Anagrelide (BL-4162A, 6,7-dichloro-1,5-dihydroimidazo[2, 1-6] quinazolin-2[3H]one monohydrochloride hydrate) is a potent and broad spectrum inhibitor of platelet aggregation. Prior studies showed that anagrelide inhibited platelet cyclic AMP (cAMP) phosphodiesterase activity but did not appreciably elevate platelet cAMP levels. We examined the effects of anagrelide on washed human platelets and found that anagrelide caused significant elevation of cAMP levels. Anagrelide treatment also resulted in activation of the platelet cAMP-dependent protein kinase at anagrelide concentrations of 0.1 to 1 microgram/ml, which inhibited platelet aggregation but caused only small increases in platelet cAMP content. When whole platelets were incubated with radiolabeled phosphate, anagrelide increased phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. The pattern of protein phosphorylation stimulated by anagrelide treatment was similar to that observed when the platelets were treated with forskolin. Anagrelide also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets. The inhibition of increased intracellular Ca++ resulted from block of thrombin-induced mobilization of intracellular Ca++, as well as prevention of Ca++ influx through the plasma membrane. Anagrelide itself had no influence on inositol 1,4,5-trisphosphate-induced Caz5++ release from isolated platelet membrane vesicles. These studies suggest that anagrelide inhibits platelet phosphodiesterase activity in intact platelets resulting in an elevation in cAMP levels sufficient to activate the cAMP-dependent protein kinase and inhibit agonist-activated Ca++ fluxes.

Blood Platelets↗

PubMed1986

Splicing of messenger RNA precursors.

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Animals↗

PubMed1984

High molecular weight proteins in cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles bind calmodulin, are phosphorylated, and are degraded by Ca2+-activated protease.

A unique set of high molecular weight proteins was identified in junctional sarcoplasmic reticulum (SR) vesicles isolated from both cardiac muscle and skeletal muscle. These high Mr proteins were not present in free SR vesicles isolated from either tissue, nor were they observed in purified sarcolemmal fractions. The junctional SR high Mr proteins migrated as doublets in sodium dodecyl sulfate-polyacrylamide gels and exhibited apparent Mr values between 290,000 and 350,000. The high Mr proteins bound calmodulin; they were the principal proteins labeled in the cardiac and skeletal muscle SR subfractions by azido-125I-calmodulin. The high Mr proteins were also substrates for an endogenous Ca2+-calmodulin-dependent protein kinase activity, as well as exogenously added catalytic subunit of cAMP-dependent protein kinase. In addition, the junctional SR high Mr proteins were the major SR proteins degraded by a Ca2+-activated protease purified from smooth muscle. Control experiments verified the separation of junctional SR vesicles and free SR vesicles from both muscle types. Junctional SR vesicles were enriched in calsequestrin, and they exhibited Ca2+ uptake which was stimulated up to 10-fold by either ryanodine or ruthenium red. Free SR vesicles were deficient in calsequestrin and were insensitive to these two agents. Localization of the cardiac and skeletal muscle high Mr proteins to the junctional SR, coupled with demonstration of their nearly identical biochemical properties, suggests that the proteins are homologous and are likely to have similar functions in both types of striated muscle.

Animals↗

PubMed1984

Alterations in the morphology of rabbit skeletal muscle plasma membrane during membrane isolation.

This study describes changes in morphology of plasmalemma from fast skeletal muscle in the course of tissue disruption and isolation. We find that conditions used to solubilize muscle contractile elements, in the isolation of plasmalemma, including the use of 0.6 M KCl or 0.4 M LiBr in the cold (0-4 degrees C), lead to altered plasmalemma morphology. The intramembrane particles, as revealed by freeze-fracture electron microscopy, become aggregated, leaving large domains devoid of particles. The square arrays in the P face and the complementary "pits" in the E face also become aggregated, sometimes forming sizeable aggregates of square arrays. Thin-section electron microscopy using tannic acid enhancement reveals plasma membrane associated components, on both cytoplasmic and extracellular faces, are largely reduced by the salt treatment. Pyrophosphate and magnesium at lower concentrations, sometimes used instead of high salt, also resulted in particle aggregation, although less pronounced than with concentrated salt solutions. The plasma membrane-associated proteins on both plasma membrane surfaces were likewise decreased by this treatment. Pyrophosphate treatment also separated the basal lamina from the plasma membrane. Incubation of muscle in isoosmotic sucrose does not alter the morphology of the plasmalemma with regard to particle aggregation, diminution of membrane associated components, or separation of the basal lamina. Our observations suggest that membrane-associated protein and/or cytoskeleton constrains the mobility of components in the plane of the membrane and that removal of this constraint leads to aggregation of intramembrane particles.

Animals↗

PubMed1984

Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle.

We have developed a procedure to isolate, from skeletal muscle, enriched terminal cisternae of sarcoplasmic reticulum (SR), which retain morphologically intact junctional "feet" structures similar to those observed in situ. The fraction is largely devoid of transverse tubule, plasma membrane, mitochondria, triads (transverse tubules junctionally associated with terminal cisternae), and longitudinal cisternae, as shown by thin-section electron microscopy of representative samples. The terminal cisternae vesicles have distinctive morphological characteristics that differ from the isolated longitudinal cisternae (light SR) obtained from the same gradient. The terminal cisternae consist of two distinct types of membranes, i.e., the junctional face membrane and the Ca2+ pump protein-containing membrane, whereas the longitudinal cisternae contain only the Ca2+ pump protein-containing membrane. The junctional face membrane of the terminal cisternae contains feet structures that extend approximately 12 nm from the membrane surface and can be clearly visualized in thin section through using tannic acid enhancement, by negative staining and by freeze-fracture electron microscopy. Sections of the terminal cisternae, cut tangential to and intersecting the plane of the junctional face, reveal a checkerboardlike lattice of alternating, square-shaped feet structures and spaces each 20 nm square. Structures characteristic of the Ca2+ pump protein are not observed between the feet at the junctional face membrane, either in thin section or by negative staining, even though the Ca2+ pump protein is observed in the nonjunctional membrane on the remainder of the same vesicle. Likewise, freeze-fracture replicas reveal regions of the P face containing ropelike strands instead of the high density of the 7-8-nm particles referable to the Ca2+ pump protein. The intravesicular content of the terminal cisternae, mostly Ca2+-binding protein (calsequestrin), is organized in the form of strands, sometimes appearing paracrystalline, and attached to the inner face of the membrane in the vicinity of the junctional feet. The terminal cisternae preparation is distinct from previously described heavy SR fractions in that it contains the highest percentage of junctional face membrane with morphologically well-preserved junctional feet structures.

Animals↗

PubMed1983

Estrogen-stimulated uptake of plasminogen by the mouse uterus.

Administration of a single low dose of estradiol to the immature (4- to 5-week-old) female mouse caused a rapid, uterine-specific increase in the uptake of radiolabeled plasminogen from plasma. A significant increase in uptake was detectable within 30 min and reached a maximum 2-4 h after administration of the hormone. After 4 h, a substantial amount (42%) of the newly taken up plasminogen was found in the uterine lumen. Half-maximal stimulation of uptake occurred at a dose of 0.20 microgram estradiol/animal. Estrogen stimulation of uptake was not blocked by puromycin, indicating that new protein synthesis was not required. Similar results were obtained with mouse plasma albumin. Estrogen-stimulated uptake was not blocked by indomethacin (10 micrograms/g BW, iv), but was blocked by prednisolone. Approximately 50% inhibition of the stimulation induced by 0.5 microgram estradiol in these 10-g animals was accomplished with 50 micrograms prednisolone. This study extends our initial findings on the estrogen-stimulated uptake of plasma proteins by the mouse uterus and provides a mechanism by which uterine plasminogen levels can be elevated before implantation.

Adenosine Diphosphate↗

PubMed1982

Isolation of plasma membrane vesicles from rabbit skeletal muscle and their use in ion transport studies.

A method has been developed for the isolation of sealed plasma membrane vesicles from rabbit white skeletal muscle. The final preparation was highly purified as indicated by enrichment of plasma membrane marker enzymes (i.e. ouabain-sensitive (Na+,K+)-ATPase, adenylate cyclase, and acetylcholinesterase). The absence of sarcoplasmic reticulum and mitochondria as contaminants was indicated by the low specific activity of marker enzymes, i.e. Ca2+-ATPase, succinate-cytochrome c reductase, and monoamine oxidase. Thin section and negative staining electron microscopy confirmed the absence of sarcoplasmic reticulum and mitochondrial contamination. The plasma membrane preparation consisted largely of sealed vesicles as observed by electron microscopy and as also demonstrated by latency of enzymic activities, which were unmasked by preincubation with detergent (sodium dodecyl sulfate). Membrane sidedness was estimated from latency of ouabain-sensitive (Na+,K+)-ATPase activity and acetylcholinesterase activity. The latency studies suggest that most of the vesicles are oriented inside out with respect to the orientation of the sarcolemma membrane in the muscle fiber. The inside-out plasma membrane vesicles actively accumulated sodium ions upon addition of ATP. The sodium ions were concentrated greater than 8-fold inside the vesicles and were released upon addition of the ionophore monensin. The sodium ions were taken up in the presence of K+ or NH4+ but not of choline. Uptake was inhibited by low concentrations of vanadate or digitoxin. The Na+ uptake was concomitant with Rb+ efflux. Therefore, the sodium ion transport and the resulting gradients formed appear to have been generated by the ouabain-sensitive (Na+,K+)-ATPase. Batrachotoxin, which opens Na+ channels in excitable tissues, prevents most of the Na+ uptake, suggesting the presence of toxin-activated Na+ channels in these plasma membrane vesicles.

Acetylcholinesterase↗

PubMed1982

Adjuvant chemoimmunotherapy with LMF plus BCG in node-negative and node-positive breast cancer - intermediate report at 4 years.

A randomized surgical adjuvant trial in 242 evaluable patients with T1-3a, N0-1, and M0 breast cancer was initiated 4 years ago. The well-tolerated, oral combination chemotherapy with six cycles of Leukeran plus methotrexate plus fluorouracil (LMF) plus repeated BCG skin scarifications was used. After 4 years, the following results were seen: (1) significant increase of relapse-free (RFS) and also overall survival (S) in both pre- and postmenopausal node-negative patients versus surgical controls (RFS 91.1 vs. 701%, P = 0.003; S 96 vs. 88%, P = 0.03); (2) no significant increase of RFS or S in pre- and postmenopausal node-positive patients versus surgical controls (RFS 50.1 versus 44%, P = 0.49; S 70 versus 68 %, P = 0.9, respectively); (3) Patients receiving greater than 90% of the planned LMF dose showed significantly better survival after 4 years; and (4) Nonrandomized comparison with concurrent Swiss adjuvant studies with LMF alone indicate no beneficial or harmful effect of BCG skin scarifications in addition to the six-cycle LMF.

Breast Neoplasms↗

PubMed1981

Estrogen-stimulated uptake of alpha 1-protease inhibitor and other plasma proteins by the mouse uterus.

Administration of a single low dose of estradiol to the immature female mouse resulted in a rapid increase in uterine trypsin inhibitory capacity. The increase was apparent within 1 h, reached a maximum in 3--4 h, and returned to base line after 18 h. No corresponding increases were observed in liver or heart. The properties of the uterine inhibitor were found to be essentially the same as those of plasma alpha 1-protease inhibitor (alpha 1-PI). When 125I-labeled mouse plasma alpha 1-PI was given iv to immature mice the administration of estradiol caused a specific stimulation of uterine uptake of the labeled protein that closely matched the estrogen-stimulated increase in uterine trypsin inhibitory capacity. Half-maximal stimulation of uptake occurred at a dose of 0.15 microgram estradiol/animal. After 3 h 97% of the labeled alpha 1-PI taken up by the uterus was in the soluble (105,000 x g) fraction of which 50% was in the lumen. Estradiol also stimulated the uptake of soybean trypsin inhibitor, bovine serum albumin, porcine fibrinogen, and human alpha 2-macroglobulin. Under conditions where puromycin blocked the synthesis of an estrogen-stimulated uterine-specific hydrolase, puromycin had no effect on the estrogen-stimulated uptake of either mouse alpha 1-PI or mouse albumin. These results suggest that the estrogen-stimulated uptake of plasma proteins by the uterus does not require new protein synthesis.

Animals↗

PubMed1980

Reconstitution of the Ca2+-transport system of human erythrocytes.

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte 'ghosts' was solubilized and reconstituted to form membranous vesicles capable of energized Ca2+ accumulation. The erythrocyte 'ghosts' for this purpose were prepared by using isoosmotic freeze-haemolysis in the presence of Tween 20 and proteinase inhibitors to stabilize the preparation. The reconstitution procedure is similar to that developed by Meissner & Fleischer [(1974) J. Biol. Chem. 249, 302-309] for skeletal-muscle sarcoplasmic-reticulum in that: (1) deoxycholate is used for the solubilization of the membrane; (2) controlled dialysis at near room temperature, rather than 0 degree C, is required in order to obtain a functional preparation capable of Ca2+ accumulation; and (3) membrane vesicles can be reassembled with protein/lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original membrane.

Biological Transport↗

PubMed1969

[Details in nursing care and their importance to the sick child's recovery].

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Pediatric Nursing↗