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1.
Clin Pharmacol Ther ; 100(5): 431-436, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27530302

RESUMO

Emerging evidence indicates that lysosome function extends beyond macromolecular degradation. Genetic and functional defects in components of the lysosomal transport machinery cause lysosomal storage disorders implicating the lysosomal solute carrier (SLC) transporters as essential to vital cell processes. The pathophysiology and therapeutic potential of lysosomal SLC transporters are highlighted here, focusing on recent discoveries in autophagic amino acid sensing (SLC38A9), phagocytic regulation in macrophages (SLC29A3, SLC15A3/A4), adenosine triphosphate (ATP) exocytosis in neurotransmission (SLC17A9), and lysosomal transport of maytansine catabolites into the cytoplasm (SLC46A3).


Assuntos
Lisossomos/fisiologia , Proteínas Carreadoras de Solutos/fisiologia , Autofagia/fisiologia , Exocitose/fisiologia , Humanos , Modelos Biológicos , Terapia de Alvo Molecular/métodos , Fagocitose/fisiologia
2.
J Membr Biol ; 187(3): 225-38, 2002 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-12163980

RESUMO

Calcium channels are important regulators of neuronal excitability and contribute to transmitter release, calcium dependent gene expression, and oscillatory behavior in many cell types. Under physiological conditions, native low-voltage (T-type)- and high-voltage-activated (HVA) currents are potently inhibited by trivalent cations. However, the presence of multiple calcium channel isoforms has hampered our ability to unequivocally assess the effects of trivalent cations on channel activity. Here, we describe the actions of nine trivalent metal ions on transiently expressed alpha1G (Cav3.1) T-type calcium channels cloned from human brain. In 2 mM external barium solution, yttrium most potently inhibited alpha1G current (IC50 = 28 nM), followed by erbium > gadolinium ~ cerium > holmium > ytterbium > neodymium > lanthanum >> scandium. With the exception of scandium, blocking affinity was loosely correlated with decreasing ionic radius. A detailed characterization of yttrium block revealed a 25-fold decrease in blocking affinity when the external concentration of charge carrier was increased from 2 mM to 20 mM. In 20 mM barium, yttrium also effectively inhibited various types of cloned HVA channels indicating that this ion is a nonselective blocker. For all calcium channels examined, yttrium preferentially inhibited inward over outward current, but block was otherwise voltage independent. In addition to peak current inhibition, P/Q- and L-type channels underwent a unique speeding of the macroscopic time course of inactivation. Whereas peak current block of alpha1A channels was highly sensitive to the external charge carrier concentration, the inactivation effects mediated by yttrium were not, suggesting that the two effects are due to distinct mechanisms. Moreover, the speeding effect was greatly attenuated by manipulations that slowed the inactivation kinetics of the channels. Thus, our evidence suggests that yttrium effects are mediated by two distinct events: peak current block likely occurring by occlusion of the pore, and kinetic speeding arising from yttrium interactions with the channel that alter the state of the inactivation gate.


Assuntos
Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Cátions/farmacologia , Metais/farmacologia , Canais de Cálcio/classificação , Células Cultivadas , Cério/farmacologia , Érbio/farmacologia , Gadolínio/farmacologia , Hólmio/farmacologia , Humanos , Rim/embriologia , Rim/fisiologia , Lantânio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neodímio/farmacologia , Escândio/farmacologia , Itérbio/farmacologia , Ítrio/farmacologia
3.
Biophys J ; 79(1): 260-70, 2000 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10866952

RESUMO

We have recently identified farnesol, an intermediate in the mevalonate pathway, as a potent endogenous modulator and blocker of N-type calcium channels (Roullet, J. B., R. L. Spaetgens, T. Burlingame, and G. W. Zamponi. 1999. J. Biol. Chem. 274:25439-25446). Here, we investigate the action of structurally related compounds on various types of voltage-dependent Ca(2+) channels transiently expressed in human embryonic kidney cells. 1-Dodecanol, despite sharing the 12-carbon backbone and headgroup of farnesol, exhibited a significantly lower blocking affinity for N-type Ca(2+) channels. Among several additional 12-carbon compounds tested, dodecylamine (DDA) mediated the highest affinity inhibition of N-type channels, indicating that the functional headgroup is a critical determinant of blocking affinity. This inhibition was concentration-dependent and relatively non-discriminatory among N-, L-, P/Q-, and R-Ca(2+) channel subtypes. However, whereas L-type channels exhibited predominantly resting channel block, the non-L-type isoforms showed substantial rapid open channel block manifested by a speeding of the apparent time course of current decay and block of the inactivated state. Consistent with these findings, we observed significant frequency-dependence of block and dependence on external Ba(2+) concentration for N-type, but not L-type, channels. We also systematically investigated the drug structural requirements for N-type channel inhibition. Blocking affinity varied with carbon chain length and showed a clear maximum at C12 and C13, with shorter and longer molecules producing progressively weaker peak current block. Overall, our data indicate that aliphatic monoamines may constitute a novel class of potent inhibitors of voltage-dependent Ca(2+) channels, with block being governed by rigid structural requirements and channel-specific state dependencies.


Assuntos
Aminas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Rim/efeitos dos fármacos , Tensoativos/farmacologia , Acetatos/farmacologia , Alcanos/farmacologia , Animais , Bário/farmacologia , Canais de Cálcio/metabolismo , Carcinógenos/farmacologia , Linhagem Celular , Dodecanol/farmacologia , Relação Dose-Resposta a Droga , Farneseno Álcool/análogos & derivados , Farneseno Álcool/farmacologia , Humanos , Rim/citologia , Rim/metabolismo , Técnicas de Patch-Clamp , Ratos , Relação Estrutura-Atividade , Transfecção
4.
J Biol Chem ; 275(9): 6388-94, 2000 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-10692440

RESUMO

The direct modulation of N-type calcium channels by G protein betagamma subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of N-type channels and Gbetagamma have recently been identified (see, i.e., Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by Gbetagamma. When syntaxin 1A was coexpressed with (N-type) alpha(1B) + alpha(2)-delta + beta(1b) channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein betagamma subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that Gbetagamma physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with Gbetagamma and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein betagamma subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Proteínas de Ligação ao GTP/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP , Proteínas de Membrana/metabolismo , Toxinas Botulínicas/farmacologia , Linhagem Celular , Humanos , Rim , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Proteínas Qa-SNARE , Proteínas R-SNARE , Proteínas Recombinantes de Fusão/metabolismo , Sintaxina 1 , Transfecção
5.
Anticancer Res ; 19(3A): 1635-40, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10470094

RESUMO

BACKGROUND: Hepatocyte growth factor (HGF) is a multifunctional peptide that binds to a specific receptor, c-met. Both HGF and c-met have been identified in normal brain and on glial tumors. The purpose of this study is to further define the biologic importance of HGF and c-met on normal and malignant glial cells grown in vitro. MATERIALS AND METHODS: Nine human malignant glioma-derived tumor cell cultures and cultures of astrocytes derived from normal brain were examined for c-met and HGF transcripts using Northern blot or RT-PCR analysis. Cellular invasiveness was quantitated by mechanical assay and mitogenesis was determined by cell count. RESULTS: C-met was expressed in five of seven malignant glioma-derived tumor cell cultures and in both normal astrocyte cultures. HGF transcript was not detected in any of the cell cultures. HGF supplementation enhanced invasiveness in c-met positive cell lines and did not alter cellular mitogenesis in the assayed cultures. CONCLUSIONS: These findings suggest that HGF is a potent stimulator of invasiveness in c-met positive malignant glioma-derived tumor cells and is not an active cytokine with regards to in vitro glial cell proliferation. HGF may therefore stimulate glioma cellular invasion in vivo through binding to its receptor and by activating tyrosine kinase secondary messengers.


Assuntos
Astrócitos/química , Astrocitoma/química , Neoplasias Encefálicas/química , Fator de Crescimento de Hepatócito/análise , Proteínas de Neoplasias/análise , Células-Tronco Neoplásicas/química , Proteínas do Tecido Nervoso/análise , Proteínas Proto-Oncogênicas c-met/análise , Adolescente , Adulto , Idoso , Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Divisão Celular/efeitos dos fármacos , Feminino , Glioblastoma/química , Glioblastoma/patologia , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , RNA Neoplásico/análise , Células Tumorais Cultivadas/efeitos dos fármacos
6.
Neuroscience ; 93(1): 349-59, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10430498

RESUMO

The subfornical organ is one of a specialized group of CNS structures devoid of a significant blood-brain barrier, collectively known as the circumventricular organs. While peptides are normally excluded from access to most regions of the CNS, the subfornical organ contains neurons with a high density of receptors for many circulating peptides, including vasopressin. There is a well-established role for the subfornical organ in stimulating the release of vasopressin, and recent evidence suggests that it may also play an important role in mediating the negative feedback actions of vasopressin. The aim of this study was to determine the direct effects of vasopressin on subfornical organ neurons through patch-clamp studies in a dissociated subfornical organ preparation. In current-clamp studies, bath application of 10 nM vasopressin caused depolarizations in 61%, hyperpolarizations in 11%, and no significant change in membrane potential in 28% of neurons tested. We then sought to determine the specific ion channels involved in regulating the vasopressin-induced excitability of subfornical organ neurons through voltage-clamp studies. Vasopressin (10 nM) decreased the peak outward current at +40 mV by 50% (n=7), which was blocked by pretreatment with a V1 receptor antagonist (n=5). Based on these findings, we carried out a systematic characterization of two subformical organ K+ channels, the delayed rectifier (I(K)) and the transient outward current (I(A)). Through voltage isolation of I(K), we found that vasopressin inhibited the steady-state current, by 33+/-7% (n=9). Vasopressin also inhibited the peak I(A) by 27+/-5% (n=8). These data provide the first evidence of a role for K+ channels in mediating the excitatory effects of vasopressin on subfornical organ neurons. The exact physiological roles and sources of vasopressin which may act on subfornical organ neurons are not completely understood at present.


Assuntos
Neurônios/metabolismo , Bloqueadores dos Canais de Potássio , Órgão Subfornical/metabolismo , Vasopressinas/farmacologia , Algoritmos , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Eletrofisiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Vasopressinas/efeitos dos fármacos , Receptores de Vasopressinas/metabolismo , Órgão Subfornical/citologia , Órgão Subfornical/efeitos dos fármacos
7.
J Neurosci Res ; 55(2): 141-6, 1999 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-9972816

RESUMO

The last two centuries, and the last fifty years in particular, have seen a dramatic increase in our understanding of the brain. More recently, philosophers have rekindled the debate about the nature of the mind and have begun to ask how and to what extent the features of the conscious mind, of the self, can be described in solely neurobiological terms. This essay describes a few ways in which neuroscientific research has changed the way philosophers think about the mind and also suggests some ways in which the methods and questions of philosophers might affect neuroscience.


Assuntos
Neurociências/tendências , Filosofia , Humanos , Redes Neurais de Computação
8.
Mol Cell Biol ; 14(11): 7046-58, 1994 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-7935420

RESUMO

Hepatocyte growth factor (HGF), a cytokine with multiple functions, exhibits cell-type-specific as well as cytokine- and steroid hormone-regulated expression. The HGF gene is known to be expressed predominately in mesenchymal but not in epithelial cells. In this study, we report the identification of a cell-type-specific transcriptional repressor in the promoter region of the mouse HGF gene, which is evidently responsible for the suppression of HGF expression in epithelial cells. Gel mobility shift assays and DNase I footprinting studies revealed that a 27-bp element (-16 to +11) around the transcription initiation site is responsible for the binding of a nuclear protein which is present in epithelial but not in mesenchymally derived cells. Further analysis of the binding activity of the DNA region with nuclear protein revealed that an approximately 19-bp sequence containing a unique palindromic structure (5'-AACCGACCGGTT-3') overlapped by a CAP box is essential for binding. Substitution of a single base (the contact site) within this region by site-directed mutagenesis resulted in total abrogation of the binding of the nuclear protein and a concomitant increase in the transcriptional activity of various lengths of HGF-chloramphenicol acetyltransferase fused genes when transfected into the epithelial cell line RL95-2 but not the mesenchymal cell line NIH 3T3. Southwestern (DNA-protein) analyses revealed that the nuclear protein which binds to this repressor element is a single polypeptide of approximately 70 kDa. Analysis of the nuclear extract prepared from regenerating mouse liver at various times after two-thirds partial hepatectomy by gel mobility shift assay revealed a substantial reduction (more than 75% within 3 h) in the binding of the repressor to its cognate binding site. Our results suggest that a cis-acting transcriptional repressor in the promoter region of the mouse HGF gene is involved in cell-type-specific regulation through binding to its cognate trans-acting protein which exists in epithelial cells but is absent in fibroblast cells.


Assuntos
Genes Reguladores , Fator de Crescimento de Hepatócito/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular , DNA/genética , DNA/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo
9.
Oncogene ; 9(7): 2045-52, 1994 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8208549

RESUMO

The c-MET proto-oncogene product is a transmembrane tyrosine kinase receptor which was recently shown to transmit an array of important cellular responses induced by Hepatocyte Growth Factor (HGF). These biological effects include induction of mitogenesis, motogenesis, morphogenesis, metastogenesis and anti-tumor activity on a variety of epithelial cells. All of these processes are known to be associated with normal and abnormal tissue growth and development. The 190 kDa c-MET protein is encoded by a major transcript of 8 kilobases (kb), which is reported to be expressed predominantly in epithelial tissues. The expression pattern of c-MET mRNA and protein are drastically modified in many tumor tissues and cell lines. Currently, no information is available on the molecular mechanisms that regulate c-MET mRNA level. In the present communication, we report for the first time that the inflammatory cytokines such as IL-1 alpha, IL-6 and TNF-alpha, as well as TGF-beta 1, EGF, HGF and the steroidal hormones (estrogen, progesterone, tamoxifen and dexamethasone) markedly influence the steady-state levels of the 8 kb c-MET mRNA in human carcinoma cell lines derived from human tissues such as ovary, breast and endometrium. We demonstrate that c-MET receptor protein is present at high levels in primary tumors of human ovaries (clear cell carcinomas). We present evidence that the 8 kb c-MET mRNA undergoes rapid degradation with a half-life of less than 30 min and that this decay can be quickly inhibited by cycloheximide. Our results suggest that the expression of the c-met proto-oncogene resembles that of an immediate early response gene.


Assuntos
Citocinas/farmacologia , Hormônios/farmacologia , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/genética , Neoplasias do Endométrio/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Precoces , Humanos , Neoplasias Ovarianas/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met , Células Tumorais Cultivadas
10.
Nature ; 325(6102): 305, 1987.
Artigo em Inglês | MEDLINE | ID: mdl-3808027
13.
Biochem J ; 142(1): 57-64, 1974 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-4140721

RESUMO

Tetrahymanol biosynthesis by the protozoan Tetrahymena pyriformis was progressively inhibited by the inclusion of cholesterol in the growth medium. Studies with labelled precursors of tetrahymanol have established that there are two major sites of inhibition in whole cells. The inhibition at the first site, between acetate and mevalonate, occurred rapidly after addition of cholesterol. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), a predominantly cytosolic enzyme in this organism, was not inhibited in cholesterol-grown cells nor by addition of cholesterol directly to the assay medium. The second major site of inhibition in whole cells is between mevalonate and squalene and this is accompanied by inhibition of the enzyme that converts farnesyl-pyrophosphate into squalene (squalene synthetase). Squalene cyclase is partially inhibited. The conversion of mevalonate into tetrahymanol in vitro was not inhibited by the addition of cholesterol to the assay medium. Tetrahymanol added to the culture medium is taken up by the cells but does not inhibit endogenous biosynthesis. It is suggested that cholesterol inhibits the later stages of tetrahymanol biosynthesis by causing a change in membrane structure and function which alters the activity of membrane-bound enzymes.


Assuntos
Colesterol/farmacologia , Tetrahymena pyriformis/metabolismo , Triterpenos/biossíntese , Acetatos/metabolismo , Oxirredutases do Álcool/antagonistas & inibidores , Radioisótopos de Carbono , Cromatografia DEAE-Celulose , Cromatografia em Camada Fina , Difosfatos/metabolismo , Farneseno Álcool/antagonistas & inibidores , Farneseno Álcool/metabolismo , Ligases/antagonistas & inibidores , Ácido Mevalônico/metabolismo , Oxigenases/antagonistas & inibidores , Ácidos Fosfóricos , Compostos Policíclicos/biossíntese , Esqualeno/metabolismo , Tetrahymena pyriformis/efeitos dos fármacos , Trítio
14.
Biochem J ; 139(1): 205-9, 1974 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-4463942

RESUMO

Mevaldate reductase was purified 70-fold from rat liver. The partly purified enzyme had a molecular weight of 27000-30000, reduced certain aromatic aldehydes and was inhibited by barbiturates. These properties are similar to those of other animal tissue aldehyde reductases (EC 1.1.1.2) and it is suggested that mevaldate reductase be reclassified as one of this group.


Assuntos
Oxirredutases do Álcool/metabolismo , Fígado/enzimologia , Animais , Barbitúricos/farmacologia , Cromatografia em Gel , Eletroforese Descontínua , Cinética , Ácido Mevalônico , Peso Molecular , Ratos , Terminologia como Assunto
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