RESUMEN
We examined the potential contribution of ventromedial (VM) tissue sparing to respiratory recovery following chronic (1 mo) unilateral C2 spinal cord injury (SCI) in rats. Preserved white matter ipsilateral to the injury was quantitatively expressed relative to contralateral white matter. The ipsilateral-to-contralateral white matter ratio was 0 after complete C2 hemisection (C2HS) and 0.23+/-0.04 with minimal VM sparing. Inspiratory (breath min(-1)) and phrenic frequency (burst min(-1)), measured by plethysmography (conscious rats) and phrenic neurograms (anesthetized rats) respectively, were both lower with minimal VM sparing (p<0.05 vs. C2HS). Tidal volume also was greater in minimal VM sparing rats during a hypercapnic challenge (p<0.05 vs. C2HS). In other C2 hemilesioned rats with more extensive VM matter sparing (ipsilateral-to-contralateral white matter ratio=0.55+/-0.05), respiratory deficits were indicated at 1 mo post-injury by reduced ventilation during hypercapnic challenge (p<0.05 vs. uninjured). Anterograde (ventral respiratory column-to-spinal cord) neuroanatomical tracing studies showed that descending respiratory projections from the brainstem are present in VM tissue. We conclude that even relatively minimal sparing of VM tissue after C2 hemilesion can alter respiratory outcomes. In addition, respiratory deficits can emerge in the adult rat after high cervical SCI even when relatively extensive VM sparing occurs.
Asunto(s)
Neuronas Motoras/fisiología , Recuperación de la Función/fisiología , Mecánica Respiratoria/fisiología , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Vértebra Cervical Axis , Masculino , Vías Nerviosas , Nervio Frénico/citología , Nervio Frénico/fisiología , Pletismografía , Ratas , Ratas Sprague-Dawley , Índice de Severidad de la EnfermedadRESUMEN
Ovarian cancer is a disease that will affect approximately 1% of American women during their lifetime, and contributes to more than 14,000 deaths annually. If not detected early, this disease has a 5-year survival rate of less than 20%. Ovarian cancer develops predominantly from the malignant transformation of a single cell type, the surface epithelium. Although the biological mechanisms of transformation remain unclear, it is probably a multistep process requiring an accumulation of genetic lesions in a number of different gene classes. Several proto-oncogenes, such as AKT2 and Ki-RAS, are activated during ovarian cancer development, with putative oncogene-containing chromosomal regions showing imbalances and DNA amplifications. A number of chromosomal regions are also lost in ovarian tumors, indicating that the inactivation of tumor suppressor genes, such as TP53, may also contribute to cancer development. An important recent advancement in the field of ovarian cancer research is the identification of the breast/ovarian cancer susceptibility genes, BRCA1 and BRCA2. Mutations in these two tumor suppressor genes are responsible for the majority of heritable forms of epithelial ovarian cancers. A second class of genes involved in DNA mismatch repair (MMR) are responsible for most cases of hereditary nonpolyposis colorectal cancer (HNPCC). HNPCC or Lynch II cancer syndrome patients are also at an increased risk for developing ovarian cancer. Individuals in cancer-prone kindreds are currently being screened for germline mutations in BRCA1, BRCA2, and several MMR genes (eg, MSH2, MLH1), and mutant allele carriers counseled for cancer risks. Issues related to counseling and management of women at high risk for developing ovarian cancer are discussed. Although BRCA1, BRCA2, and a number of MMR genes have been identified, many more genes involved in gynecologic malignancies remain to be discovered and the clinical significance of the cancer genes already known is still in its infancy.
Asunto(s)
Proteína BRCA1/genética , Proteínas de Neoplasias/genética , Neoplasias Ováricas/genética , Proteínas Proto-Oncogénicas , Factores de Transcripción/genética , Proteína BRCA2 , Aberraciones Cromosómicas/genética , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Femenino , Genes ras , Pruebas Genéticas , Humanos , Proteínas Oncogénicas/genética , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Neoplasias Ováricas/prevención & control , Linaje , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-aktRESUMEN
Investigators have shown that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can inhibit certain estrogenic events in vivo and in vitro. To further investigate this phenomenon, the effects of estradiol (E2) alone or TCDD plus estradiol on several estrogen-dependent parameters were evaluated in weanling female Sprague-Dawley rats. E2 (10 micrograms/kg/day, Postnatal Days (PND) 21 and 22) caused significant increases in relative uterine weight and keratinization of the vaginal epithelium (PND 23). E2 significantly reduced uterine estrogen receptor (ER) protein levels and serum FSH levels, with a trend toward reduction of ER mRNA levels. None of these parameters were affected by pretreatment with 20, 40, or 80 micrograms/kg TCDD (PND 19). Uterine progesterone receptor levels were not affected by E2 or TCDD in the present study. In contrast, TCDD significantly decreased body weight (40 or 80 micrograms/kg) by PND 21, significantly decreased relative thymic weights, and significantly increased relative hepatic weights (20, 40, and 80 micrograms/kg, by PND 23). In addition, TCDD dramatically induced CYPIA1 hepatic mRNA levels, indicating that TCDD was properly delivered and could mediate other well-documented Ah receptor-dependent events. Thus, weanling female Sprague-Dawley rats are not sensitive to the antiestrogenic effects of TCDD at doses which cause overt toxicity. The results provide evidence that the previously reported antiestrogenic effects of TCDD are probably species, strain, and age dependent.
Asunto(s)
Estradiol/farmacología , Dibenzodioxinas Policloradas/toxicidad , Receptores de Estrógenos/efectos de los fármacos , Útero/efectos de los fármacos , Vagina/efectos de los fármacos , Animales , Sitios de Unión , Citocromo P-450 CYP1A1 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Epitelio/efectos de los fármacos , Epitelio/patología , Femenino , Hormona Folículo Estimulante/sangre , Hígado/metabolismo , Tamaño de los Órganos/efectos de los fármacos , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Dibenzodioxinas Policloradas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Útero/metabolismo , Vagina/patología , DesteteRESUMEN
Trichloroethene was degraded in expanded-bed bioreactors operated with mixed-culture methanotrophic attached films. Biomass concentrations of 8 to 75 g volatile solids (VS) per liter static bed (L(sb)) were observed. Batch TCE degradation rates at 35 degrees C followed the Michaelis-Menten model, and a maximum TCE degradation rate (q(max)) of 10.6 mg TCE/gVS . day and a half velocity coefficient (K(S)) of 2.8 mg TCE/L were predicted. Continuous-flow kinetics also followed the Michaelis-Menten model, but other parameters may be limiting, such as dissolved copper and dissolved methane-q(max) and K(S) were 2.9 mg TCE/gVS . day and 1.5 mg TCE/L, respectively, at low copper concentrations (0.003 to 0.006 mg Cu/L). The maximum rates decreased substantially with small increases in dissolved copper. Methane consumption during continuous-flow operation varied from 23 to 1200 g CH(4)/g TCE degraded. Increasing the influent dissolved methane concentration from 0.01 mg/L to 5.4 mg/L reduced the TCE degradation rate by nearly an order of magnitude at 21 degrees C. Exposure of biofilms to 1.4 mg/L tetrachloroethene (PCE) at 35 degrees C resulted in the loss of methane utilization ability. Tests with methanotrophs grown on granular activated carbon indicated that lower effluent TCE concentrations could be obtained. The low efficiencies of TCE removal and low degradation rates obtained at 35 degrees C suggest that additional improvements will be necessary to make methanotrophic TCE treatment attractive.
RESUMEN
TCDD decreases ER levels in vitro and in vivo, possibly through down-regulation of the ER structural gene, although the actual mechanism is unknown. We report the identification of six partial and two full length DREs in the 5' flanking and coding regions of the human ER gene. To test the ability of the coding region DRE (+102) to bind the activated AhR, two complementary oligonucleotides corresponding to the DRE-containing region of the human ER gene were synthesized, termed hERO, and used in a gel shift assay. TCDD-activated nuclear extracts from both mouse Hepa 1c1c7 cells and human MCF-7 cells specifically bound to the hERO with relative binding affinities of 15.5 nM and 5.6 nM, respectively. This binding was dependent upon a functional DRE. The hERO also specifically competed for AhR binding with a DRE-containing oligonucleotide from the mouse CYP1A1 gene. The results suggest a possible mechanism by which TCDD could be acting to modulate ER levels.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Genes , Dibenzodioxinas Policloradas/farmacología , Receptores de Droga/metabolismo , Receptores de Estrógenos/efectos de los fármacos , Receptores de Estrógenos/genética , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Núcleo Celular/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Humanos , Cinética , Neoplasias Hepáticas Experimentales , Ratones , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Receptores de Hidrocarburo de Aril , Receptores de Estrógenos/metabolismo , Células Tumorales CultivadasRESUMEN
The effects of raising cyclic AMP levels, by forskolin stimulation, beta-adrenoceptor activation or cyclic AMP phosphodiesterase inhibition, on inositol phospholipid hydrolysis and increases in intracellular free [Ca2+] ([Ca2+]i) elicited by a range of agonists have been investigated in the hamster vas deferens smooth-muscle cell line DDT1MF-2. Isoprenaline (log [EC50 (M)] = -7.7 +/- 0.2), forskolin and the type IV cyclic AMP phosphodiesterase inhibitor rolipram elicited significant increases in the accumulation of cyclic [3H]AMP. Pretreatment with forskolin (10 microM) attenuated histamine (100 microM)- and N6-cyclopentyladenosine (CPA; 300 nM)-induced release of intracellular Ca2+, observed when cells are stimulated in Ca(2+)-free buffer containing 0.1 mM EGTA. Forskolin had no effect on ATP (100 microM)- or bradykinin (1 microM)-stimulated release of intracellular Ca2+. Histamine-induced intracellular Ca2+ release was also inhibited by pretreatment with rolipram (100 microM) or the membrane-permeant cyclic AMP analogue (Sp)-adenosine 3',5'-monophosphothioate (100 microM). Isoprenaline (1 microM) pretreatment (in the presence of 10 microM rolipram, a concentration which on its own did not decrease the histamine response) attenuated histamine-induced intracellular Ca2+ release. Forskolin inhibited histamine (100 microM)- and CPA (100 nM) stimulated accumulation of [3H]-inositol phosphates, but was without effect on ATP or bradykinin responses. Addition of forskolin (in the presence of 100 microM rolipram) after the cells had been stimulated with histamine (in experiments initiated in Ca(2+)-free buffer) inhibited the rise in [Ca2+]i observed when extracellular Ca2+ (2 mM) was re-applied (owing to receptor-mediated Ca2+ influx). Finally, the refilling of intracellular Ca2+ stores (after receptor-mediated Ca2+ influx is blocked by mepyramine) can be demonstrated in the presence of raised cyclic AMP levels.
Asunto(s)
Calcio/metabolismo , AMP Cíclico/fisiología , Músculo Liso/metabolismo , Fosfatidilinositoles/metabolismo , Receptores Histamínicos H1/metabolismo , Animales , Línea Celular , Colforsina/farmacología , Cricetinae , AMP Cíclico/metabolismo , Hidrólisis , Fosfatos de Inositol/metabolismo , Isoproterenol/farmacología , Masculino , Inhibidores de Fosfodiesterasa/farmacología , Pirrolidinonas/farmacología , Receptores Adrenérgicos beta/efectos de los fármacos , Receptores Adrenérgicos beta/metabolismo , Receptores Histamínicos H1/efectos de los fármacos , RolipramRESUMEN
This study examined the advantages and limitations of upright bicycle exercise echocardiography in the evaluation of a large series of patients with known or suspected coronary artery disease. The study population consisted of 309 patients (231 men, mean age 57 +/- 11 years) who underwent exercise echocardiography within 8.5 +/- 16.1 days of coronary angiography. All stress electrocardiographic, echocardiographic, and angiographic data were reinterpreted in a blinded manner by the investigators. No patient was excluded because of poor echocardiographic image quality. Wall motion was analyzed at baseline, peak exercise, and immediately after exercise with a 16-segment model, and a regional wall motion score index was calculated at each stage. Abnormalities were ascribed to the distribution of the three coronary arteries and correlated with qualitative angiography. There were 126 patients with wall motion abnormalities at rest and 211 (75%) with coronary artery disease. The stress electrocardiogram (ECG) was negative in 61, positive in 144, and nondiagnostic in 104, yielding a sensitivity of 40% and a specificity of 89%. Echocardiography was normal in 76 of 98 patients without coronary disease (78% specificity) and abnormal in 193 of 211 patients with disease (91% sensitivity). Sensitivity was higher among patients with multivessel disease compared with those with single vessel disease (95% versus 86%, respectively, p = 0.03). Among patients with normal wall motion at rest (n = 183), sensitivity was 83% and specificity was 84%. Of the 104 patients with a nondiagnostic stress ECG, echocardiography correctly identified 95% of those with coronary disease and 75% of those without disease. Among 82 patients with a wall motion abnormality at rest, an additional exercise-induced wall motion abnormality developed in 32 of 46 patients (70%) with multivessel disease and seven of 32 (22%) with single-vessel disease. Overall, echocardiography detected 258 of 392 (66%) individual coronary lesions. Accuracy was higher for lesions in the left anterior descending and right coronary arteries (both 79%) compared with the left circumflex artery (36%, p < 0.001). In conclusion, upright bicycle exercise echocardiography is an accurate technique for the evaluation of patients with known or suspected coronary artery disease and is especially valuable in patients with a nondiagnostic stress ECG. The test provides supplemental information on the extent and location of coronary lesions and is useful in patients with and without prior myocardial infarction.
Asunto(s)
Enfermedad Coronaria/diagnóstico por imagen , Ecocardiografía , Prueba de Esfuerzo , Angiografía Coronaria , Enfermedad Coronaria/diagnóstico , Electrocardiografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y EspecificidadRESUMEN
1. The effect of histamine H1-receptor stimulation on inositol phospholipid hydrolysis has been investigated in the hamster vas deferens smooth muscle cell line, DDT1MF-2. 2. Histamine (EC50 = 27 microM) stimulated the accumulation of [3H]-inositol phosphates in DDT1MF-2 cells prelabelled with [3H]-myo-inositol. 2-Thiazolylethylamine (EC50 42 microM) produced a maximal response of similar magnitude to histamine while the maximal response obtained with N alpha-methylhistamine (EC50 = 72 microM) and 2-pyridylethylamine (EC50 = 85 microM) were much lower (circa 65%, histamine = 100%). 3. The H1-selective agonists 2-(3-fluorophenyl)-histamine (2-FPH) and 2-(3-chlorophenyl)-histamine (2-CPH) both appeared to act as partial H1-agonists in this system. Both compounds produced maximal responses of only 30% (with respect to histamine = 100) and were able to antagonize the inositol phosphate response to histamine (estimated Kp = 10.4 and 18.9 microM for 2-FPH and 2-CPH respectively). 4. The response to histamine was antagonized by the H1-antagonists, mepyramine (KD 0.4 nM), (+)-chlorpheniramine (KD 1.2 nM) and promethazine (KD 0.3 nM). Furthermore, the (-)-isomer of chlorpheniramine was approx. three orders of magnitude less potent than the corresponding (+)-isomer. 5. The response to histamine (0.1 mM) was not altered by prior treatment of cells with pertussis toxin (100 ng ml-1; 24 h) whereas the inositol phosphate response to adenosine A1-receptor stimulation in this cell line was significantly attenuated under these conditions. 6. These data indicate that histamine-stimulated inositol phospholipid hydrolysis in DDT1MF-2 cells is mediated via a classical H1-receptor. Furthermore, the results also suggest that histamine HI- and adenosine A,-receptors activate phospholipase C in DDTMF-2 cells via two different G-protein-coupled pathways.
Asunto(s)
Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Histamina/farmacología , Músculo Liso/metabolismo , Fosfatidilinositoles/metabolismo , Receptores Histamínicos H1/fisiología , Animales , Ácido Araquidónico/metabolismo , Calcio/farmacología , Línea Celular , Cricetinae , Histamina/análogos & derivados , Hidrólisis , Masculino , Músculo Liso/citología , Músculo Liso/efectos de los fármacos , Toxina del Pertussis , Receptores Histamínicos H1/efectos de los fármacos , Estereoisomerismo , Conducto Deferente , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Implantation is the process that leads from blastocyst attachment to its embedding in the uterine wall. It is widely believed that failure of implantation is a common cause of pregnancy loss. Toxic agents can interfere directly with the process of implantation and therefore may account for unexplained implantation failures. Our knowledge of human implantation remains limited, mainly due to the lack of adequate experimental models. Studies of mechanisms underlying implantation in humans are by nature and for ethical reasons restricted to in vitro models. The aim of this review is to provide a critical evaluation of various in vitro models of implantation in humans, as well as essential background knowledge required for application of these models to the assessment of peri-implantation toxicity. Particular attention has been devoted to cell-cell and cell-matrix interactions as possible endpoints in the screening of toxic agents.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Implantación del Embrión/efectos de los fármacos , Implantación del Embrión/fisiología , Modelos Biológicos , Toxicología/métodos , Trofoblastos/efectos de los fármacos , Trofoblastos/fisiología , Animales , Células Cultivadas , Femenino , Humanos , Técnicas In Vitro , Trofoblastos/citologíaRESUMEN
1. The effect of adenosine receptor-stimulation on inositol phospholipid hydrolysis and calcium mobilization has been investigated in the hamster vas deferens smooth muscle cell line DDT1 MF-2. 2. Adenosine receptor stimulation increased the accumulation of total [3H]-inositol phosphates in DDT1 MF-2 cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was N6-cyclopentyladenosine greater than 5'-N-ethylcarboxamidoadenosine greater than 2-chloroadenosine greater than adenosine. 3. The response to 2-chloroadenosine was antagonized by the antagonists 8-cyclopentyl-1,3-dipropylxanthine (KD 1.2 nM), PD 115,199 (KD 39 nM) and 8-phenyltheophylline (KD 31 nM). 4. The inositol phosphate response to 2-chloradenosine (10 microM) was not significantly altered when the extracellular Ca2+ ion concentration was reduced from 2.4 mM to 1.2 mM or 0.6 mM. Under calcium-free conditions, however, a reduced but still significant response to 2-chloroadenosine was evident (39 +/- 10% of the response in calcium-containing medium). 5. The 5-lipoxygenase inhibitor AA861 (10 and 100 microM) inhibited the inositol phosphate response to 2-chloroadenosine by 40 +/- 9% and 60 +/- 4% respectively. The cyclo-oxygenase inhibitor, indomethacin, however, was without significant effect at 1 microM. 6. 2-Chloroadenosine stimulated an increase in intracellular free Ca2+ ion concentration in fura-2 loaded DDT1 MF-2 cells in calcium-free medium containing 0.1 mM EGTA, which could be inhibited by the adenosine A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM). 7. These data suggest that adenosine A1-receptor stimulation results in inositol phospholipid hydrolysis and calcium mobilization from intracellular stores in DDT1 MF-2 cells.
Asunto(s)
Calcio/metabolismo , Músculo Liso/metabolismo , Fosfatidilinositoles/metabolismo , Receptores Purinérgicos/metabolismo , 2-Cloroadenosina/farmacología , Adenosina/farmacología , Animales , Benzoquinonas/farmacología , Línea Celular , Cricetinae , Hidrólisis , Indometacina/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Masculino , Músculo Liso/efectos de los fármacosRESUMEN
During embryogenesis, maternal administration of cadmium (Cd) produces teratogenic effects, including hydrocephalus (HC), whereas later in gestation (during the fetal period), such effects have not been reported. Since there is little placental transfer of Cd late in gestation, such differences in response could be due to a lower Cd concentration in the fetus compared with the embryo after maternal Cd exposure, or could be due to a decreased sensitivity of the fetal central nervous system (CNS) to Cd. To test the susceptibility of the late gestational CNS to Cd, day 19 (sperm plug = day 0) rat fetuses were directly injected i.p. with CdCl2 (165, 100, 50 nmoles/fetus in 5 microliters saline). All fetuses in one horn were treated with Cd, while fetuses in the other horn were treated with saline. Fetuses were collected on day 21, grossly examined, weighed, fixed in Bouin's fixative, and later razor sectioned. Cd did not affect fetal viability or body weight. However, Cd caused a dose-dependent increase in hydrocephalus, with the total number of fetuses showing moderate to severe HC being 0/45, 0/11, 6/10, and 18/20 for controls, low, medium, and high doses, respectively. Mild HC was noted in one control and two low Cd fetuses. Brain necrosis was correlated with hydrocephalus, being observed in 0/45, 0/11, 5/10, and 16/20 fetuses, respectively. In medium-dose fetuses without HC or brain necrosis, extravasation of erythrocytes was noted histologically within the cortical parenchyma, suggesting that hemorrhaging may lead to brain necrosis and hydrocephalus in Cd-exposed fetuses. Thus, the fetal CNS is susceptible to the toxic effects of Cd.
Asunto(s)
Intoxicación por Cadmio/complicaciones , Feto/efectos de los fármacos , Hidrocefalia/inducido químicamente , Animales , Cadmio/administración & dosificación , Cloruro de Cadmio , Intoxicación por Cadmio/patología , Relación Dosis-Respuesta a Droga , Femenino , Hidrocefalia/patología , Necrosis , Ratas , Ratas Endogámicas , TeratógenosRESUMEN
Normal human endometrial cells were grown on an extracellular matrix containing type IV collagen, laminin, heparan sulfate proteoglycan, and entactin (Matrigel). On the extracellular matrix, dispersed endometrial cells remained rounded, and aggregated to form mounds of cells, which continued to grow in this arrangement. At 10 d, light microscopy demonstrated that these mounds were comprised of an eosinophilic substance, containing individual fusiform stromal cells. About 50% of the mounds were covered with a single layer of polarized cuboidal to columnar cells with basal nuclei, whereas 60% contained columnar cells forming glandular structures with open lumina. These polarized cuboidal and columnar cells were epithelial, based on their positive staining for cytokeratins and the possession of microvilli, tonofilaments, abundant glycogen, ribosomes, and primitive junctional complexes. Kreyberg's stain showed the presence of mucin within the lumina of the glands, indicating that they were functional. Thus, human endometrial cells grown on an extracellular matrix form a simple cuboidal to columnar epithelium, a stromal component, and glandular structures, thereby mimicking the in vivo morphology of the endometrium.
Asunto(s)
Endometrio/citología , Matriz Extracelular/fisiología , División Celular , Células Cultivadas , Técnicas de Cultivo/métodos , Endometrio/ultraestructura , Células Epiteliales , Femenino , Humanos , Inmunohistoquímica , Queratinas/análisis , Microscopía Electrónica , Microvellosidades/ultraestructuraRESUMEN
Platelet inhibition by agents that increase intracellular levels of cAMP is associated with cAMP-dependent phosphorylation of specific intracellular proteins, including a membrane-associated 22-kDa microsomal protein called thrombolamban. In view of recent studies suggesting that platelets also contain 22-kDa GTP-binding proteins that are homologous with ras-encoded p21 proteins, the present work was undertaken to examine the possibility that thrombolamban and the Ras-like proteins were the same. Platelet microsomes were labeled with [gamma-32P]ATP and the labeled proteins were examined by autoradiography of sodium dodecyl sulfate/polyacrylamide gels. On Western blots of both one-dimensional and two-dimensional gels, thrombolamban immunoreacted with M90, a monoclonal antibody that recognizes the GTP-binding domain of Ras p21 proteins, but not with Y13-259, a monoclonal antibody that recognizes another domain and is specific for Ras proteins. Overlay experiments with unlabeled platelet microsomes demonstrated numerous low molecular weight proteins that bound [alpha-32P]GTP, although none could be identified as thrombolamban. Finally, thrombolamban was immunoprecipitated by M90. These studies show that thrombolamban is a low molecular weight protein that is immunologically related to the Ras family of GTP-binding proteins.
Asunto(s)
Anticuerpos Monoclonales , Proteínas Sanguíneas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Epítopos/análisis , Proteínas de Unión al GTP/metabolismo , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Quinasas/metabolismo , Adenosina Trifosfato/sangre , Plaquetas/metabolismo , Proteínas Sanguíneas/inmunología , Western Blotting , Electroforesis en Gel Bidimensional , Proteínas de Unión al GTP/inmunología , Humanos , Focalización Isoeléctrica , Microsomas/metabolismo , Peso Molecular , Proteína Oncogénica p21(ras)/inmunología , FosforilaciónRESUMEN
Cyclic AMP inhibits platelet activation, at least in part, by reducing intracellular levels of ionic calcium. Previous studies using platelet microsomal fractions have suggested that one mechanism for this effect is stimulation by cyclic AMP and its protein kinase of calcium uptake into microsomal storage sites. In the present study, the effect of cyclic AMP and its protein kinase on calcium uptake by microsomal membranes has been re-examined using the active catalytic subunit of cyclic AMP-dependent protein kinase. The catalytic subunit increased calcium uptake two-fold, but this effect was not inhibited by boiling the catalytic subunit or by recombination with the regulatory subunit of cyclic AMP-dependent protein kinase, conditions that inhibited catalytic subunit activity. Conversely, dialysis of the catalytic subunit preparation against low phosphate buffer, which did not inhibit catalytic subunit activity, inhibited the stimulation of calcium uptake by the catalytic subunit preparation. Finally, the addition of high phosphate buffer, similar in phosphate concentration to that of the catalytic subunit preparation, stimulated calcium uptake. We conclude that the catalytic subunit does not directly stimulate calcium uptake by platelet microsomes.
Asunto(s)
Plaquetas/metabolismo , Calcio/sangre , Microsomas/metabolismo , Proteínas Quinasas/fisiología , Transporte Biológico/fisiología , Proteínas Sanguíneas/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Técnicas In Vitro , FosforilaciónRESUMEN
Two new proteins with apparent molecular masses of 53 kDa and 190 kDa have been identified in both sarcoplasmic reticulum and human blood platelets using a monoclonal antibody, FII1b5. The sarcoplasmic reticulum FII1b5 antigens were present in the terminal cisternae fraction, but were absent from light sarcoplasmic reticulum. The platelet and skeletal muscle proteins were not sensitive to digestion with endoglycosidase H under conditions that removed carbohydrate from the 53 kDa glycoprotein in sarcoplasmic reticulum or GPIIIa in platelet microsomes and did not bind 45Ca in a nitrocellulose overlay calcium-binding assay. These results distinguished the FII1b5 antigens from the 53 kDa glycoprotein and calsequestrin of sarcoplasmic reticulum. The 190 kDa platelet and sarcoplasmic reticulum proteins were extracted from membranes with high concentrations of NaCl, indicating that the high molecular mass FII1b5 antigens are peripherally associated with the bilayers. In contrast, the platelet and muscle 53 kDa proteins remained membrane-bound in the presence of high salt concentrations, suggesting that they are integral proteins.
Asunto(s)
Plaquetas/análisis , Proteínas Sanguíneas/análisis , Calcio/metabolismo , Proteínas Musculares/análisis , Retículo Sarcoplasmático/análisis , Acetilglucosaminidasa/metabolismo , Animales , Anticuerpos Monoclonales , Proteínas Sanguíneas/metabolismo , Humanos , Immunoblotting , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa , Proteínas de la Membrana/análisis , Proteínas de la Membrana/metabolismo , Peso Molecular , Proteínas Musculares/metabolismo , Músculos/análisis , ConejosRESUMEN
JAr choriocarcinoma cells grew as multicellular spheroids, and exhibited a logarithmic pattern of growth, reaching geometric mean diameters of at least 1200 microns after 21 days in culture. HCG, E2 and P4 were secreted into the culture medium throughout the entire culture period, in proportion to spheroid size. This, along with the presence of lipid droplets, non-staining glycogen, Golgi apparatus and microvilli on the spheroid surface and in intercellular spaces indicated that the JAr cells within spheroids were functionally active. While spheroids of less than 800 microns GMD attached to culture dishes and produced cellular outgrowth, larger spheroids showed impaired attachment, and a delay in the subsequent production of outgrowth, which was not correlated with the development of a necrotic core. This outgrowth contained more multinucleated cells and cellular projections than that of smaller spheroids, suggesting that the cells in larger spheroids had undergone early differentiative changes. The spheroid culture system will be applied to the study of processes, such as implantation, which may require a three-dimensional arrangement of placental cells.
Asunto(s)
Coriocarcinoma/ultraestructura , Coriocarcinoma/metabolismo , Gonadotropina Coriónica/biosíntesis , Estradiol/biosíntesis , Humanos , Microscopía Electrónica , Progesterona/biosíntesis , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/fisiología , Células Tumorales Cultivadas/ultraestructuraRESUMEN
Competitive binding and fluorescence energy transfer experiments were used to examine the binding of 2'[3']-O-(2,4,6-trinitrophenyl) adenosine-5'-diphosphate (TNP-ADP) to the catalytic site of Ca ATPase. Fluorescein isothiocyanate (FITC), which is known to covalently bind near the catalytic site (13), was shown to exclude all TNP-ADP binding. TNP-ADP, in turn, will protect against FITC labeling of the Ca ATPase in its native state and in both phosphoenzyme forms. The competitive nature of these probes indicates that TNP-ADP binds to the catalytic site exclusively. Fluorescence energy transfer studies using TNP-ADP as an energy acceptor and 1,N6-ethenoadenosine-5'-diphosphate (epsilon-ADP) as an energy donor were used to estimate the distance between nucleotide binding sites in the enzyme complex. A lower limit for the distance measured was 44 A. This is interpreted to be the distance between catalytic sites on adjacent monomers of a dimer unit. The results of this work are consistent with a single nucleotide site per ATPase monomer.
Asunto(s)
Adenosina Difosfato/análogos & derivados , ATPasas Transportadoras de Calcio/metabolismo , Fluoresceínas , Tiocianatos , Adenosina Difosfato/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Fluoresceína-5-Isotiocianato , Fluoresceínas/metabolismo , Modelos Moleculares , Músculos/enzimología , Conejos , Tiocianatos/metabolismoRESUMEN
We have measured the ability of extracts of tissues from several species of mammals, birds, reptiles, amphibia and fish to demethylate adducts of O6-methylguanine in exogenous DNA by transfer of the methyl group to an acceptor protein. Our study also encompassed tissues from a smaller number of invertebrates, from arthropods, molluscs and annelids. The vertebrate tissues used were liver, brain, spleen and kidney. In the case of the invertebrates we sampled liver, neural tissue, gonads, digestive tract and hepatopancreas. There was no consistent change in the amount of acceptor activity per unit of protein or DNA going from cold-blooded to warm-blooded vertebrates. Liver invariably had the highest amount; this finding was not unexpected since metabolic processes in the liver are high, and good cellular protective mechanism important. Inter-class comparisons within the vertebrates are highly speculative, and hindered by the fact that there is little information on carcinogenesis in animals other than rodents and humans. O6-methylguanine acceptor activity was found in all the invertebrate tissues tested. The amounts were variable, 0.003-0.0051 fmol/micrograms cellular DNA, but the values fell within the range of those found in the tissues of vertebrates.