RESUMEN
The methylation state of DNA from human colon tissue displaying neoplastic growth was determined by means of restriction endonuclease analysis. When compared to DNA from adjacent normal tissue, DNA from both benign colon polyps and malignant carcinomas was substantially hypomethylated. With the use of probes for growth hormone, gamma-globin, alpha-chorionic gonadotropin, and gamma-crystallin, methylation changes were detected in all 23 neoplastic growths examined. Benign polyps were hypomethylated to a degree similar to that in malignant tissue. These results indicate that hypomethylation is a consistent biochemical characteristic of human colonic tumors and is an alteration in the DNA that precedes malignancy.
Asunto(s)
Neoplasias del Colon/metabolismo , ADN de Neoplasias/metabolismo , Gonadotropina Coriónica/genética , Neoplasias del Colon/genética , Pólipos del Colon/genética , Pólipos del Colon/metabolismo , Cristalinas/genética , Globinas/genética , Hormonas Glicoproteicas de Subunidad alfa , Hormona del Crecimiento/genética , Humanos , Metilación , Fragmentos de Péptidos/genéticaRESUMEN
Interferons (IFNs) induce early-response genes by stimulating Janus family (Jak) tyrosine kinases, leading to tyrosine phosphorylation of Stat transcription factors. Previous studies implicated protein-tyrosine phosphatase (PTP) activity in the control of IFN-regulated Jak/Stat signaling, but the specific PTPs responsible remained unidentified. We have found that SH2 domain-containing PTP1 (SHPTP1; also called PTP1C, HCP, or SHP) reversibly associates with the IFN-alpha receptor complex upon IFN addition. Compared with macrophages from normal littermate controls, macrophages from motheaten mice, which lack SHPTP1, show dramatically increased Jak1 and Stat1 alpha tyrosine phosphorylation, whereas Tyk2 and Stat2 activation is largely unaffected. These findings correlate with selectively increased complex formation on a gamma response element, but not an IFN-stimulated response element, in motheaten macrophages. Our results establish that SHPTP1 selectively regulates distinct components of Jak/Stat signal transduction pathways in vivo.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Interferón Tipo I/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiología , Transactivadores/metabolismo , Animales , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/aislamiento & purificación , Péptidos y Proteínas de Señalización Intracelular , Janus Quinasa 1 , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Mutantes , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Regiones Promotoras Genéticas , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/aislamiento & purificación , Proteínas Tirosina Quinasas/aislamiento & purificación , Proteínas Tirosina Fosfatasas con Dominio SH2 , Factor de Transcripción STAT2 , Transducción de Señal/efectos de los fármacos , Transactivadores/aislamiento & purificaciónRESUMEN
Previous studies have indicated that the expression of viral oncoproteins, cell transformation, or phorbol ester treatment of cells can inhibit alpha/beta interferon (IFN-alpha/beta)-induced gene expression. The mechanisms by which these promoters of cell growth exert their inhibitory effects vary, but in most instances they involve a disruption of the IFN-alpha/beta-induced transcription complex ISGF3 such that the DNA-binding component of this complex (the 48-kDa ISGF3gamma protein) does not bind to the interferon-stimulated response element (ISRE). In this report, we demonstrated that phorbol ester treatment of human peripheral blood monocytes dramatically inhibits activation of IFN-alpha/B-stimulated early response genes but by a mechanism which does not involve abrogation of the ISRE binding of ISGF3gamma. Phorbol ester treatment of monocytes inhibited IFN alpha-stimulated tyrosine phosphorylation of the transcription factors Stat1alpha, Stat2, and Stat3 and of the tyrosine kinase Tyk2 but had no effect on IFN-gamma activation of Stat1alpha. IFNalpha-stimulated tyrosine phosphorylation of Jak1 and the alpha subunit of the IFN-alpha receptor were unaffected by phorbol 12-myristate 13-acetate (PMA). Moreover, PMA caused the dephosphorylation of Tyk2 but not of Jak1, which was activated by IFN. Pretreatment of cells with vanadate prevented the effects of PMA with regard to PMA-induced Tyk2 dephosphorylation. These observations suggest that PMA exerts its inhibitory effects by activation of a tyrosine phosphatase which selectively regulates Tyk2 but not Jak1 activity.
Asunto(s)
Interferón-alfa/antagonistas & inhibidores , Interferón gamma/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Transducción de Señal/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Secuencia de Bases , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Factor 3 de Genes Estimulados por el Interferón , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón , Interferón-alfa/metabolismo , Interferón gamma/metabolismo , Datos de Secuencia Molecular , Monocitos/metabolismo , Fosforilación , Receptores de Interferón/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Type I interferons (IFNs) bind and signal through cell surface receptors that share at least one common component. One candidate for such a component is the interferon-alpha receptor (IFNAR). Genetic studies have shown that the IFNAR gene product is required for response to many type I interferons. However, these studies also suggest that the IFNAR protein interacts with an additional receptor component(s) to form functionally complete type I IFN receptors. Although these genetic studies have contributed significantly to understanding the type I IFN receptors. Although these genetic studies have contributed significantly to understanding the type I IFN receptors, little biochemical characterization of IFNAR and its function has been reported. To facilitate biochemical studies of the IFNAR gene product, a monoclonal antibody, GB8, recognizing the extracellular domain of IFNAR was prepared. The epitope for GB8 maps to the second extracellular domain of IFNAR between amino acids 278 and 293. GB8 identifies IFNAR in western blots of cell membranes as a broad band with molecular mass ranging from 100 to 150 kD in membranes from CHO cells overexpressing the human IFNAR gene to 136-150 kD in Daudi cell membranes. Such variations in the mean value and the range of molecular mass between IFNAR in different cell lines suggest differences in glycosylation. The majority of glycosylation is N-linked, although there may also be a small amount O-linked oligosaccharide. Deglycosylation of IFNAR in Daudi cell membranes results in a 70 kD IFNAR species, indicating that nearly half of the apparent molecular mass of Daudi cell IFNAR is contributed by carbohydrate moieties.
Asunto(s)
Proteínas de la Membrana/metabolismo , Receptores de Interferón/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Bases , Western Blotting , Conformación de Carbohidratos , Epítopos , Glicosilación , Humanos , Datos de Secuencia Molecular , Peso Molecular , Células Tumorales CultivadasRESUMEN
Small, reversibly biotinylated RNAs as described here are versatile ligands for affinity chromatography of RNA-binding components. These RNAs can be attached to a solid support by binding to avidin and used as ligands, or they may be hybridized to another RNA which acts as the ligand. The incorporation of a disulfide bond in the linker arm connecting biotin to the RNA makes it possible to dissociate the RNA from avidin under mild conditions. Our results regarding the binding and elution of the biotinylated RNA may be applied to other, reversibly biotinylated molecules.
Asunto(s)
Precursores del ARN/aislamiento & purificación , ARN de Hongos/aislamiento & purificación , ARN/aislamiento & purificación , Actinas/genética , Secuencia de Bases , Biotina , Cromatografía de Afinidad/métodos , Exones , Indicadores y Reactivos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Empalme del ARN , ARN Mensajero/genética , Ribonucleoproteínas/aislamiento & purificación , Saccharomyces cerevisiae/genéticaRESUMEN
Recent studies are described concerning the physiological role and tissue distribution of Protein I, a synapse-specific phosphoprotein. This protein is present in both the central and the peripheral nervous system and appears to be enriched in synaptic vesicles. It is a major substrate for both Ca2(-)- and cyclic AMP-dependent phosphorylation in the brain. In slices of the facial motor nucleus from rat brainstem, serotonin (5-HT) added to the incubation medium stimulates the phosphorylation of Protein I. This effect is potentiated by low concentrations of isobutylmethylxanthine and is prevented by the serotonin antagonist mianserin.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Animales , AMP Cíclico/metabolismo , Mianserina/farmacología , Sistema Nervioso/metabolismo , Neurotransmisores/farmacología , Fosforilación , Serotonina/fisiología , Sinapsis/fisiología , Sinapsinas , Membranas Sinápticas/metabolismoRESUMEN
OBJECTIVE: To investigate the rate of seropositivity of anti-JC virus (JCV) antibodies in a German multiple sclerosis (MS) cohort treated with natalizumab in the postmarketing setting and to assess anti-JCV serostatus in samples obtained before diagnosis of progressive multifocal leukoencephalopathy (PML). METHODS: This was a blinded, retrospective cross-sectional and longitudinal analysis for anti-JCV antibodies using a confirmatory 2-step ELISA on 2,782 blood samples obtained from 2,253 patients nationwide for routine testing for anti-natalizumab antibodies during open-label treatment between 2007 and 2010. RESULTS: Of the natalizumab-treated patients with MS, 58.8% tested positive for anti-JCV antibodies. The rate of seropositivity was higher in males and increased with age, with a plateau between age intervals 20-29 and 30-39 years. In longitudinal analyses, 19 of 194 (9.8%) patients converted from anti-JCV antibody-negative to seropositive status over 7.7 months; 4.7% reverted from antibody-positive to seronegative status over 7.9 months. Antibody levels, especially in the latter group, were low, indicating fluctuations around the lower cut point of the assay. Neither anti-JCV serostatus nor antibody levels were associated with immunosuppressive pretreatment, duration of natalizumab treatment, or anti-natalizumab antibodies. All samples obtained from 10 patients who developed PML were seropositive (13 samples before PML diagnosis [2.0-37.6 months]; 2 samples at diagnosis). Antibody levels in these samples were higher than those in samples from seropositive patients who did not develop PML. CONCLUSIONS: These data argue for the potential clinical utility of JCV serology for PML risk stratification. However, further investigations of fluctuations in serostatus and of antibody levels for a more precise understanding of the predictive value are warranted.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Antivirales/sangre , Inmunosupresores/uso terapéutico , Virus JC/inmunología , Leucoencefalopatía Multifocal Progresiva/diagnóstico , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/virología , Adulto , Ensayos Clínicos como Asunto , Estudios de Cohortes , Estudios Transversales , Femenino , Alemania/epidemiología , Humanos , Leucoencefalopatía Multifocal Progresiva/epidemiología , Leucoencefalopatía Multifocal Progresiva/inmunología , Leucoencefalopatía Multifocal Progresiva/virología , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/epidemiología , Natalizumab , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Método Simple CiegoRESUMEN
BACKGROUND: Accelerating the clearance of therapeutic monoclonal antibodies (mAbs) from the body may be useful to address uncommon but serious complications from treatment, such as progressive multifocal leukoencephalopathy (PML). Treatment of PML requires immune reconstitution. Plasma exchange (PLEX) may accelerate mAb clearance, restoring the function of inhibited proteins and increasing the number or function of leukocytes entering the CNS. We evaluated the efficacy of PLEX in accelerating natalizumab (a therapy for multiple sclerosis [MS] and Crohn disease) clearance and alpha4-integrin desaturation. Restoration of leukocyte transmigratory capacity was evaluated using an in vitro blood-brain barrier (ivBBB). METHODS: Twelve patients with MS receiving natalizumab underwent three 1.5-volume PLEX sessions over 5 or 8 days. Natalizumab concentrations and alpha4-integrin saturation were assessed daily throughout PLEX and three times over the subsequent 2 weeks, comparing results with the same patients the previous month. Peripheral blood mononuclear cell (PBMC) migration (induced by the chemokine CCL2) across an ivBBB was assessed in a subset of six patients with and without PLEX. RESULTS: Serum natalizumab concentrations were reduced by a mean of 92% from baseline to 1 week after three PLEX sessions (p < 0.001). Although average alpha4-integrin saturation was not reduced after PLEX, it was reduced to less than 50% when natalizumab concentrations were below 1 mug/mL. PBMC transmigratory capacity increased 2.2-fold after PLEX (p < 0.006). CONCLUSIONS: Plasma exchange (PLEX) accelerated clearance of natalizumab, and at natalizumab concentrations below 1 mug/mL, desaturation of alpha4-integrin was observed. Also, CCL2-induced leukocyte transmigration across an in vitro blood-brain barrier was increased after PLEX. Therefore, PLEX may be effective in restoring immune effector function in natalizumab-treated patients.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Esclerosis Múltiple/tratamiento farmacológico , Intercambio Plasmático/métodos , Adolescente , Adulto , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados , Quimiotaxis de Leucocito/efectos de los fármacos , Quimiotaxis de Leucocito/fisiología , Femenino , Humanos , Inmunosupresores/administración & dosificación , Inmunosupresores/efectos adversos , Inmunosupresores/farmacocinética , Cadenas alfa de Integrinas/efectos de los fármacos , Cadenas alfa de Integrinas/metabolismo , Integrina alfa4/efectos de los fármacos , Integrina alfa4/metabolismo , Leucocitosis/inducido químicamente , Leucocitosis/fisiopatología , Leucocitosis/terapia , Estudios Longitudinales , Masculino , Tasa de Depuración Metabólica/fisiología , Persona de Mediana Edad , Esclerosis Múltiple/inmunología , Natalizumab , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: There is considerable interest in tissue-protective treatments for multiple sclerosis (MS). METHODS AND OBJECTIVES: We convened a group of MS clinical trialists and related researchers to discuss designs for proof of concept studies utilizing currently available data and assessment methods. RESULTS: Our favored design was a randomized, double-blind, parallel-group study of active treatment versus placebo focusing on changes in brain volume from a post-baseline scan (3-6 months after starting treatment) to the final visit 1 year later. Study designs aimed at reducing residual deficits following acute exacerbations are less straightforward, depending greatly on the anticipated rapidity of treatment effect onset. CONCLUSIONS: The next step would be to perform one or more studies of potential tissue-protective agents with these designs in mind, creating the longitudinal data necessary to refine endpoint selection, eligibility criteria, and sample size estimates for future trials.
Asunto(s)
Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/patología , Fármacos Neuroprotectores/uso terapéutico , Humanos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Fumaric acid esters (FAE) have proven their therapeutic efficacy in psoriasis, a Th1 mediated skin disease. More recently, preliminary data have suggested an activity in multiple sclerosis (MS) as well. To investigate further possible mechanisms of action of these compounds in inflammatory diseases, we studied the FAE methyl hydrogen fumarate (MHF) and dimethyl fumarate (DMF) in chronic experimental autoimmune encephalomyelitis (EAE) induced by immunization of C57BL/6 mice with MOG peptide aa 35-55. Preventive treatment with these FAE was delivered twice a day by oral gavage. Both esters had a significant therapeutic effect on the disease course and histology showed a strongly reduced macrophage inflammation in the spinal cord. Multiparameter cytokine analysis from blood detected an increase of IL-10 in the treated animals. We conclude that the underlying biological activity of FAE in EAE is complex and, to elucidate the molecular mechanisms, further investigation is needed.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Fumaratos/uso terapéutico , Inmunosupresores/uso terapéutico , Animales , Biomarcadores/sangre , Recuento de Células , Citocinas/sangre , Dimetilfumarato , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Inflamación , Interleucinas/sangre , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Médula Espinal/inmunología , Médula Espinal/patología , Linfocitos T/inmunologíaRESUMEN
OBJECTIVE: To determine the incidence and clinical significance of neutralizing antibody (NAb) formation in patients with relapsing multiple sclerosis (MS) who participated in the European Interferon Beta-1a IM Dose-Comparison Study. METHODS: Patients were randomized to treatment with interferon beta-1a (IFNbeta-1a) 30 microg or 60 microg IM once weekly for up to 4 years. Serum samples obtained at baseline and every 3 months thereafter were screened for the presence of IFN binding antibodies by ELISA. Patients whose results were seropositive on ELISA were screened for the presence of NAbs using an antiviral cytopathic effect assay. Patients were considered to be positive for NAbs (NAb+) if the baseline NAb titer was 0 and two or more consecutive postbaseline titers were > or = 20. Patients were considered to be negative for NAbs (NAb-) if the baseline NAb titer was 0 and all postbaseline NAb titers were < 5. RESULTS: The proportion of patients who became NAb+ was lower in patients who received 30 microg of IFNbeta-1a than in those who received 60 microg (7/400 [1.8%] vs 19/395 [4.8%]; p = 0.02). The mean time to NAb+ status was 14.5 +/- 6.2 months. Compared with patients who remained NAb-, NAb+ patients showed the following: higher relapse rates from months 12 to 48 (p = 0.04), higher rate of mean change (worsening) in Expanded Disability Status Scale score from baseline to month 48 (p = 0.01), greater number of T1 gadolinium-enhanced lesions at months 24 and 36 (p = 0.02 and 0.03), and greater accrual of new or enlarging T2 lesions from month 12 to months 24 and 36 (p = 0.05 and 0.09). CONCLUSIONS: Neutralizing antibodies (NAbs) to interferon beta-1a (IFNbeta-1a), as observed with other IFNbetas used in the treatment of multiple sclerosis, reduce the therapeutic benefits measured by relapses and MRI activity. Data from this study also suggest NAbs to IFNbeta-1a reduce treatment benefits as measured by change in Expanded Disability Status Scale score.
Asunto(s)
Autoanticuerpos/inmunología , Interferón beta/efectos adversos , Interferón beta/inmunología , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/inmunología , Adulto , Autoanticuerpos/sangre , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Evaluación de la Discapacidad , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interferón beta-1a , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Prevención Secundaria , Resultado del TratamientoRESUMEN
Large changes in the intracellular concentration of sn-glycerol 3-phosphate had no effect on the acyl chain distribution of the phospholipids of Escherichia coli. This result directly contradicts the prediction by other workers based on in vitro experiments.
Asunto(s)
Escherichia coli/análisis , Ácidos Grasos/análisis , Glicerofosfatos/metabolismo , Fosfolípidos/análisis , Escherichia coli/metabolismo , Fosfolípidos/biosíntesisRESUMEN
Previous genetic and biochemical experiments have suggested that the adenylate kinase of Escherichia coli may be directly involved in phospholipid synthesis through formation of a complex with sn-glycerol-3-phosphate acyltransferase, the membrane-bound enzyme that catalyzes the first step in phospholipid synthesis. In this paper we report direct experiments to test this hypothesis. A mutation within the adenylate kinase structural gene is described that results in a temperature-sensitive phospholipid synthesis (assayed in vivo) and a temperature-sensitive acyltransferase. The adenylate kinase activity of this strain is only minimally altered either in vitro or [as assayed by adenosine 5'-triphosphate (ATP) levels] in vivo. This result demonstrates that the inhibition of phospholipid synthesis is not the result of reduced ATP levels. We report the purification of E. coli adenylate kinase to homogeneity; and find that the addition of homogeneous wild-type adenylate kinase to membranes containing a mutationally altered temperature-sensitive acyltransferase results in thermal stabilization of the acyltransferase activity. Ovalbumin has no such protective effect. Purified E. coli inner membranes contain several proteins that are precipitated by addition of anti adenylate kinase antibody to detergent-solubilized membranes.
Asunto(s)
Adenilato Quinasa/metabolismo , Escherichia coli/enzimología , Fosfolípidos/biosíntesis , Fosfotransferasas/metabolismo , Adenosina Trifosfato/metabolismo , Adenilato Quinasa/genética , Adenilato Quinasa/aislamiento & purificación , Membrana Celular/enzimología , Estabilidad de Medicamentos , Escherichia coli/genética , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Calor , Técnicas Inmunológicas , MutaciónRESUMEN
As a component of bacteriophage Qbeta replicase, S1 is required both for initiation of Qbeta minus strand RNA synthesis and for translational repression, which has been traced to the ability of the enzyme to bind to an internal site in the Qbeta RNA molecule. Previously, Senear and Steitz (Senear, A. W., and Steitz, J. A. (1976) J. Biol. Chem. 251, 1902-1912) found that isolated S1 protein binds specifically to an oligonucleotide spanning residues -38 to -63 from the 3' terminus of Qbeta RNA. Here we report that S1 also interacts strongly with a second oligonucleotide in Qbeta RNA, which is derived from the region recognized by replicase just 5' to the Qbeta coat protein cistron. Both sequences exhibit pyrimidine-rich regions.
Asunto(s)
Colifagos/metabolismo , Escherichia coli/metabolismo , Q beta Replicasa/metabolismo , ARN Nucleotidiltransferasas/metabolismo , ARN Viral , Proteínas Ribosómicas/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Oligorribonucleótidos/análisis , Unión Proteica , ARN Viral/metabolismoRESUMEN
We have partially purified the yeast spliceosome by differential sedimentation in glycerol gradients. By electron microscopy we have identified a particle in these fractions that is the spliceosome. In 100 mM KCl buffer, the yeast spliceosome is an ovoid disc with the dimensions of 20 x 23.5 nm with a central indentation. To verify that these ovoid particles were spliceosomes, specific labels were used to tag them. These tagged spliceosomes were then identified in the electron microscope. The salt dependent shift of sedimentation rate for the spliceosome can be explained by a change in size of the particle.
Asunto(s)
Precursores de Ácido Nucleico/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Ribonucleoproteínas/ultraestructura , Proteínas Bacterianas/metabolismo , Biotina/metabolismo , Cationes Monovalentes/farmacología , Fraccionamiento Celular/métodos , Centrifugación por Gradiente de Densidad , Técnicas Inmunológicas , Sustancias Macromoleculares , Microscopía Electrónica , Saccharomyces cerevisiae , EstreptavidinaRESUMEN
Two kinetically distinguishable sn-glycerol 3-phosphate (glycerol-P) acryltransferase activities were detected in Escherichia coli inner membranes using acyl-acyl carrier protein (ACP) substrates. The first system was characterized as having a Michaelis constant (Km) for glycerol-P of 90 microM and utilized palmitoyl-ACP to form primarily 1-acylglycerol-P. Palmitoyl-CoA and cis-vaccenoyl-ACP were also utilized by this system but, with these substrates, significantly more phosphatidic acid was formed as compared to palmitoyl-ACP. Although palmitoyl-ACP and palmitoyl-CoA had kinetically indistinguishable glycerol-P sites, distinct acyl donor binding sites were inferred from kinetic experiments using acyl carrier protein as an acyltransferase inhibitor. A second enzyme system, characterized as having a Km for glycerol-P of 700 microM, was found using palmitoyl-ACP as a substrate. This acyltransferase had a slightly higher pH optimum than the low Km acyltransferase activity, and phosphatidic acid was the major product. Two degradative reactions were identified in this system. One reaction yielded diacylglycerol when palmitoyl-ACP was the substrate. The other degradative reaction produced glycerol. Glycerol was formed in all incubations but was most pronounced when palmitoyl-ACP was the substrate.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Aciltransferasas/metabolismo , Escherichia coli/enzimología , Fosfolípidos/biosíntesis , Ácidos Grasos/metabolismo , Glicerofosfatos/metabolismo , Cinética , Albúmina Sérica Bovina/farmacología , Especificidad por SustratoRESUMEN
We have used the reversible, bifunctional reagent ethylene glycol bis[3-(2-ketobutyraldehyde) ether] to cross-link RNA to protein within intact ribosomal subunits from Escherichia coli. Here we describe the synthesis of this compound (termed bikethoxal) and demonstrate its ability to form covalent attachments between RNA and protein in the 5S RNA-L18 complex and within 30S and 50S ribosomal subunits. The reagent is a symmetrical dicarbonyl compound and reacts with guanine in single-stranded RNA and with arginine in protein. RNA-protein cross-links generated with this reagent are stable, as demonstrated by the comigration of 35S-labeled ribosomal proteins with ribosomal RNA on neutrally buffered sodium dodecyl sulfate (SDS)-agarose gels. However, the cross-linked product is unstable in mildly basic conditions, allowing the identification of the linked macromolecules by conventional techniques. The reagent is potentially capable of cross-linking any combination of single-stranded RNA, single-stranded DNA, or protein; it should prove a useful probe of the RNA-protein proximities within the E. coli ribosome, since the SDS-agarose gel system we describe provides a rapid method of optimizing this RNA--protein cross-linking reaction.
Asunto(s)
Aldehídos/farmacología , Reactivos de Enlaces Cruzados/farmacología , Escherichia coli/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Aldehídos/síntesis química , Concentración de Iones de Hidrógeno , Cinética , Ribosomas/efectos de los fármacos , Ribosomas/ultraestructuraRESUMEN
A phylogenetic survey of proteins immunologically related to Synapsin I, a major synaptic vesicle-associated phosphoprotein in mammals was carried out. Proteins antigenically related to Synapsin I were found by use of radioimmunoassay and other radioimmunochemical techniques in the nervous systems of several vertebrate and invertebrate species, which included birds, reptiles, amphibians, fish, echinoderms, arthropods, and mollusks. Four proteins present in fish brain, antigenically related to Synapsin I, were further studied and found to resemble mammalian Synapsin I in several respects. Like Synapsin I, the fish proteins were present in high amounts in nervous tissue, were enriched in synaptosomal fractions of brain where they were substrates for endogenous protein kinases, were acid extractable, and were sensitive to digestion by collagenase. In addition, two-dimensional peptide-mapping analysis revealed some homology between major phosphopeptide fragments of Synapsin I and the fish proteins. The results indicate that proteins related to Synapsin I are wide-spread in the animal kingdom.
Asunto(s)
Cyprinidae/metabolismo , Carpa Dorada/metabolismo , Proteínas del Tejido Nervioso/análisis , Animales , Anuros , Aplysia , Artrópodos , Química Encefálica , Bovinos , Pollos , AMP Cíclico/farmacología , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Técnicas Inmunológicas , Técnicas de Inmunoadsorción , Colagenasa Microbiana/metabolismo , Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Conejos , Radioinmunoensayo , Ratas , Estrellas de Mar , Sinapsinas , TortugasRESUMEN
A Hartmann-Shack wave-front sensor is used to measure the wave aberrations of the human eye by sensing the wave front emerging from the eye produced by the retinal reflection of a focused light spot on the fovea. Since the test involves the measurements of the local slopes of the wave front, the actual wave front is reconstructed by the use of wave-front estimation with Zernike polynomials. From the estimated Zernike coefficients of the tested wave front the aberrations of the eye are evaluated. It is shown that with this method, using a Hartmann-Shack wave-front sensor, one can obtain a fast, precise, and objective measurement of the aberrations of the eye.
Asunto(s)
Distorsión de la Percepción , Percepción Visual/fisiología , Adaptación Ocular , Fóvea Central/fisiología , Humanos , Procesamiento de Imagen Asistido por Computador , Luz , Matemática , Trastornos de la Percepción/diagnósticoRESUMEN
The ability to isolate DNA from preserved human tissues would provide numerous experimental opportunities. In this report it is shown that DNA can be extracted from tissues prepared for routine histopathological examination (i.e., fixed with formaldehyde and embedded in paraffin). Although the extracted DNA is not intact, it is double stranded, cleavable with restriction endonucleases, and suitable for a variety of standard techniques used in molecular biology.