RESUMEN
The genus Cosmos is native of America and is constituted by 34 species; 28 of them are endemic of Mexico. The cosmos are used as a nematicide, antimalarial, and antioxidative agent. The aim of this study was to estimate the genetic diversity among 7 cosmos species based on random amplified polymorphic DNA (RAPD) and inter-simple sequences repeats (ISSR) markers. With RAPD markers, the obtained polymorphism was 91.7 % and the genetic diversity was 0.33, whereas these values were 65.6%, and 0.22 from ISSR markers, respectively, indicating the presence of high genetic diversity among the Cosmos species that were analyzed. The unweighted pair group method with arithmetic mean dendrograms that were obtained with both markers were notably similar, revealing 2 clusters and indicating a clear genetic differentiation among the Cosmos species that were assessed. The first cluster comprised the species Cosmos sulphureus, Cosmos pacificus, and Cosmos diversifolius, while the second cluster included the species Cosmos purpureus, Cosmos crithmifolius, Cosmos bipinnatus, and Cosmos parviflorus. Besides this, the Cosmos species were clustered according to their collection sites. The Mantel test corroborates the correlation between the genetic distance and the geographic altitude of each Cosmos species. The results suggest that it is necessary to preserve the Cosmos species in their natural habitat in addition to the germoplasm collection for ex situ conservation.
Asunto(s)
Asteraceae/genética , Genes de Plantas , Repeticiones de Microsatélite , Polimorfismo Genético , Análisis por Conglomerados , Marcadores Genéticos , Filogenia , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
An aetiopathogenetic analysis of non-endemic nasopharyngeal carcinoma (NPC) in European and Southern American patient groups was performed. Specifically, the study sought to determine the proportion of Epstein-Barr Virus (EBV)-positive tumour cells in NPC patients in two very different populations (Europe and South America) in areas not associated with a high incidence of NPC. Clinical data (age, sex and onset of clinical disease) were also analyzed. A total of 50 NPC samples, 24 from a European hospital (EH) and 26 from two South American hospitals (SAH), were included. Nuclear staining for Epstein-Barr virus-encoded small RNA (EBER) was performed by in situ hybridization (ISH). Latent membrane protein 1 (LMP1) expression was measured by immunohistochemical (IHC) analysis. A higher incidence of NPC was observed in patients > 40 years of age in EH; in SAH, by contrast, the incidence was higher in patients aged ≤ 40 years. Cervical lymph node metastasis was detected in 31 patients (of whom 84.6% were from SAH). A total of 72% of samples were EBERpositive; the incidence of EBER positivity was greater in type 3 NPCs. EBV was detected in a large proportion of epithelial cells in samples from both EH and SAH (75% vs. 69.2%, respectively). An association was found between EBER detection in lymphocytes and patient origin (p = 0.0001). LMP1 expression was detected in 64% of patients. ISH for the detection of EBER is the most sensitive technique for demonstrating EBV in tumour tissue. The incidence of EBV was not significantly greater in either of the study populations, but was significantly higher in patients with type 3 NPC. Definitive histological diagnosis of NPC was reached earlier in EH than in SAH, where metastases were more frequently diagnosed, suggesting that the disease had reached a more advanced stage by the time treatment was started.
Asunto(s)
Carcinoma/virología , Infecciones por Virus de Epstein-Barr/complicaciones , Neoplasias de Cabeza y Cuello/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma/epidemiología , Niño , Infecciones por Virus de Epstein-Barr/epidemiología , Europa (Continente)/epidemiología , Femenino , Neoplasias de Cabeza y Cuello/epidemiología , Herpesvirus Humano 4/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , América del Sur/epidemiología , Adulto JovenRESUMEN
Tigridia pavonia is the most popular species in the Tigridia genus, and is currently marketed in Europe, Asia, and Australia as a landscape plant. Although it is native to Mexico, there are no breeding programs for it. In this study, we attempted to increase its flower color spectrum and growth habit by interspecific hybridization with T. augusta. Interspecific hybrids between T. pavonia and T. augusta were successfully obtained for the first time using the cut-style pollination and ovary slice culture techniques. On the contrary, no hybrids were obtained from a reciprocal cross. At three, four, and five days after pollination (DAP) ovaries were sliced and cultured on Murashige and Skoog medium without growth regulators and ammonium nitrate, but were supplemented with 6% sucrose, 50 mg/L yeast extract, and 0.25% Gelrite. After 80 days of culture initiation, the germination of only 10 embryos was observed in ovary slices cultured at three DAP. After transfer to identical fresh medium, six hybrid embryos developed into seedlings. All obtained hybrid seedlings were transplanted successfully to soil, and grew normally. The progenies investigated were identified as true hybrids based on randomly amplified polymorphic DNA analysis.
Asunto(s)
Cruzamiento/métodos , Flores/genética , Plantas/genética , Plantones/genética , Cruzamientos Genéticos , Técnicas de Cultivo , Flores/crecimiento & desarrollo , Hibridación Genética , Polinización , Técnica del ADN Polimorfo Amplificado Aleatorio/métodos , Plantones/crecimiento & desarrolloRESUMEN
In this work, the temperature dependence of the sarco-endoplasmic reticulum Ca(2+) -ATPase (SERCA2) activity from rainbow trout Oncorhynchus mykiss cardiac ventricles was measured and compared with the mammalian SERCA2 isoform. The rate of ATP-dependent Ca(2+) transport catalysed by O. mykiss vesicles was totally abolished by thapsigargin and the Ca(2+) ionophore A(23187) . At warm temperatures (25 and 30° C), the SERCA2 from O. mykiss ventricles displayed the same rate of Ca(2+) uptake. At 35° C, the activity of the O. mykiss enzyme decreased after 20 min of reaction time. The rate of Ca(2+) uptake catalysed by the mammalian SERCA2 was temperature dependent exhibiting its maximal activity at 35° C. In contrast to the rate of Ca(2+) uptake, the rate of ATP hydrolysis catalysed by O. mykiss SERCA2 was not significantly different at 25 and 35° C, but the rate of ATP hydrolysis catalysed by the rat Rattus norvegicus SERCA2 isoform at 35° C was two-fold higher than at 25° C. At low temperatures (5 to 20° C), the rate of Ca(2+) uptake from O. mykiss SR was less temperature dependent than the R. norvegicus isoform, being able to sustain a high activity even at 5° C. The mean ±s.e. Q(10) values calculated from 25 to 35° C for ATP hydrolysis were 1·112 ± 0·026 (n = 3) and 2·759 ± 0·240 (n = 5) for O. mykiss and R. norvegicus, respectively. Taken together, the results show that the O. mykiss SERCA2 was not temperature dependent over the 10 to 25° C temperature interval commonly experienced by the animal in vivo. The Q(10) value of SERCA2 was significantly lower in O. mykiss than R. norvegicus which may be key for cardiac function over the wide environmental temperatures experienced in this eurythermal fish.
Asunto(s)
Ventrículos Cardíacos/enzimología , Oncorhynchus mykiss/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Temperatura , Animales , Femenino , Proteínas de Peces/metabolismo , Ratas , Ratas WistarRESUMEN
The 13q33-34 region harbours a susceptibility locus to Ascaris lumbricoides, although the underlying genes are unknown. Immunoglobulin (Ig)E and IgG confer protective immunity and here we sought to investigate in an endemic population whether LIG4, TNFSF13B and IRS2 genes influence IgE and IgG levels against Ascaris and the ABA-1 allergen as a putative resistance marker. Mite-allergic asthmatic patients were analysed for potential relationships between Ascaris predisposition and allergy. One thousand and sixty-four subjects from Cartagena, Colombia, were included. Single nucleotide polymorphisms (SNPs) were genotyped using TaqMan assays. Antibody levels were measured by enzyme-linked immunosorbent assay. Linear and logistic regressions were used to model effects of genotypes on antibody levels. The GG genotype of LIG4 (rs1805388) was associated with higher IgE levels to Ascaris compared with other genotypes. TNFSF13B (rs10508198) was associated positively with IgG levels against Ascaris extract and IgE levels against ABA-1. In asthmatics, IRS2 (rs2289046) was associated with high total IgE levels. Associations held up after correction by population stratification using a set of 52 ancestry markers, age, sex and disease status. There was no association with asthma or mite sensitization. In a tropical population, LIG4 and TNFSF13B polymorphisms are associated with specific IgE and IgG to Ascaris, supporting previous linkage studies implicating the 13q33 region. Our results suggest that genes protecting against parasite infections can be different to those predisposing to asthma and atopy.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Ascariasis/inmunología , Ascaris lumbricoides , Asma/genética , Inmunoglobulina E/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Ascariasis/genética , Ascaris lumbricoides/inmunología , Asma/inmunología , Asma/microbiología , Factor Activador de Células B/genética , Estudios de Casos y Controles , Niño , ADN Ligasa (ATP) , ADN Ligasas/genética , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Proteínas Sustrato del Receptor de Insulina/genética , Modelos Lineales , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Adulto JovenRESUMEN
Here we investigated whether the depletion of CD4+ lymphocytes, observed in mononuclear cells incubated with Taenia solium metacestode E/S products or with living cysts was due to apoptosis. Using the deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), electron microscopy and DNA gel electrophoresis, we found signs of apoptosis in these cells. Results showed that cysteine protease activity was responsible for this effect, since E-64 prevented cell death in all cases. Electron microscopy studies showed that lymphocytes exhibited features of apoptosis such as cellular membrane integrity, strangling and fragmentation of nuclei, chromatin condensation, apoptotic bodies and loss of microvilli. In contrast, lymphocytes co-cultured with living metacestodes plus E-64 exhibited integrity of their structures. DNA fragmentation was detected by TUNEL assays and DNA gel electrophoresis. The results suggested that cell death induced by the cysteine protease from the T. solium metacestode may be involved in down-regulation of cell-mediated responses in infected hosts.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos T CD4-Positivos/parasitología , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Leucina/análogos & derivados , Leucina/farmacología , Taenia solium/enzimología , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/ultraestructura , Humanos , Cinética , Taenia solium/crecimiento & desarrollo , Taenia solium/ultraestructuraRESUMEN
La biopsia de próstata guiada por ecografía transrectal ha revolucionado la detección del cáncer de próstata, sin embargo, existen pocos ensayos a nivel nacional del impacto de este procedimiento en la calidad de vida de los pacientes, más aún, si consideramos que estudios recientes han sugerido incrementar el número de muestras para aumentar la sensibilidad de este examen. El objetivo de este trabajo fue determinar el impacto en la calidad de vida de los pacientes sometidos a biopsia de próstata guiada por ecografía transrectal en 6 y 12 muestras y demostrar la utilidad de la sedación durante el examen. Se randomizaron 60 pacientes, del Servicio de Urología del Hospital San José, en dos grupos: 30 pacientes con sedación (Midazolam 2,5 mg endovenoso) y 30 pacientes con placebo. Estos grupos se subdividieron en grupos de 6 y 12 muestras de tejido prostático. Se constató edad, antígeno prostático, tacto rectal, comorbilidad y complicaciones post biopsia. Todos los pacientes contestaron un cuestionario realizado post examen y un segundo cuestionario 4 semanas después. La calidad de vida fue evaluada utilizando 2 escalas (Short form 36 item health survey). La edad promedio de los pacientes fue de 70 años, con valores de antígeno prostático entre 4,6- 281 ng/dl. Al comparar los grupos sometidos a sedación (6 y 12 muestras) versus los grupos sin sedación sometidos a biopsia transrectal guiada por ecografía, se encuentran diferencias estadísticamente significativas (p<0,0005). Las complicaciones de la sedación en el procedimiento fueron 0 por ciento. Se discuten los resultados.
Asunto(s)
Humanos , Masculino , Próstata , Biopsia , Calidad de Vida , Sedación Consciente/métodos , UltrasonografíaRESUMEN
Vesicles derived from the endoplasmic reticulum of sea cucumber smooth muscle retain a membrane bound Ca(2+)-ATPase that is able to transport Ca(2+) into the vesicles at the expense of ATP hydrolysis. In contrast with vesicles obtained from rabbit muscles, the activity of the Ca(2+)-dependent ATPase from sea cucumber is dependent on monovalent cations (K(+)>Na(+)>Li(+)). With the addition of highly sulfated polysaccharide to vesicle preparations from rabbit muscle, Ca(2+) uptake decreases sharply and becomes highly sensitive to monovalent cations, as observed with vesicles from sea cucumber muscle. These results led us to investigate the possible occurrence of a highly sulfated polysaccharide on vesicles from the endoplasmic reticulum of sea cucumber smooth muscle, acting as an "endogenous" Ca(2+)-ATPase inhibitor. In fact, vesicles derived from the invertebrate, but not from rabbit muscle, contain a highly sulfated polysaccharide. This compound inhibits Ca(2+) uptake in vesicles obtained from rabbit muscle and the inhibition is antagonized by monovalent cation. In addition, sea cucumber muscles contain high concentrations of another polysaccharide, which surrounds the muscle fibers, and was characterized as a fucosylated chondroitin sulfate. Possibly the occurrence of sulfated polysaccharides in the sea cucumber muscles is related with unique properties of the invertebrate body wall, which can rapidly and reversibly alter its mechanical properties, with change in length by more than 200%.
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ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Músculo Liso/química , Polisacáridos/farmacología , Animales , Calcio/metabolismo , Inhibidores Enzimáticos/aislamiento & purificación , Transporte Iónico , Peso Molecular , Músculo Liso/metabolismo , Polisacáridos/química , Polisacáridos/aislamiento & purificación , Potasio/metabolismo , Conejos , Retículo Sarcoplasmático/química , Retículo Sarcoplasmático/metabolismo , Pepinos de Mar , Sodio/metabolismo , Ácidos Sulfúricos/químicaRESUMEN
Although several Ca(2+)-ATPase isoforms have been described in vertebrates, little is known about Ca(2+)-transport in the muscle of invertebrates. In the microsomal fraction obtained from the sea cucumber (Ludwigothurea grisea) longitudinal body wall smooth muscle, we identified a Ca(2+)-transport ATPase that is able to transport Ca(2+) at the expense of ATP hydrolysis. This enzyme has a high affinity for both Ca(2+) and ATP, an optimum pH around 7.0, and - different from the vertebrate sarcoplasmic reticulum Ca(2+)-ATPases isoforms so far described - is activated 3- to 5-fold by K(+) but not by Li(+), at all temperatures, Ca(2+) and ATP concentrations tested. Calcium accumulation by the sea cucumber microsomes is inhibited by Mg/ATP concentrations >1 mM and the accumulated Ca(2+) is released to the medium when the ATP concentration is raised from 0.1 to 4.0 mM.
Asunto(s)
Adenosina Trifosfato/fisiología , ATPasas Transportadoras de Calcio/metabolismo , Músculo Liso/enzimología , Potasio/fisiología , Retículo Sarcoplasmático/enzimología , Animales , Calcio/metabolismo , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Permeabilidad de la Membrana Celular , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Cinética , Magnesio/farmacología , Microsomas/enzimología , Ácido Oxálico/farmacología , Fosfatos/farmacología , Pepinos de Mar , Especificidad por Sustrato , TemperaturaRESUMEN
The patch-clamp technique was used to study the effect of intracellularly added inactivating "ball" peptide (BP) of the Shaker B K+ channel upon Ca(2+)-dependent inwardly rectifying K+ channels of the intermediate conductance type expressed in HeLa cells. Intracellular BP caused only moderate inhibition of outward K+ currents when assayed at an intracellular Ca2+ concentration of 100 nmol/l. Increasing intracellular Ca2+ levels led in itself to some voltage-dependent blockade of K+ currents, which was absent when high extracellular K+ was used. An additional strong blockade by intracellular BP was nevertheless observed both in Na(+)- and K(+)-rich extracellular solutions. A non-inactivating BP analogue had no effect. At this higher intracellular Ca2+ concentration the inhibition of these intermediate conductance Ca(2+)-dependent channels by BP was voltage-dependent, being absent at hyperpolarizing potentials, and could be relieved by increasing extracellular K+. These data suggest that BP acts at an internal pore site in Ca(2+)-dependent intermediate conductance K+ channels of HeLa cells, and that these might possess a receptor site for the peptide similar to that of other K+ channels such as Ca(2+)-activated maxi-K+ channels.
Asunto(s)
Calcio/fisiología , Péptidos/farmacología , Canales de Potasio de Rectificación Interna , Canales de Potasio/efectos de los fármacos , Calcio/metabolismo , Conductividad Eléctrica , Electrofisiología , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Bloqueadores de los Canales de Potasio , Canales de Potasio/fisiologíaRESUMEN
The effects of heparin and dextran sulfate 8,000 on two isoforms of the sarco/endoplasmic reticulum Ca(2+)-ATPase of different animal tissues and on the corn root H(+)-ATPase were examined. In the absence of sulfated polysaccharides the pH profile's of the three transport ATPases were quite different, but after the addition of heparin or dextran sulfate 8,000 the pH profiles of the three enzymes became similar, all showed maximal activity at pH 7.0. Potassium and sodium antagonized the effects of sulfated polysaccharides on the three transport ATPases, but the antagonism was considerably reduced at acidic pH values.
Asunto(s)
ATPasas Transportadoras de Calcio/efectos de los fármacos , Sulfato de Dextran/farmacología , Heparina/farmacología , ATPasas de Translocación de Protón/efectos de los fármacos , Animales , Transporte Biológico , Encéfalo/enzimología , ATPasas Transportadoras de Calcio/metabolismo , Concentración de Iones de Hidrógeno , Fibras Musculares de Contracción Rápida/enzimología , Raíces de Plantas/enzimología , ATPasas de Translocación de Protón/metabolismo , Conejos , Retículo Sarcoplasmático/enzimología , Ésteres del Ácido Sulfúrico/farmacología , Zea mays/enzimologíaRESUMEN
Vesicles derived from maize roots retain a membrane bound H(+)-ATPase that is able to pump H+ at the expense of ATP hydrolysis. In this work it is shown that heparin, fucose-branched chondroitin sulfate and dextran sulfate 8000 promote a shift of the H(+)-ATPase optimum pH from 6.0 to 7.0. This shift is a result of a dual effect of the sulfated polysaccharides, inhibition at pH 6.0 and activation at pH 7.0. At pH 6.0 dextran 8000 promotes an increase of the apparent K(m) for ATP from 0.28 to 0.95 mM and a decrease of the Vmax from 14.5 to 7.1 mumol Pi/mg x 30 min-1. At pH 7.0 dextran 8000 promotes an increase in Vmax from 6.7 to 11.7 mumol Pi/mg x 30 min-1. In the presence of lysophosphatidylcholine the inhibitory effect of the sulfated polysaccharides observed at pH 6.0 was not altered but the activation of pH 7.0 decreased. It was found that in the presence of sulfated polysaccharides the ATPase became highly sensitive to K+ and Na+. Both the inhibition at pH 6.0 and the activation promoted by the polysaccharide were antagonized by monovalent cations (K+ > Na+ > > Li+).
Asunto(s)
Polisacáridos/farmacología , ATPasas de Translocación de Protón/efectos de los fármacos , Membrana Celular/enzimología , Sulfato de Dextran/farmacología , Concentración de Iones de Hidrógeno , Cinética , Lisofosfatidilcolinas/farmacología , Raíces de Plantas/enzimología , Potasio/farmacología , ATPasas de Translocación de Protón/metabolismo , Sodio/farmacología , Espermidina/farmacología , Espermina/farmacología , Sulfatos/farmacología , Temperatura , Zea mays/enzimologíaRESUMEN
A total of 1211 Cuban multibacillary leprosy patients treated for at least 5 years were clinically and bacteriologically examined. They were being treated according to a 2-phase monotherapy regimen with RMP first and DADDS afterwards. On skin-smear examination 50 patients were found positive, of which 9 showed a BI of 3+ or higher at any site. With regard to the clinical status the only cases found with clinical signs of relapse were 5 out of 7 long-standing patients with BI of 4+ and 5+. A 6th patient of this high BI group who showed a good clinical condition, except for a heavy infiltration of both earlobes, was receiving a second RMP course when examined and biopsied for this research. These 9 patients were biopsied and susceptibility tests to RMP and DDS performed. The results showed that in 1 case the Mycobacterium leprae were resistant to both drugs; the organisms from 2 other patients were susceptible to RMP but low-grade resistant to DDS. Those from another patient were susceptible to RMP and fully resistant to DDS. In 3 other cases the bacilli did not multiply in any of the mice but 1 of these strains was from the patient taking a second RMP course, therefore this strain might also be susceptible to RMP and resistant to DDS. In the last 2 cases multiplication was only observed in 2 of the controls and in 1 of the 0.0001% DDS treated mice; therefore, these experiments were not conclusive, and the AFB recovered were inoculated into fresh mice to repeat the tests but these failed to multiply.
Asunto(s)
Dapsona/farmacología , Lepra/microbiología , Mycobacterium leprae/efectos de los fármacos , Rifampin/farmacología , Animales , Farmacorresistencia Microbiana , Femenino , Humanos , Lepra/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
The distribution and down-regulation of the muscarinic acetylcholine receptor (mAChR) were studied in dissociated cells from right (RCC) and left (LCC) cerebral cortex. For this purpose [3H]quinuclidinyl benzilate (QNB) and [3H]pirenzepine (Pz), two muscarinic antagonists, were used. The mAChR binding sites detected with [3H]QNB were asymmetrically distributed between the two hemispheres, the majority being found in the RCC. Asymmetry was also evident in the distribution of the mAChR subtypes (M1 and M2) detected with [3H]Pz. Under basal conditions the RCC had roughly 50% more M1 subtype than the LCC. The pharmacological and kinetic parameters were similar for both antagonists in RCC and LCC, indicating that the observed lateralization was due to a different density of the receptor rather than to different kinetics of binding of the two radioligands. After sustained stimulation with the agonist carbamoylcholine, the receptor sites detected with [3H]Pz, i.e. the M1 subtype of mAChR, decreased at a higher rate in the RCC (44%) than in the LCC (25% of controls), demonstrating that the down-regulation process is more active in the right than in the left cortex, and thus implying that there is better coupling between the stimulated mAChR and its effector system in the former.
Asunto(s)
Corteza Cerebral/metabolismo , Regulación hacia Abajo , Receptores Muscarínicos/metabolismo , Animales , Cinética , Masculino , Pirenzepina/metabolismo , Quinuclidinil Bencilato/metabolismo , Ratas , Ratas WistarRESUMEN
1. Polyphosphoinositide content and phosphorylation of lipids and proteins were analyzed in oocytes of the toad Bufo arenarum Hensel. 2. Plasma membrane-enriched fractions obtained from full-grown, prophase-arrested oocytes incorporated 32P into both phospholipids and proteins after incubation with [gamma-32P]ATP in an Mg(2+)-containing medium. Phosphatidylinositol 4-phosphate (PIP), phosphatidate (PA) and phosphatidylinositol-4,5-bisphosphate (PIP2) were the only labelled lipids. The 32P incorporation depended on incubation time, the amount of protein, and the ATP concentration. 3. Autoradiography of polyacrylamide gel electropherograms and scintillation counting showed that the radioactivity was mainly associated with a group of membrane proteins having an M(r) of 87,000. 4. This paper provides evidence for the capacity of prophase-arrested oocytes from Bufo arenarum to synthesize polyphosphoinositides and to phosphorylate distinct membrane proteins.
Asunto(s)
Proteínas de la Membrana/metabolismo , Oocitos/metabolismo , Fosfatidilinositoles/biosíntesis , Adenosina Trifosfato/metabolismo , Animales , Bufo arenarum , Membrana Celular/metabolismo , Femenino , Técnicas In Vitro , Lípidos de la Membrana/metabolismo , Ácidos Fosfatidicos/metabolismo , Fosfatos de Fosfatidilinositol , FosforilaciónRESUMEN
The widely used alkaline treatment of acetylcholine-receptor (AChR)-rich membranes from Torpedo marmorata (electric fish) and Discopyge tschudii (a marine ray) results not only in the extraction of non-receptor peripheral proteins but also in that of glycerophospholipids (approximately 13%). Minor acidic phospholipids, notably phosphatidic acid and polyphosphoinositides, are particularly enriched in the NaOH extracts. When electrocytes or receptor-rich membranes are incubated with [32P]Pi or [gamma-32P]ATP, polyphosphoinositides accumulate most of the label (approximately 45% in D. tschudii; 96% in T. marmorata) and exhibit the highest specific radioactivity. Furthermore, more than 50% of these phosphorylated lipids are extracted by NaOH together with the peripheral membrane proteins. NaOH treatment also results in modification of the phosphorylation pattern of AChR membrane proteins. Phosphorylation decreases in the Mr-43,000 group of peripheral proteins and in the gamma-subunit of the receptor. The results indicate that polyphosphoinositides constitute a metabolically very active lipid pool in the postsynaptic membrane, and that a substantial proportion of these phospholipids are preferentially released from the membrane together with other acidic phospholipids upon peripheral-protein extraction. The conclusion is drawn that membranes submitted to the above treatments can no longer be considered equivalent to native ones in terms of their phospholipid composition and phosphorylation characteristics.