an erroneous and tedious process, especially in the absence of the amino-acid sequence. Using the DNA sequence information and the current version of ARP/wARP, 281 residues, or 93% of the polypeptide chain (including side chains), were built and refined to an R factor of 16.5% without any manual intervention.


Assuntos
Manosidases/química , Sequência de Aminoácidos , Sequência de Bases , DNA , Elétrons , Dados de Sequência Molecular , Conformação Proteica , beta-Manosidase
4.
J Neurosci Res ; 60(6): 733-42, 2000 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-10861785

RESUMO

The dysfunction of the blood-brain barrier (BBB) occurring after traumatic brain injury (TBI) is mediated by intracerebral neutrophil accumulation, chemokine release (e.g., interleukin (IL)-8) and upregulation of adhesion molecules (e.g., intercellular adhesion molecule (ICAM)-1). In patients with severe TBI, we previously found that elevated cerebrospinal fluid (CSF) IL-8 and soluble (s)ICAM-1 correlate with BBB dysfunction, and this prompted us to concomitantly monitor IL-8, sICAM-1 and their stimulator tumor necrosis factor (TNF)-alpha in CSF. Potential mechanisms for upregulation of the IL-8 analogue, murine macrophage inflammatory protein (MIP)-2, and sICAM-1 at the BBB were studied using cultured mouse astrocytes and brain microvascular endothelial cells (MVEC). In CSF of seven patients, IL-8 and sICAM-1 were elevated for 19 days after severe TBI, whereas TNF-alpha exceeded normal values on 9 days. Stimulation of MVEC and astrocytes with TNF-alpha simultaneously induced the release of MIP-2 reaching saturation by 4-8 hr and of sICAM-1 increasing continuously from 2-4 hr to 12 hr. Augmented sICAM-1 production correlated with enhanced membrane-bound (m)ICAM-1 expression in both cell types (r(s) = 0.96 and 0.90, P < 0.0001), but was markedly higher in astrocytes. The release of sICAM-1 was not influenced by IL-8 or MIP-2, although astrocytes and MVEC expressed the IL-8/MIP-2 receptor (CXCR-2) as determined by FACS analysis. Instead, we found that sICAM-1 strongly induced MIP-2 secretion by both cell types with kinetics differing from those evoked by TNF-alpha. If added together, sICAM-1 and TNF-alpha synergistically induced MIP-2 production suggesting the involvement of two different pathways for MIP-2 regulation.


Assuntos
Astrócitos/metabolismo , Circulação Cerebrovascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/farmacologia , Monocinas/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Adulto , Animais , Astrócitos/efeitos dos fármacos , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Células Cultivadas , Quimiocina CXCL2 , Sinergismo Farmacológico , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Humanos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos , Microcirculação/efeitos dos fármacos , Pessoa de Meia-Idade , Monocinas/fisiologia , Solubilidade
5.
J Neurosci Res ; 60(1): 45-57, 2000 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-10723067

RESUMO

Galectin-3 is a member of the galectin family of beta-galactoside-specific animal lectins. Here we show that galectin-3 is constitutively expressed in 15 out of 16 glioma cell lines tested, but not by normal or reactive astrocytes, oligodendrocytes, glial O-2A progenitor cells and the oligodendrocyte precursor cell line Oli-neu. Galectin-3 is also expressed by one oligodendroglioma cell line, but not by primitive neuroectodermal tumor and 4 neuroblastoma cell lines tested so far. In all galectin-3 expressing cell lines, the lectin is predominantly, if not exclusively, localized intracellularly and carries an active carbohydrate recognition domain (shown for C6 rat glioma cells). Moreover, in contrast to primary astrocytes, glioma cells do not or only weakly adhere to substratum-bound galectin-3, probably reflecting an unusual glycosylation pattern. Our findings indicate that the expression of galectin-3 selectively correlates with glial cell transformation in the central nervous system and could thus serve as a marker for glial tumor cell lines and glial tumors.


Assuntos
Antígenos de Diferenciação/metabolismo , Lectinas/metabolismo , Neuroglia/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Western Blotting , Adesão Celular , Células Cultivadas , Galectina 3 , Glioma , Humanos , Imuno-Histoquímica , Camundongos , Neuroglia/patologia , Oligodendroglia/metabolismo , Ratos , Células Tumorais Cultivadas
6.
Parasitology ; 120 ( Pt 2): 135-42, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10726275

RESUMO

Over a period of 26 months from January 1996 to February 1998, 388 foxes from the city of Zürich, Switzerland, were examined for intestinal infections with Echinococcus multilocularis and other helminths. The prevalence of E. multilocularis in foxes sampled during winter increased significantly from 47% in the urban to 67% in the adjacent recreational area, whereas prevalence rates of other helminths were similar in both areas. Seasonal differences in the prevalence of E. multilocularis were only found in urban subadult male foxes which were significantly less frequently infected in summer than in winter. The distribution of the Echinococcus biomass, as expressed by worm numbers per fox was overdispersed in 133 infected foxes randomly sampled in winter. Ten of these foxes (8%) were infected with more than 10,000 specimens and carried 72% of the total biomass of E. multilocularis (398,653 worms). Prevalences did not differ significantly in these foxes in regard to age and sex but worm burdens were significantly higher in subadult foxes as compared with adult foxes. In voles (Arvicola terrestris) trapped in a city park of Zürich, E. multilocularis metacestodes were identified by morphological examination and by PCR. The prevalence was 20% among 60 rodents in 1997 and 9% among 75 rodents in 1998. Protoscoleces occurred in 2 of the cases from 1997. The possible risk for human infection is discussed with respect to the established urban E. multilocularis cycle.


Assuntos
Arvicolinae/parasitologia , Equinococose/veterinária , Echinococcus/crescimento & desenvolvimento , Raposas/parasitologia , Doenças dos Roedores/epidemiologia , Animais , Anticorpos Monoclonais , Equinococose/epidemiologia , Feminino , Imunofluorescência/veterinária , Humanos , Mucosa Intestinal/parasitologia , Intestino Delgado/parasitologia , Fígado/parasitologia , Masculino , Prevalência , Doenças dos Roedores/parasitologia , Estações do Ano , Suíça/epidemiologia , População Urbana
7.
FEBS Lett ; 436(3): 466-70, 1998 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-9801170

RESUMO

cGMP has been shown to either activate or inhibit Na,K-ATPase activity. Using mouse brain endothelial cells which express both ouabain-resistant alpha1 and ouabain-sensitive alpha2 and alpha3 isoforms, we show that cGMP reduces total Na,KATPase activity to about 58%. The inhibition is prevented by the protein kinase G (PKG)-specific inhibitor KT5823, indicating that cGMP-mediated activation of PKG leads to inhibition of the pump. A similar extent of inhibition is obtained with nitric oxide. cGMP-induced inhibition acts mainly on alpha1 isoforms but hardly affects alpha2/alpha3 isoforms. These data suggest that inhibition of Na,K-ATPase activity by cGMP occurs in an isoform-selective manner in brain endothelial cells.


Assuntos
Carbazóis , Circulação Cerebrovascular , GMP Cíclico/farmacologia , Endotélio Vascular/enzimologia , Indóis , Isoenzimas/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Alcaloides/farmacologia , Animais , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Cinética , Camundongos , Óxido Nítrico/farmacologia , Ouabaína/farmacologia , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes/antagonistas & inibidores
8.
Biochem Biophys Res Commun ; 239(3): 804-9, 1997 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-9367850

RESUMO

Interleukin-1 alpha (IL-1 alpha) and interleukin-6 (IL-6), both known to be able to open the blood-brain barrier (BBB), downregulated plasma membrane-associated tyrosine phosphatase activity in primary porcine brain endothelial cells (PBEC). In contrast, transforming growth factor beta (TGF-beta) upregulated PTP activity and tumor necrosis factor alpha (TNF-alpha) had no effect. Plasma membrane-associated PTP activity of PBEC was upregulated at contact inhibited growth arrest. Tightly confluent cells reduced 3H-inulin permeability by 34% compared with just confluent cells indicating the formation of barrier properties. The decrease in permeability temporally correlated with the elevated PTP activity of the cells at growth arrest and was reversed to control by IL-1 alpha. Vanadate, a broad-specificity PTP inhibitor, also enhanced 3H-inulin permeability. These data suggest that IL-1 alpha-induced endothelial permeability could be controlled through lowering PTP activity.


Assuntos
Encéfalo/enzimologia , Encéfalo/fisiologia , Permeabilidade Capilar/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiologia , Interleucina-1/farmacologia , Proteínas Tirosina Fosfatases/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/citologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/farmacologia , Endotélio Vascular/citologia , Ativação Enzimática/efeitos dos fármacos , Proteínas Tirosina Fosfatases/efeitos dos fármacos , Suínos
9.
FEBS Lett ; 412(1): 1-4, 1997 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-9257678

RESUMO

The ion gradients generated by the Na,K-ATPase are essential for Na+-coupled transport systems, osmoregulation and restoration of ion concentrations in excitable tissues. Indirectly, the sodium pump controls intracellular Ca2+ concentration through the Na/Ca exchanger. In the nervous system various neurotransmitters can modulate Na,K-ATPase activity. The great diversity of Na,K-ATPase subunit isoforms, their complex spatial and temporal regulation of expression and their cellular localisation imply a functional role of the sodium pump in different regulatory pathways. Among these, potassium homeostasis and modulation of synaptic transmission are discussed here.


Assuntos
Homeostase , Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/fisiologia , Transmissão Sináptica/fisiologia , Animais , Isoenzimas/fisiologia
10.
Proc Natl Acad Sci U S A ; 94(4): 1136-41, 1997 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-9037019

RESUMO

The functional unit of the Na,K-ATPase consists of a catalytic alpha subunit noncovalently linked with a glycoprotein subunit, beta. Using ouabain binding assays and immunoprecipitation of rodent alpha/beta complexes, we show here that all six possible isozymes between three alpha and two beta isoforms can be formed in Xenopus oocytes. Two isoform-specific differences in alpha/beta interactions are observed: (i) alpha1/beta1 and alpha2/beta2 complexes, in contrast to alpha1/beta2 complexes, are stable against Triton X-100-mediated dissociation, and (ii) beta2 subunits must carry N-glycans to combine with alpha1 but not with alpha2. The interacting surfaces are mainly exposed to the extracellular side because coexpression of a truncated beta1 subunit comprising the ectodomain results in assembly with alpha1 and alpha2, but not with alpha3; the beta2 ectodomain combines with alpha2 only. A chimera consisting of 81% and 19% of the alpha1 N terminus and alpha2 C terminus, respectively, behaves like alpha2 and coprecipitates with the beta2 ectodomain. In contrast, the reciprocal chimera does not coprecipitate with the beta2 ectodomain. These results provide evidence for a selective interaction of Na,K-ATPase alpha and beta subunits.


Assuntos
Glicoproteínas/metabolismo , Isoenzimas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Polaridade Celular , Inibidores Enzimáticos/metabolismo , Estabilidade Enzimática , Glicoproteínas/ultraestrutura , Isoenzimas/ultraestrutura , Camundongos , Modelos Moleculares , Ouabaína/metabolismo , Ligação Proteica , Conformação Proteica , Ratos , ATPase Trocadora de Sódio-Potássio/ultraestrutura
11.
Biochem Biophys Res Commun ; 231(3): 755-9, 1997 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-9070887

RESUMO

Both subunits of the Na,K-ATPase are encoded by several genes giving rise to at least six isozymes. To examine whether beta isoforms assemble with alpha 1 in a selective manner, we have overexpressed wild-type and chimeric beta subunits in L929 cells and examined assembly as a function of resistance towards detergent-mediated dissociation. In the presence of digitonin all beta chimeras coimmunoprecipitate the endogenous alpha 1 subunit. Only beta proteins with the ectodomain of beta 1 coimmunoprecipitate alpha 1 in the presence of Triton X-100. All beta chimeras stimulate Na,K-ATPase activity in L929 cells. These data indicate that the beta subunit ectodomains mediate interactions with alpha 1 and influence the stability of this complex.


Assuntos
Isoenzimas/química , ATPase Trocadora de Sódio-Potássio/química , Animais , Linhagem Celular , Digitonina/química , Espaço Extracelular , Substâncias Macromoleculares , Camundongos , Proteínas Recombinantes de Fusão , Relação Estrutura-Atividade , Transfecção
12.
Toxicol In Vitro ; 11(5): 513-8, 1997 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20654344

RESUMO

The availability of an in vitro blood-brain barrier model would represent a powerful alternative to experimental animals in pharmacological and toxicological research. This overview collects the various current approaches to build an in vitro model of the blood-brain barrier for these purposes. Purified bovine, porcine and human brain microcapillary endothelial cells as well as several immortalized cell lines have been used to model the blood-brain barrier in vitro, partly in co-culture with astrocytes of various species, or various cell lines such as C6 glioma or N2a neuroblastoma cells. The collected data indicate that functional parameters often can be induced by soluble and membrane-bound factors in such cell systems. Relevant barrier-specific parameters are reviewed: electrical resistance, and structure and function of the multidrug resistance P-glycoprotein and the y-glutamyl transpeptidase. Both P-glycoprotein and gamma-glutamyl transpeptidase have great influence on the pharmacodynamics, toxicology and metabolic capacity of the blood-brain barrier (drug efflux, oxidative damage, detoxification of endotoxins, etc.). Several available in vitro models appear to be suited for pharmacotoxicological screening, if the functional parameters gamma-glutamyl transpeptidase, P-glycoprotein as well as transendothelial resistance are monitored.

13.
J Neurosci Res ; 46(5): 551-64, 1996 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-8951667

RESUMO

Zebrafish beta 3, a full length cDNA clone encoding a zebrafish Na,K-ATPase beta subunit, was isolated. The protein shares highest homology with the beta 3 subunits of amphibians and mammals, slightly less homology with the beta 2 subunits, and is distinct from the beta 1 subunits. The fish beta subunit co-assembled with alpha subunits to form Na,K-ATPase enzymes when expressed in Xenopus oocytes. Embryonic expression was first detected by whole-mount in situ hybridization between 8-12 hr post-fertilization (hpf) in the head mesoderm. Subsequently, and up to 24 hpf, the mRNA was confined to four dorsal domains in the anterior neural tube. After a transient downregulation during the second day, expression was again conspicuous in the nervous system of 3-day-old larvae. Based on its distribution pattern, the fish beta subunit could be involved in setting up regional identities in the developing fish CNS and in the differentiation of distinct cell types.


Assuntos
Proteínas do Tecido Nervoso/genética , ATPase Trocadora de Sódio-Potássio/genética , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Diferenciação Celular , DNA Complementar/genética , Genes , Hibridização In Situ , Larva , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/química , Oócitos , RNA Mensageiro/biossíntese , Alinhamento de Sequência , ATPase Trocadora de Sódio-Potássio/biossíntese , ATPase Trocadora de Sódio-Potássio/química , Xenopus laevis , Peixe-Zebra/genética
14.
Gene ; 160(2): 213-7, 1995 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-7543868

RESUMO

We describe a plasmid, pNKS2-myc, designed for convenient in-frame fusion of an antibody-specific epitope sequence to the N terminus of a desired cDNA and subsequent synthesis of transcripts that direct the synthesis of the tagged polypeptide in Xenopus laevis (Xl) oocytes. pNKS2-myc contains an SP6 promoter, followed by the translation initiation sequence of the Na,K-pump beta 3 subunit of Xl and the sequence encoding an epitope derived from the human c-myc proto-oncogene product. Appropriate restriction sites allow one to insert virtually any desired cDNA fragment directly behind the epitope-specific sequence and before a long poly(A) tail. After linearization with EcoRI or NotI, polyadenylated cRNA can be synthesized that is efficiently translated in Xl oocytes. The utility of pNKS2-myc is demonstrated by cloning cDNAs coding for Na,K-pump subunits into this vector and injecting the corresponding cRNAs into oocytes. The tagged mouse beta 1 and beta 2 subunit isoforms could be purified from detergent extracts of these cells by immunoprecipitation with a generally available monoclonal antibody (mAb) to the tag, 9E10, as well as with specific mAb that recognize individual beta subunit isoforms. Under native conditions, endogenous and coexpressed exogenous alpha 1 subunits (the catalytic subunit of the Na,K-pump) were co-precipitated, indicating that the N-terminal addition of the decapeptide epitope has no adverse effect on the folding of beta subunits nor on their assembly with alpha subunits. Furthermore, the Myc-specific mAb likewise precipitated a Myc-tagged Na,K-pump alpha 1 subunit together with any of the co-synthesized beta subunits.


Assuntos
Clonagem Molecular/métodos , Epitopos/genética , Genes myc , Vetores Genéticos , Oócitos/metabolismo , RNA Complementar/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Xenopus laevis/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , DNA Complementar/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Epitopos/imunologia , Vetores Genéticos/genética , Glicosilação , Complexo de Golgi/metabolismo , Humanos , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Proto-Oncogene Mas , RNA Complementar/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , ATPase Trocadora de Sódio-Potássio/genética , Torpedo/genética
15.
J Biol Chem ; 268(35): 26260-7, 1993 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-7504672

RESUMO

We have previously provided evidence for a dual function of the adhesion molecule on glia (AMOG/beta 2), the beta 2 subunit of the murine Na,K-ATPase, both as neural recognition molecule mediating neuron-glia interactions and as functional beta subunit of the sodium pump. To analyze the functional role of AMOG/beta 2 in neurite outgrowth, AMOG/beta 2-expressing L-cells were generated by transfection and used as substrates for neurite outgrowth of cerebellar and hippocampal neurons. AMOG/beta 2-transfected L-cells led to an increase in neurite length after 6 h, which was specifically inhibited by antibodies to AMOG/beta 2 and a neuronal membrane fraction. Moreover, the extracellular domain of AMOG/beta 2 generated as a soluble recombinant protein in Chinese hamster ovary cells partially inhibited the increase in neurite outgrowth on AMOG/beta 2-transfected L-cells. L-cells transfected with the mouse beta 1 subunit had no effect on neurite extension. Our observations show for the first time differences in functional properties for different beta isoforms of the Na,K-ATPase and suggest that AMOG/beta 2 but not beta 1 is able to interact with an unknown neuronal receptor leading to increased neurite outgrowth, most likely via signal transduction.


Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuritos , ATPase Trocadora de Sódio-Potássio/fisiologia , Adenosina Trifosfatases , Animais , Proteínas de Transporte de Cátions , Células Cultivadas , Células L/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Transfecção
16.
Gene ; 120(2): 307-12, 1992 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-1383096

RESUMO

A hybrid gene consisting of the sequences coding for the signal peptide and N terminus of a type-I membrane protein, the neural cell adhesion molecule (N-CAM), and the extracellular domain of the adhesion molecule on glia (AMOG/beta 2), a type-II membrane protein, was constructed. The sequence was inserted into a eukaryotic expression vector containing the human cytomegalovirus promoter and the glutamine synthetase selection marker, and used to transfect Chinese hamster ovary cells. The resulting stably transformed cell lines produced large amounts of soluble recombinant AMOG/beta 2 (reAMOG/beta 2), which was secreted into the culture medium as a heavily glycosylated 40-55-kDa protein. N-terminal sequence analysis revealed that the protein is not cleaved at the natural signal peptide cleavage site of N-CAM, but two amino acids (aa) further downstream. Treatment of reAMOG/beta 2 with N-glycosidase F (GlycoF) reduced the molecular mass to 27 kDa, corresponding to the calculated mass of the unglycosylated form. In contrast to AMOG/beta 2 isolated from mouse brain, which is sensitive to endoglycosidase H, the immunoaffinity-purified re-protein is more resistant to this treatment, indicating that the sugars attached to reAMOG/beta 2 are mainly of the complex type. Our results demonstrate the feasibility of secreting the extracellular domain of a type-II membrane protein, which is usually inserted into the membrane with the C terminus facing the extracellular side.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Adenosina Trifosfatases , Sequência de Aminoácidos , Animais , Células CHO , Proteínas de Transporte de Cátions , Moléculas de Adesão Celular Neuronais/biossíntese , Moléculas de Adesão Celular Neuronais/isolamento & purificação , Linhagem Celular Transformada , Cricetinae , Eletroforese em Gel de Poliacrilamida , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/isolamento & purificação , Vetores Genéticos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/isolamento & purificação , Sinais Direcionadores de Proteínas/biossíntese , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/isolamento & purificação , Mapeamento por Restrição , Transfecção
17.
J Biol Chem ; 267(28): 20212-6, 1992 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-1383200

RESUMO

The adhesion molecule on glia, AMOG, an integral cell surface glycoprotein highly expressed by cerebellar astrocytes and involved in neuron to astrocyte adhesion and granule neuron migration (Antonicek, H., Persohn, E., and Schachner, M. (1987) J. Cell Biol. 104, 1587-1595) has been identified as a beta 2 subunit isoform of the mouse sodium pump (Gloor, S., Antonicek, H., Sweadner, K.J., Pagliusi, S., Frank, R., Moos, M., and Schachner, M. (1990) J. Cell Biol. 110, 165-174). Here we demonstrate that AMOG/beta 2 expressed by cRNA injection in Xenopus oocytes is capable of combining with endogenous Xenopus alpha 1 subunits or coexpressed Torpedo alpha 1 subunits to yield a functional alpha 1/AMOG sodium pump isozyme. Determinations of the number of ouabain binding sites and ouabain-sensitive 86Rb+ uptake suggest that the alpha 1/AMOG isozyme has slightly lower maximum transport rate and apparent affinity for external K+ than the alpha 1/beta 1 isozyme. Immunoprecipitation of alpha 1/AMOG complexes from digitonin extracts of [35S]methionine-labeled oocytes with a monoclonal anti-AMOG antibody provides direct evidence for a stable association between AMOG and the alpha 1 subunits of Xenopus and Torpedo.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , ATPase Trocadora de Sódio-Potássio , Adenosina Trifosfatases , Animais , Proteínas de Transporte de Cátions , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Digitonina , Eletroforese em Gel de Poliacrilamida , Proteínas da Matriz Extracelular/genética , Isoenzimas/metabolismo , Camundongos , Oócitos , Ouabaína/metabolismo , Plasmídeos , Testes de Precipitina , RNA/genética , RNA Complementar , Rubídio/metabolismo , Torpedo , Xenopus
18.
Biochem J ; 279 ( Pt 2): 329-36, 1991 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-1719955

RESUMO

Recent evidence suggests that the beta subunit of the Na+ pump is essential for the alpha subunit to express catalytic activity and for assembly of the holoenzyme in the plasma membrane. We report here that injection into Xenopus laevis oocytes of cRNAs specific for beta 1 subunit isoforms of the Na+ pump of four species (Torpedo californica, chicken, mouse and rat) causes a time-dependent increase in the number of ouabain-binding sites, both in the plasma membrane and in internal membranes. Expression of the beta 1 subunit of the Na+ pump of mouse and rat in the oocytes could be substantiated by immunoprecipitation using a polyclonal antiserum against the mouse beta 1 subunit. Scatchard analysis in permeabilized cells disclosed that the affinity for ouabain is unchanged after expression of each of the beta 1 subunits. A proportional increase in ouabain-sensitive 86Rb+ uptake indicates that the additionally expressed ouabain-binding sites on the cell surface represent functional Na+ pumps. The findings support the concept of Geering. Theulaz, Verrey, Häuptle & Rossier [(1989) Am. J. Physiol. 257, C851-C858] that beta 1 subunits expressed in oocytes associate with an excess of endogenous alpha subunits of the Na+ pump to form a hybrid enzyme. In addition, all of the beta 1 isoforms investigated in the present study were also capable of combining with the co-expressed alpha 1 subunit of the Torpedo Na+ pump to produce a functional enzyme. Injection of cRNA encoding for the Torpedo alpha 1 subunit alone had no effect on the ouabain-binding capacity of the surface and intracellular membranes of the oocyte.


Assuntos
Oócitos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/fisiologia , Xenopus laevis , Animais , Galinhas , Feminino , Expressão Gênica , Técnicas de Imunoadsorção , Cinética , Substâncias Macromoleculares , Masculino , Ouabaína/metabolismo , Multimerização Proteica , RNA/genética , RNA Complementar , Ratos , Radioisótopos de Rubídio , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , Especificidade da Espécie , Torpedo , Transfecção
19.
Somat Cell Mol Genet ; 16(4): 401-5, 1990 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1699290

RESUMO

AMOG, identified as an adhesion molecule that mediates neuron-astrocyte interaction, has structural similarity to the beta-subunit of Na,K ATPase. We have mapped the AMOG gene to human chromosome 17 and mouse chromosome 11 by somatic cell hybrid analysis. Recombinant inbred strain mapping has placed the Amog locus close to genes for zinc finger protein-3 and the asialoglycoprotein receptor in a region of mouse chromosome 11 that is homologous to human 17p.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Cromossomos Humanos Par 17 , Proteínas da Matriz Extracelular/genética , ATPase Trocadora de Sódio-Potássio/genética , Adenosina Trifosfatases , Animais , Southern Blotting , Proteínas de Transporte de Cátions , Mapeamento Cromossômico , Humanos , Células Híbridas , Camundongos , Camundongos Endogâmicos , Polimorfismo Genético
20.
J Cell Biol ; 110(1): 165-74, 1990 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1688561

RESUMO

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Proteínas do Tecido Nervoso/genética , ATPase Trocadora de Sódio-Potássio/genética , Adenosina Trifosfatases , Sequência de Aminoácidos , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Sequência de Bases , Western Blotting , Encéfalo/enzimologia , Encéfalo/metabolismo , Proteínas de Transporte de Cátions , Adesão Celular , Células Cultivadas , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Imunofluorescência , Biblioteca Gênica , Cinética , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Rubídio/metabolismo , Homologia de Sequência do Ácido Nucleico
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