Lipid analysis and freeze-fracture studies on isolated transverse tubules and sarcoplasmic reticulum subfractions of skeletal muscle.
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Biomedical subjects
Publications and source records attributed to M Garcia.
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The affinity and number of binding sites of [3H]ouabain to isolated transverse (T) tubules were determined in the absence and presence of deoxycholate. In both conditions the KD was approximately 53 nM while deoxycholate increased the number of binding sites from 3.5 to 37 pmol/mg protein. We concluded that the ouabain binding sites were located primarily on the inside of the isolated vesicle and that the vesicles were impermeable to ouabain. ATP induced a highly active Na+ accumulation by the T tubules which increased Na+ in the T tubular lumen by almost 200 nmol/mg protein. The accumulation had an initial fast phase lasting 2-3 min and a subsequent slow phase which continued for at least 40 min. The rate of the initial fast phase indicated a turnover number of 20 Na+/s. The Na+ accumulation was prevented by monensin but was unaffected by valinomycin. Ouabain did not influence Na+ uptake, but digitoxin inhibited it. At low K+ the accumulation of Na+ was reduced 3.7-fold below the value at 50 mM K+. 86Rb, employed as a tracer to detect K+, showed a first phase of K+ release while Na+ was accumulated. After 2-3 min, K+ was reaccumulated while Na+ continued to increase in the lumen. T tubules accumulated Cl- on addition of ATP. This suggested that ATP initiated an exchange of Na+ for K+ followed by uptake of Na+ and K+ accompanied by Cl-.
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After in vivo and in vitro administration of aestradiol, the chromatin of lamb endometrium and of hormone-dependent 7,12-dimethylbenzanthracene Rat mammary tumors is rapidly transformed from a condensed into a diffuse chromatin. A stimulation of 3H leucine incorporation into protein was also observed. These modifications were specific for estrogen receptor ligands and for estrogen responsive tissues. In vitro tamoxifen induced pycnosis. These changes could be used to improve the prediction of hormone dependency of human breast and uterine cancers.
Experiments were carried out to clarify the sites of action of beta-adrenergic agonists in skeletal muscle microsomes. Microsomes were fractionated into longitudinal reticulum, terminal cisternae, and isolated transverse tubules. Transverse tubules were selectively labeled and tracked with [3H]ouabain. beta-adrenergic receptor was identified by [3H]dihydroalprenolol binding. Assays of beta-adrenergic receptor, adenylate cyclase, and protein kinase-stimulated phosphorylation showed: 1) beta-adrenergic receptor was detected in transverse tubules with a receptor density of 0.61 pmol/mg of protein. No significant binding was detected in longitudinal reticulum or in terminal cisternae. 2) Isoproterenol-stimulated adenylate cyclase was present in microsomes but was similarly confined to the transverse tubular fraction. The activity of F- stimulated cyclase in transverse tubules was 2.3 nmol/mg of protein/min. 3) No phosphorylation of microsomes by cyclic AMP and protein kinase could be detected. We conclude that the action of epinephrine on skeletal muscle is mediated through receptors and adenylate cyclase in the external membrane.
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In the immature rat uterus, occupation of the androgen and estrogen receptor sties after injection of 5 alpha dihydrotestosterone (DHT = 17beta - hydroxy-5alpha-androstan-3-one) were compared to the biological responses induced by the androgen on protein synthesis. Injection of 100 mug DHT induced a maximal occupation of androgen receptor sites (RA) but was totally ineffective in translocating the estrogen receptor sites and in increasing general protein synthesis. Conversely, high doses of androgen (greater than or equal to 3 mg) translocated the estrogen receptor (RE) and stimulated general protein synthesis. In addition, these high doses induced a specific uterine protein undistinguishable from that induced by estradiol treatment (IP). These results strongly suggest that the uterotrophic response of the uterus to androgen is correlated with the nuclear transreceptor which is present in much smaller amounts.
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In the immature rat uterus, high concentrations of androgens competed specifically with estradiol on the estrogen receptor (RE). This competition was stereospecific for C19 steroids bearing a 17beta and/or 3 hydroxyl group. Very low affinity ligands, such as testosterone, could not compete with estradiol at equilibrium but decreased the association rate of estradiol on its receptor. High doses (greater than 0.4mg) of 5dihydrotestosterone provoked in vivo as in vitro the nuclear translocation of RE. The nuclear receptor thus formed displayed the same 5.2 S sedimentation constant as that induced by estradiol. We conclude that the weak affinity binding of androgens to the estrogen receptor is sufficient to induce its nuclear translocation in vivo provided androgen concentration is high enough in uterus to occupy the estradiol binding site. Conversely, progesterone which does not bind RE could not provoke its nuclear translocation.
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Six-letter arrays containing symmetrical, e.g., H, M, T, or asymmetrical letters, e.g., J, K, R, were tachistoscopically exposed bilaterally for 100 msec. to 100 college students. Significantly more asymmetrical than symmetrical letters were identified, and significantly more Ss identified more asymmetrical than symmetrical items. This experiment, which incorporated methodological considerations suggested by Harcum (1964) and Bryden (1968), corroborates their findings. Their ideas and other findings were used to account for the data. Asymmetrical letters have more intrinsic left-to-right directionality than symmetrical elements. The rapidly fading after-stimulation of tachistoscopically presented alphabetical material is usually scanned from left to right. Individual array letters might also be scanned in the same direction. Confluence of directionality of letter and scan, which obtains only with asymmetrical letters, might have typically allowed asymmetrical targets to be scanned more rapidly and, consequently, more efficiently than symmetrical displays.
Trimethoprim and sulfamethoxazole were tested alone and in combination against 227 recently isolated Shigella strains. Variations in medium constituents and inoculum size were used to determine the optimal testing conditions. The plate dilution method with addition of 5% lysed horse blood to the susceptibility test medium and an inoculum size of 10(2) organisms was found to provide satisfactory results. All 227 strains were inhibited by low concentrations of trimethoprim, and all were susceptible to the combination of 0.06 mug of trimethoprim per ml and 1.25 mug of sulfamethoxazole per ml. Sixteen percent of these strains were resistant to ampicillin, 33% to tetracycline, 15% to chloramphenicol, and 27% to cephalothin. Based on these in vitro observations, trimethoprim and sulfamethoxazole appear worth evaluating for treatment of shigellosis due to multiply antibiotic-resistant strains.
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