Comparison of the control and pathways for degradation of the acetylcholine receptor and average protein in cultured muscle cells

In the studies reported here, we investigated whether the degradation of the acetylcholine receptor (AChR) in cultured muscle cells involves similar mechanisms as and is controlled in a manner similar to, the catabolism of the bulk of cell protein. We compared these processes after labeling cell protein with radioactive leucine or phenylalanine for 24 hours, or labeling the acetylcholine receptor with (¹²⁵I)-bungarotoxin. The apparent average half-life of cell protein was 38 ± 2 hours and that of the receptor-toxin complex was 25 ± 1 hours. Incubation in media lacking serum and embryo extract accelerated the degradation of both average protein and the receptor-toxin complex. Insulin reduced the rate of catabolism of both average protein and the receptor-toxin complex toward levels seen in the presence of serum. However, although these two degradative processes seem to be controlled similarly, they probably involve different mechanisms. The protease inhibitors leupeptin and chymostatin, which slowed overall proteolysis in nongrowing muscles and hepatocytes, reduced the degradation of the ACh receptor by 2–11-fold, but had no, or only slight, effects on the catabolism of average protein, even when overall proteolysis was accelerated by omitting serum and embryo extract. Chloroquine, an inhibitor of lysosomal function, also reduced the degradation of AChR, by about 10-fold, but decreased overall protein breakdown by only 20–30%. Incubation of myotubes at lower temperatures reduced both degradative processes, but affected the breakdown of the receptor to a greater extent. Thus the rate-limiting steps in these processes have different activation energies. Incubation with 2-deoxyglucose, an inhibitor of glycolysis, decreased the breakdown of average protein but not that of the receptor-toxin complex. However, the two degradative processes were sensitive to azide, an inhibitor of oxidative phosphorylation. Although the lysosome is the primary site for AChR degradation and perhaps for degradation of other surface proteins, the breakdown of most proteins in myotubes seems to involve a distinct proteolytic system requiring metabolic energy.     

An evaluation of the phylogenetic position of the dinoflagellateCrypthecodinium cohnii based on 5S rRNA characterization

Summary Partial nucleotide sequences for the 5S and 5.8S rRNAs from the dinoflagellateCrypthecodinium cohnii have been determined, using a rapid chemical sequencing method, for the purpose of studying dinoflagellate phylogeny. The 5S RNA sequence shows the most homology (75%) with the 5S sequences of higher animals and the least homology (< 60%) with prokaryotic sequences. In addition, it lacks certain residues which are highly conserved in prokaryotic molecules but are generally missing in eukaryotes. These findings suggest a distant relationship between dinoflagellates and the prokaryotes. Using two different sequence alignments and several different methods for selecting an optimum phylogenetic tree for a collection of 5S sequences including higher plants and animals, fungi, and bacteria in addition to theC. cohnii sequence, the dinoflagellate lineage was joined to the tree at the point of the plant-animal divergence, well above the branching point of the fungi. This result is of interest because it implies that the well-documented absence in dinoflagellates of histones and the typical nucleosomal subunit structure of eukaryotic chromatin is the result of secondary loss. and not anindication of an extremely primitive state, as was previously suggested. Computer simulations of 5S RNA evolution have been carried out in order to demonstrate that the above-mentioned phylogenetic placement is not likely to be the result of random sequence convergence.We have also constructed a phylogeny for 5.8S RNA sequences in which plants, animals, fungi and the dinoflagellates are again represented. While the order of branching on this tree is the same as in the 5S tree for the organisms represented, because it lacks prokaryotes, the 5.8S tree cannot be considered a strong independent confirmation of the 5S result. Moreover, 5.8S RNA appears to have experienced very different rates of evolution in different lineages indicating that it may not be the best indicator of evolutionary relationships.We have also considered the existing biological data regarding dinoflagellate evolution in relation to our molecular phylogenetic evidence.     

The tetracycline resistance transposons Tn1721 and Tn1771 have three 38-base-pair repeats and generate five-base-pair direct repeats

Summary The 10.7 kilobase (kb) tetracycline resistance transposons Tn1721 and Tn1771, isolated from disparate sources, are completely homologous on the basis of heteroduplex analyses. Both transposable elements are capable of forming multiple duplications of a 5.3 kb portion encompassing the resistance genes (tet region). A model accounting for both, recA-independent translocation and recA-dependent amplification, postulates two direct and one inverted repeat as essential constituents of the transposons. DNA sequence analyses of Tn1721 and Tn1771 have substantiated this model. They demonstrated three identical 38 base pair repeats identically in both transposons dividing them into a minor transposon and a tet region. Identical sequences of at least 87 base pairs providing recombination hot spots for gene duplication have been found at the ends of the repetitious tet region. Translocation of Tn1721 and Tn1771 generates five base pair direct repeats at the respective sites of insertion. On the basis of the heteroduplex molecules and sequences analyzed the two transposons are identical.To Professor Wolfram Heumann on the occasion of his 65th birthday     

Temperature dependence of the barley root plasma membrane-bound Ca2+- and Mg2+-dependent ATPase

Arrhenius plots of the maximal velocities for the Ca²⁺-and Mg²⁺-dependent ATPase activities found in a plasma membrane-rich microsome fraction isolated from the roots of barley ( Hordeum vulgare L. cv. Conquest) were nonlinear. Arrhenius plot analyses using a relation which produced curvilinear Arrhenius plots accurately fit the data and allowed the calculation of the activation enthalpies and molar heat capacities of activation. The temperature dependence of the computed Km values for the Ca²⁺- and Mg²⁺-dependent ATPase activities was complex, with the highest enzyme-substrate affinities being obtained near the barley seedling growth temperature (16°C). Using electron paramagnetic resonance spectroscopy with amphiphilic cationic and anionic spin probes, it was possible to demonstrate that temperature changes and increasing Ca²⁺ concentrations could alter the mobility of the membrane lipid polar head groups. Inhibition of the ATPase activities by high levels of Ca²⁺ may result from a Ca2+-induced reduction in the lipid polar head group mobility. The possible role of lipid polar head group-protein interactions in the complex temperature dependence of the barley root ATPase kinetic constants is discussed.     

O(3)-(2-Acetylamino-2-deoxy-β-d-glucopyranosyl)-oleanolic acid,a novel triterpenoid glycoside from two Pithecellobium species

A new triterpenoid glycoside containing an amino sugar moiety has been isolated from Pithecellobium cubense and P. arboreum and identified as O(3)- (2-acetylamino-2-deoxy-β-d-glucopyranosyl)oleanolic acid. β-d-Glucopyranosyl-α-spinasterol was also obtained from P. cubense.     

Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine 3′:5′-cyclic monophosphate

Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [(3)H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.     

Activation of old cuticle chitin as a substrate for chitinase in the molt of Manduca

During the molt, chitin in the old cuticle of $\text{Manduca}$ is digested by chitinase taken up from molting fluid, but the chitin in intact (= premolt) cuticle is not accessible to chitinase. As a prerequisite of digestion, old cuticle chitin is rendered competent to serve as chitinase substrate in a reaction attributable to trypsin-like proteolytic activity of molting fluid.     

TECHNICAL CONSIDERATIONS IN CANNULATING THE ASCENDING AORTA

A successfully used technique of aortic arch cannulation is reported. This cannula is inexpensive and can be made easily in the hospital. The position and advantages of the cannula are discussed.