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1.
Nat Commun ; 12(1): 4855, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381044

RESUMO

The vertebrate brain consists of diverse neuronal types, classified by distinct anatomy and function, along with divergent transcriptomes and proteomes. Defining the cell-type specific neuroproteomes is important for understanding the development and functional organization of neural circuits. This task remains challenging in complex tissue, due to suboptimal protein isolation techniques that often result in loss of cell-type specific information and incomplete capture of subcellular compartments. Here, we develop a genetically targeted proximity labeling approach to identify cell-type specific subcellular proteomes in the mouse brain, confirmed by imaging, electron microscopy, and mass spectrometry. We virally express subcellular-localized APEX2 to map the proteome of direct and indirect pathway spiny projection neurons in the striatum. The workflow provides sufficient depth to uncover changes in the proteome of striatal neurons following chemogenetic activation of Gαq-coupled signaling cascades. This method enables flexible, cell-type specific quantitative profiling of subcellular proteome snapshots in the mouse brain.


Assuntos
Ascorbato Peroxidases/metabolismo , Núcleo Celular/metabolismo , Corpo Estriado/metabolismo , Proteoma/metabolismo , Animais , Ascorbato Peroxidases/genética , Corpo Estriado/citologia , Citosol/metabolismo , Espectrometria de Massas , Camundongos , Vias Neurais , Neurônios/citologia , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Coloração e Rotulagem , Fluxo de Trabalho
2.
Mol Psychiatry ; 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526823

RESUMO

Schizophrenia (Sz) is a highly polygenic disorder, with common, rare, and structural variants each contributing only a small fraction of overall disease risk. Thus, there is a need to identify downstream points of convergence that can be targeted with therapeutics. Reduction of microtubule-associated protein 2 (MAP2) immunoreactivity (MAP2-IR) is present in individuals with Sz, despite no change in MAP2 protein levels. MAP2 is phosphorylated downstream of multiple receptors and kinases identified as Sz risk genes, altering its immunoreactivity and function. Using an unbiased phosphoproteomics approach, we quantified 18 MAP2 phosphopeptides, 9 of which were significantly altered in Sz subjects. Network analysis grouped MAP2 phosphopeptides into three modules, each with a distinct relationship to dendritic spine loss, synaptic protein levels, and clinical function in Sz subjects. We then investigated the most hyperphosphorylated site in Sz, phosphoserine1782 (pS1782). Computational modeling predicted phosphorylation of S1782 reduces binding of MAP2 to microtubules, which was confirmed experimentally. We generated a transgenic mouse containing a phosphomimetic mutation at S1782 (S1782E) and found reductions in basilar dendritic length and complexity along with reduced spine density. Because only a limited number of MAP2 interacting proteins have been previously identified, we combined co-immunoprecipitation with mass spectrometry to characterize the MAP2 interactome in mouse brain. The MAP2 interactome was enriched for proteins involved in protein translation. These associations were shown to be functional as overexpression of wild type and phosphomimetic MAP2 reduced protein synthesis in vitro. Finally, we found that Sz subjects with low MAP2-IR had reductions in the levels of synaptic proteins relative to nonpsychiatric control (NPC) subjects and to Sz subjects with normal and MAP2-IR, and this same pattern was recapitulated in S1782E mice. These findings suggest a new conceptual framework for Sz-that a large proportion of individuals have a "MAP2opathy"-in which MAP function is altered by phosphorylation, leading to impairments of neuronal structure, synaptic protein synthesis, and function.

3.
Mol Psychiatry ; 20(9): 1091-100, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25330739

RESUMO

Numerous investigations support decreased glutamatergic signaling as a pathogenic mechanism of schizophrenia, yet the molecular underpinnings for such dysregulation are largely unknown. In the post-mortem dorsolateral prefrontal cortex (DLPFC), we found striking decreases in tyrosine phosphorylation of N-methyl-D aspartate (NMDA) receptor subunit 2 (GluN2) that is critical for neuroplasticity. The decreased GluN2 activity in schizophrenia may not be because of downregulation of NMDA receptors as MK-801 binding and NMDA receptor complexes in postsynaptic density (PSD) were in fact increased in schizophrenia cases. At the postreceptor level, however, we found striking reductions in the protein kinase C, Pyk 2 and Src kinase activity that in tandem can decrease GluN2 activation. Given that Src serves as a hub of various signaling mechanisms affecting GluN2 phosphorylation, we postulated that Src hypoactivity may result from convergent alterations of various schizophrenia susceptibility pathways and thus mediate their effects on NMDA receptor signaling. Indeed, the DLPFC of schizophrenia cases exhibit increased PSD-95 and erbB4 and decreased receptor-type tyrosine-protein phosphatase-α (RPTPα) and dysbindin-1, each of which reduces Src activity via protein interaction with Src. To test genomic underpinnings for Src hypoactivity, we examined genome-wide association study results, incorporating 13 394 cases and 34 676 controls. We found no significant association of individual variants of Src and its direct regulators with schizophrenia. However, a protein-protein interaction-based network centered on Src showed significant enrichment of gene-level associations with schizophrenia compared with other psychiatric illnesses. Our results together demonstrate striking decreases in NMDA receptor signaling at the postreceptor level and propose Src as a nodal point of convergent dysregulations affecting NMDA receptor pathway via protein-protein associations.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Quinases da Família src/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Estudos de Casos e Controles , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Camundongos Knockout , Plasticidade Neuronal , Fosforilação , Densidade Pós-Sináptica/genética , Densidade Pós-Sináptica/metabolismo , Córtex Pré-Frontal/metabolismo , Mapas de Interação de Proteínas , Esquizofrenia/enzimologia , Esquizofrenia/patologia , Transdução de Sinais , Quinases da Família src/genética
4.
Neuroscience ; 158(2): 642-53, 2009 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-18996445

RESUMO

The olfactory epithelium constitutes the sole source of regenerating neural cells that can be obtained from a living human. As such, primary cultures derived from human olfactory epithelial biopsies can be utilized to study neurobiological characteristics of individuals under different conditions and disease states. Here, using such human cultures, we report in vitro generation of cells that exhibit a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. Using in situ hybridization, we demonstrate for the first time the native expression of olfactory receptors in cultured cells derived from human olfactory epithelial tissue. We further establish the presence and function of olfactory transduction molecules in these cells using immunocytochemistry, calcium imaging and molecular methods. Western blot analysis revealed the expression of neurotransmitter receptors for dopamine (D2R), 5-HT (5HT2C) and NMDA subtypes 1 and 2A/2B. Stimulation with dopamine or 5-HT enhanced receptor G protein activation in a subtype specific manner, based on 35S-guanosine triphosphate incorporation assay. Functional characteristics of the cultured cells are demonstrated through enhanced tyrosine phosphorylation of NMDAR 2A/2B and recruitment of signaling partners in response to NMDA stimulation. The array of neuronal characteristics observed here establishes that proliferating cells derived from the human olfactory epithelium differentiate in vitro to express functional and molecular attributes of mature olfactory neurons. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders. Their ready availability from living humans thus provides a new tool to link functional and molecular features of neural cells with clinical characteristics of individual living patients.


Assuntos
Células Epiteliais/metabolismo , Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Mucosa Olfatória/citologia , Neurônios Receptores Olfatórios/metabolismo , Adulto , Animais , Células Cultivadas , Dopaminérgicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Feminino , Glicina/farmacologia , Humanos , Imunoprecipitação/métodos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Proteína de Marcador Olfatório/genética , Proteína de Marcador Olfatório/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Serotoninérgicos/farmacologia , Adulto Jovem
5.
Clin Genet ; 71(4): 311-9, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17470132

RESUMO

Congenital indifference to pain (CIP) is a rare condition in which patients have severely impaired pain perception, but are otherwise essentially normal. We identified and collected DNA from individuals from nine families of seven different nationalities in which the affected individuals meet the diagnostic criteria for CIP. Using homozygosity mapping and haplotype sharing methods, we narrowed the CIP locus to chromosome 2q24-q31, a region known to contain a cluster of voltage-gated sodium channel genes. From these prioritized candidate sodium channels, we identified 10 mutations in the SCN9A gene encoding the sodium channel protein Nav1.7. The mutations completely co-segregated with the disease phenotype, and nine of these SCN9A mutations resulted in truncation and loss-of-function of the Nav1.7 channel. These genetic data further support the evidence that Nav1.7 plays an essential role in mediating pain in humans, and that SCN9A mutations identified in multiple different populations underlie CIP.


Assuntos
Mutação , Insensibilidade Congênita à Dor/genética , Canais de Sódio/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 2/genética , Códon sem Sentido , Análise Mutacional de DNA , Feminino , Efeito Fundador , Mutação da Fase de Leitura , Genética Populacional , Haplótipos , Humanos , Masculino , Canal de Sódio Disparado por Voltagem NAV1.7 , Linhagem , Deleção de Sequência
6.
Neuroscience ; 145(1): 42-55, 2007 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-17239545

RESUMO

Environmental deprivation contributes in important ways to the development of a wide range of psychiatric disorders. Isolation rearing of rodents, a model for environmental deprivation in humans, consistently produces hyperlocomotion, which provides a measurable parameter to study the underlying mechanisms of early adverse psychosocial stressors. Male Sprague-Dawley rat pups were separated from dams at postnatal (PN) day 20 and reared either in groups of three or in isolation. On PN 38, locomotion was assessed in the open field. On PN 46, rats were killed and gene expression patterns examined in the medial prefrontal cortex (mPFC). Isolation-reared rats displayed increased locomotor activity and decreased resting time in the open field. Specific gene expression patterns in the mPFC were associated with both isolation rearing and hyperlocomotive behavior in the open field. Genes involved in these expression patterns included immediate early genes (IEGs) and genes that regulate cell differentiation and apoptosis. The study of these genes could provide important insights into how abnormal early psychosocial events affect brain function and behavior.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Precoces/fisiologia , Locomoção/fisiologia , Córtex Pré-Frontal/metabolismo , Isolamento Social , Animais , Animais Recém-Nascidos , Comportamento Animal , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ratos , Ratos Sprague-Dawley , Tempo de Reação
8.
Gene Ther ; 6(5): 922-30, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10505118

RESUMO

Herpes simplex virus (HSV) is a new platform for gene therapy. We cloned the human herpesvirus HSV-1 strain F genome into a bacterial artificial chromosome (BAC) and adapted chromosomal gene replacement technology to manipulate the viral genome. This technology exploits the power of bacterial genetics and permits generation of recombinant viruses in as few as 7 days. We utilized this technology to delete the viral packaging/cleavage (pac) sites from HSV-BAC. HSV-BAC DNA is stable in bacteria and the pac-deleted HSV-BAC (p45-25) is able to package amplicon plasmid DNA as efficiently as a comparable pac-deleted HSV cosmid set when transfected into mammalian cells. Moreover, the utility of bacterial gene replacement is not limited to HSV, since most herpesviruses can be cloned as BACs. Thus, this technology will greatly facilitate genetic manipulation of all herpesviruses for their use as research tools or as vectors in gene therapy.


Assuntos
Deleção de Genes , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Herpesvirus Humano 1/genética , Animais , Chlorocebus aethiops , Cromossomos Bacterianos , Genoma Viral , Mutagênese Sítio-Dirigida , Células Vero
9.
Biochem Pharmacol ; 50(2): 235-41, 1995 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-7632168

RESUMO

Calphostin C is an anti-tumor agent that binds to the regulatory domain of protein kinase C and inhibits the binding of phorbol dibutyrate. Recent studies suggest that there may be structural similarities between protein kinase C (PKC) and diacylglycerol kinase (DGK). Both enzymes bind diacylglycerol and phosphatidylserine, and sequencing of the 80 kDa diacylglycerol kinase shows that it contains zinc finger-like sequences, similar to those occurring in PKC. Similarities in some enzymatic properties of PKC and DGK led us to examine whether regulatory-site inhibitors of PKC also might inhibit DGK. For these studies, the membrane-bound DGK was partially purified from porcine testis membranes. Calphostin C inhibited DGK with an IC50 in the micromolar range. The inhibition of DGK by calphostin C was competitive with respect to diacylglycerol and was not affected by the presence or absence of phosphatidylserine. Other inhibitors of protein kinase C were without effect, with the exception of Adriamycin, which inhibited at millimolar concentrations. Staurosporine, which binds to the catalytic domain of protein kinase C, did not inhibit DGK. The results suggest that there are functional similarities between the substrate binding site of DGK and the regulatory site of protein kinase C.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Naftalenos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Testículo/enzimologia , Alcaloides/farmacologia , Animais , Sítios de Ligação , Diacilglicerol Quinase , Doxorrubicina/farmacologia , Ferro/farmacologia , Cinética , Masculino , Compostos Organometálicos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Estaurosporina , Suínos
10.
Cutis ; 54(4): 275-8, 1994 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7805414

RESUMO

A severely debilitated patient showed primary cutaneous mucormycosis with a Mucor species at a tape erosion site. The pathogenic nature and epidemiologic features of this unusual fungal infection are reviewed to emphasize its recognition in the differential diagnosis of ischemic lesions in immunocompromised patients. Iron overload may be a risk factor for mucormycosis.


Assuntos
Dermatomicoses/etiologia , Ferro/efeitos adversos , Mucormicose/etiologia , Dermatomicoses/microbiologia , Feminino , Humanos , Pessoa de Meia-Idade , Fatores de Risco
11.
J Appl Physiol (1985) ; 72(4): 1375-9, 1992 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-1592729

RESUMO

Visceral movement due to impact loading is believed to play a role in the locomotor-respiratory coupling (LRC) that has been detected in a number of mammalian species. In the bird and bat species in which LRC has been described, the effect of the wing muscles on the timing of respiration appears to be a dominant influence. To test the hypothesis that LRC occurs in humans propelling wheelchairs (where there is no impact loading and the arms are used for locomotion), we studied 10 wheelchair athletes on a motorized treadmill at three speeds. Each subject's data were analyzed by spectral analysis (based on the fast Fourier transform), which detected apparent LRC (rates within 1% of a single-digit integer ratio) in 12 (40%) of the 30 test settings. However, a control analysis, in which each subject's arm-thrust rates were compared with another subject's breathing rates, revealed apparent (but false) coupling in 8 (27%), not significantly less often (using the chi 2 test). These findings appear to refute the hypothesis that LRC occurs during wheelchair propulsion. These data are consistent with the theory that the visceral piston is important to LRC and suggest that rhythmic arm movements are insufficient to induce the phenomenon in this setting.


Assuntos
Locomoção/fisiologia , Periodicidade , Mecânica Respiratória/fisiologia , Adulto , Braço , Fenômenos Biomecânicos , Humanos , Masculino , Modelos Biológicos , Movimento/fisiologia , Cadeiras de Rodas
12.
Obstet Gynecol ; 77(1): 63-8, 1991 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1701526

RESUMO

The sensitivity and specificity of maternal serum screening for Down syndrome with different biochemical markers were evaluated. Detection rates with different combinations of maternal serum alpha-fetoprotein (MSAFP), hCG, and unconjugated estriol (uE3) were established by retrieving and analyzing 54 serum specimens from women with confirmed Down syndrome pregnancies, compared with 657 specimens from women with normal outcomes. With a risk cutoff of 1:270 at the second trimester, the detection rate with MSAFP, hCG, and uE3 was two to three times higher than with MSAFP alone. With all three markers, the detection rate for Down syndrome increased from 50 to 77% as maternal age increased, and was 60% in a representative screened population. If uE3 was omitted, the detection rate decreased from 60 to 48%. One thousand women were screened prospectively, either with MSAFP or with all three markers prospectively, either with MSAFP or with all three markers and 4.1% with MSAFP. With the three markers, the positive predictive value for Down syndrome was 2.2% overall and as high as 5.9% in older women. Therefore, the addition of hCG and uE3 to the maternal serum screen increases the positive predictive value by 50-300%, depending on maternal age. These results confirm the efficacy of screening for Down syndrome using maternal age and three serum markers.


Assuntos
Síndrome de Down/diagnóstico , Estriol/sangue , Idade Materna , Gravidez de Alto Risco , Diagnóstico Pré-Natal , alfa-Fetoproteínas/análise , Biomarcadores/sangue , Doenças Fetais/diagnóstico , Humanos , Valor Preditivo dos Testes , Estudos Prospectivos , Fatores de Risco , Sensibilidade e Especificidade
13.
Biochem J ; 266(1): 291-9, 1990 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-2155609

RESUMO

We investigated the diacyglycerol kinase species present in several baboon tissues using the substrates sn-1-stearoyl-2-arachidonoyl diacylglycerol and sn-1,2-didecanoyl diacylglycerol. Chromatography of octyl glucoside extracts of the baboon (Papio cynocephalus papio) tissues on hydroxyapatite columns revealed the presence of three diacylglycerol kinase species with different substrate preferences. One species markedly 'preferred' the substrate sn-1-stearoyl-2-arachidonoylglycerol, the two other species preferred sn-1,2-didecanoylglycerol. Measurement of the activity of the baboon brain diacylglycerol kinases toward diacylglycerols with a range of different fatty acid chains revealed a strict preference of the arachidonoyl diacylglycerol kinase for sn-1-acyl-2-arachidonoyl diacylglycerol, whereas the other enzymes showed no preference toward several long-chain-fatty-acid-containing diacylglycerols. The arachidonoyl diacylglycerol kinase was particularly abundant in brain and testis, whereas liver was practically devoid of this enzyme. The arachidonoyl diacylglycerol kinase from baboon brain was found to be predominantly associated with the particulate fraction and exhibited an apparent molecular mass of 130 kDa.


Assuntos
Isoenzimas/análise , Fosfotransferases/análise , Animais , Encéfalo/enzimologia , Diacilglicerol Quinase , Diglicerídeos/metabolismo , Isoenzimas/metabolismo , Rim/enzimologia , Fígado/enzimologia , Masculino , Músculos/enzimologia , Papio , Ácidos Fosfatídicos/metabolismo , Fosfotransferases/metabolismo , Baço/enzimologia , Especificidade por Substrato , Testículo/enzimologia , Distribuição Tecidual
14.
J Biol Chem ; 263(3): 1575-83, 1988 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-2826485

RESUMO

We studied the influence of platelet-derived growth factor (PDGF) on diacylglycerol phosphorylation in Swiss 3T3 cells. Rates of incorporation of 32P into phosphatidic acid (PA) and phosphatidylinositol (PtdIns) were determined in prelabeled cells into which sn-1,2-didecanoylglycerol (diC10) had been introduced. PDGF stimulated the formation of [32P]PA and -PtdIns from endogenous substrates but decreased the formation of [32P]PA10 and -PtdIns10. Direct measurements of diacylglycerol phosphorylation in lysates of quiescent and stimulated cells showed that PDGF stimulated the phosphorylation of endogenous diacylglycerol 2-fold in parallel with diacylglycerol accumulation but decreased by 50% the phosphorylation of diC10. Total diacylglycerol kinase activity, measured in a mixed micellar assay, was not changed by PDGF treatment. The maximum activity of diacylglycerol kinase exceeded that needed to phosphorylate all of the endogenous diacylglycerol, suggesting that the PDGF-dependent increase in diacylglycerol mass would account for the increase in PA formation. The increased mass of diacylglycerol also could explain the inhibition of diC10 phosphorylation, via substrate competition. The predominant species of endogenous diacylglycerol was 1-stearoyl-2-arachidonoyl-glycerol (18:0/20:4 diacylglycerol). In mixed micelles, the rate of phosphorylation of 18:0/20:4 diacylglycerol was 8-fold higher than that of diC10, and the 18:0/20:4 species competed with diC10 for phosphorylation. Studies showed that a membrane-bound enzyme accounted for the PDGF effect on PA formation; there was no evidence for translocation of cytosolic enzyme to the membrane. The results support these conclusions: 1) PDGF stimulates the phosphorylation of cellular diacylglycerol by promoting a transient accumulation of this lipid. 2) The stimulated phosphorylation is catalyzed by a diacylglycerol kinase that preferentially phosphorylates 18:0/20:4 diacylglycerol over diC10. 3) The diacylglycerol kinase responsible for the PDGF effect is membrane-bound.


Assuntos
Diglicerídeos/metabolismo , Glicerídeos/metabolismo , Fosfotransferases/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Linhagem Celular , Diacilglicerol Quinase , Camundongos , Ácidos Fosfatídicos/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , Fosforilação
15.
J Biol Chem ; 263(3): 1584-92, 1988 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-2826486

RESUMO

The membrane-bound diacylglycerol kinase from Swiss 3T3 cells (M-DG kinase) was characterized with a mixed micellar assay system, and compared with the cytosolic diacylglycerol kinase from 3T3 cells and with the membrane-bound diacylglycerol kinase from Escherichia coli. M-DG kinase selectively phosphorylated arachidonoyl-diacylglycerols, at a rate 2- to 8-fold higher than that for other naturally occurring long-chain diacylglycerols. In contrast, the cytosolic 3T3 enzyme exhibited little or no selectivity among long-chain diacylglycerols but had higher activity with more soluble substrates such as 1,2-didecanoylglycerol. Comparison of the properties of M-DG kinase with those of the bacterial membrane-bound enzyme revealed that selectivity for arachidonoyl-diacylglycerol was unique to the mammalian enzyme. All three kinases were activated by phosphatidylserine, but activation did not alter the arachidonoyl selectivity of M-DG kinase. Phosphatidylserine activated M-DG kinase by increasing Vm and decreasing the apparent Km for diacylglycerol. High concentrations of diacylglycerol reduced the Ka for phosphatidylserine, but did not abolish the phosphatidylserine requirement for maximum activity. Examination of the thermal lability of M-DG kinase revealed that this enzyme was rapidly and selectively inactivated by preincubation with its preferred substrate. This novel effect may have obscured previous attempts to discern substrate selectivity. Taken together, the results provide evidence that M-DG kinase is an arachidonoyl-diacylglycerol kinase that may participate in the formation of arachidonoyl-enriched species of phosphatidylinositol.


Assuntos
Diglicerídeos/metabolismo , Escherichia coli/enzimologia , Glicerídeos/metabolismo , Fosfotransferases/metabolismo , Animais , Linhagem Celular , Citosol/enzimologia , Diacilglicerol Quinase , Ativação Enzimática , Temperatura Alta , Membranas/enzimologia , Camundongos , Fosfatidilserinas/metabolismo , Fosfolipídeos/metabolismo , Fosforilação
16.
J Youth Adolesc ; 17(4): 349-69, 1988 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24277652

RESUMO

Deviation from age-appropriate identity-developmental stage and problem drinking in 75 undergraduate women was investigated. A quantity/frequency scale assessed problem drinking. Two measures of identity, one projective and one observational, were used. May's Deprivation/Enhancement fantasy pattern measure assessed sexual identity. The College Women's Assertion Sample assessed the cognitive-style component of identity. The results supported the hypotheses that younger (freshman/sophomore) women deviating from their age-appropriate identity stage of dedifferentiation, and older (junior/senior) women deviating from their age-appropriate stage of identity integration, were significantly more likely to experience drinking problems than were women who had entered their age-appropriate identity stages. The findings support psychodynamic theories of identity development in late adolescence, and suggest that problem-drinking women in different age/ developmental stages of identity drink for different reasons and should be treated differently.

17.
J Biol Chem ; 262(3): 1105-10, 1987 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-3027078

RESUMO

The regulation of phosphoinositide phosphorylation was studied in Swiss 3T3 cells that were stimulated by platelet-derived growth factor (PDGF). Studies with intact cells showed that the mitogen increased the incorporation of 32P into phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns-P), and phosphatidylinositol 4,5-bisphosphate (PtdIns-P2) during the cell cycle, with distinct peaks of incorporation for all three phosphoinositides after 1 h, and for PtdIns and PtdIns-P2 after 20 h. Direct measurements of the activities of PtdIns kinase and PtdIns-P kinase in freeze-thawed cells revealed that the activity of PtdIns kinase was rate-limiting for the synthesis of PtdIns-P2. Maximal activities of PtdIns kinase and PtdIns-P kinase, with exogenous substrates, were unchanged during the 1st h of PDGF treatment, but doubled during the next 24 h. The increase in PtdIns kinase activity began within 2-4 h, exceeded the increase in cell protein, and was abolished by cycloheximide, which suggests that the enzyme was induced specifically in response to PDGF. The increase in activity of PtdIns-P kinase paralleled the increase in cell protein. Dose-response curves for PDGF showed that the activities of PtdIns kinase and PtdIns-P kinase at 24 h increased in proportion to the extent of mitogenic stimulation of the cells. Our results support the conclusion that the activities of PtdIns kinase and PtdIns-P kinase increase in response to PDGF, but only after several hours of cell cycle traverse.


Assuntos
Fosfatos de Fosfatidilinositol , Fosfatidilinositóis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Fator de Crescimento Derivado de Plaquetas/farmacologia , 1-Fosfatidilinositol 4-Quinase , Animais , Ciclo Celular , Linhagem Celular , Cinética , Camundongos , Ácidos Fosfatídicos/metabolismo , Fosfatidilinositol 4,5-Difosfato , Fosforilação , Fosfotransferases/metabolismo
18.
J Appl Behav Anal ; 19(4): 337-48, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-3804867

RESUMO

We evaluated the effectiveness of a comprehensive training program for enhancing the conversational skills of socially isolated, impaired elderly nursing home residents. A multiple baseline design across behaviors was used to train four subjects (aged 87, 85, 68, and 66), on four content-related conversational components: expressing common courtesies, making positive self-disclosures, asking questions, and making interjections and acknowledgements. Training procedures included instructions, modeling, behavior rehearsal, feedback, and reinforcement. Results showed positive effects with all four subjects; in two cases, changes were significant enough to affect untrained observers' perceptions of the elders' conversational skills.


Assuntos
Terapia Comportamental/métodos , Relações Interpessoais , Casas de Saúde , Idoso , Feminino , Humanos , Comportamento Imitativo , Masculino , Desempenho de Papéis , Isolamento Social , Telefone
19.
Physiol Behav ; 35(3): 371-5, 1985 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-4070410

RESUMO

Young, specific-pathogen-free cats were fed purified diets containing different sources of fat. Food intake was depressed and cats lost weight when the diet contained either hydrogenated coconut oil (HCO) or medium-chain triglycerides (MCT). With an MCT preparation enriched in 8:0 (MCT8), cats would not eat after first tasting the diet. When cats were offered a choice of two high-fat diets, they chose the basal diet over a diet containing 30% HCO, by a ratio of 4.5:1. Low levels of MCT8 (5% or 10% by weight) were also rejected, whereas cats did not reject 5% or 15% MCT12. Caprylic acid, at 0.1-1.0% of the diet, was rejected. In other studies, food intake and body weight decreased when HCO was added to a fat-free diet. Cats fed 25% or 35% HCO lost weight. When 5% safflower seed oil was added to the HCO diets, body weights and food intake improved, but were still less than optimal. These studies indicate that the food intake depression in cats fed dietary HCO and MCT is primarily a result of impalatability, and that the fatty acid moiety may be responsible for the aversion.


Assuntos
Caprilatos , Gorduras na Dieta , Preferências Alimentares , Óleos Vegetais , Triglicerídeos , Animais , Peso Corporal , Gatos , Óleo de Coco , Hidrogenação , Especificidade da Espécie , Paladar
20.
Proc Natl Acad Sci U S A ; 82(12): 3993-7, 1985 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-2987958

RESUMO

Two transformed rodent cell lines (RS-1 and LSTRA) were studied in vitro to determine if their major protein tyrosine kinases catalyzed the phosphorylation of phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), or diacylglycerol. RS-1 cells, transformed by Rous sarcoma virus, contain high levels of pp60src; LSTRA cells, transformed by Moloney murine leukemia virus, contain a tyrosine kinase (pp56) that is the product of an unknown cellular gene. Rates of phosphorylation of peptide tyrosine were elevated more than 20-fold in RS-1 and LSTRA particulate fractions compared to fractions from suitable control cells (N2 and YAC-1), but there was not a proportional increase in rates of phosphorylation of PtdIns, PtdIns4P, or diacylglycerol. Heat (34 degrees C) completely inactivated the LSTRA tyrosine kinase, while it enhanced the phosphorylation of PtdIns and PtdIns4P and had no effect on the phosphorylation of diacylglycerol. PtdIns4P inhibited the phosphorylation of PtdIns but had no effect on tyrosine kinase activity. An antibody, raised against a peptide with a sequence homologous to the autophosphorylation site of pp60src, immunoprecipitated tyrosine kinase activity from RS-1 and LSTRA extracts but had no effect on PtdIns kinase or PtdIns4P kinase activity. These results provide evidence that the phosphorylations of tyrosine and PtdIns are catalyzed by different proteins. An additional observation was that a monoclonal antibody that binds to pp60src and pp56 removed PtdIns kinase as well as tyrosine kinase activity from RS-1 and LSTRA particulate extracts. This antibody also removed PtdIns kinase from N2 and YAC-1 extracts, in which tyrosine kinase activity was low or undetectable. Thus, the anti-pp60src monoclonal antibody may recognize the PtdIns kinase in addition to pp60src and pp56.


Assuntos
Transformação Celular Neoplásica/metabolismo , Fosfatidilinositóis/biossíntese , Fosfatidilinositóis/metabolismo , Fosfotransferases/metabolismo , Proteínas Quinases/metabolismo , 1-Fosfatidilinositol 4-Quinase , Animais , Anticorpos Monoclonais , Membrana Celular/enzimologia , Galinhas , Diglicerídeos/metabolismo , Temperatura Alta , Fosfatidilinositóis/farmacologia , Fosforilação , Fosfotransferases/antagonistas & inibidores , Proteínas Tirosina Quinases , Especificidade por Substrato
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