STUDIES OF THE TRIAD : II. Penetration of Tracers into the Junctional Gap

Ferritin and Imferon molecules were introduced as tracers inside "skinned" muscle fibers to test which part of the triadic junction gap is freely exchangeable with the sarcoplasm. At least 50% of the T-system surface is freely accessible from the sarcoplasm. Of the remainder, 30% of the total T-system surface is covered by the junctional feet, and 20% in the center of the junction may or may not be accessible. The possibility is discussed that the triadic junction may not function as an electrical coupling.     

The effect of the plating medium on the recovery of nonsense suppressors in Saccharomyces cerevisiae

The recovery of nonsense suppressors in a strain of Saccharomyces cerevisiae carrying five ochre mutations, tr, hi, ly, ar, and ad, is affected by the plating medium. The highest frequency is observed on tryptophanless medium, while the lowest is observed on adenineless medium. Experiments showed that exogenous histidine inhibits suppressor expression and that exogenous adenine relieves this inhibition. In histidine-independent strains, mutation expression requires adenine. A model, based on the role of RNA in supersuppression and on the biosynthetic pathways of histidine and adenine, is proposed to account for the observed data. It cannot, however, account for the high frequency of suppressors on tryptophanless medium. The tentative conclusion is drawn either that mis-reading of the tryptophan nonsense codon by mutated tRNA is facilitated by the neighboring bases or that the type of acceptable amino acid is less rigorously limited in the mutated site of the tryptophan locus than in those of the other suppressible loci.This work was in part supported by the Calouste Gulbenkian Foundation and by the Medical Research Council (Grant No. G969/24/B).     

Antagonism between growth and flocculation inPichia stipitis NRRL Y-7124:Influence of Ca +2 and Mg +2 ions

Summary The effects of Mg⁺² and Ca⁺² ion concentrations onPichia stipitis growth and flocculation were studied by a 2×2 factorial design. Negative interaction was detected between these ions (p<0.01): maximum growth rate was attained with high Mg⁺² (3 mM) and low Ca⁺² (0.34 mM) concentration levels, whereas the inverse ionic ratio was required to reach the maximum flocculation extent. Neither stimulatory or inhibitory effects on growth or flocculation were observed by increasing other cations (Zn⁺², Mn⁺², Co⁺², Fe⁺²) or vitamins concentration. An antagonistic relationship between growth and flocculation was concluded.     

AN ELECTRON MICROSCOPE STUDY OF DENERVATION ATROPHY IN RED AND WHITE SKELETAL MUSCLE FIBERS

A study, mainly by electron microscopy, has been made on two leg muscles of rat, in the course of atrophy experimentally induced by total denervation. As a preliminary the chief distinctive features of the soleus, chosen as a representative of pure red muscle, and of the gastrocnemius, representative of pure white muscle, are described. Two major phases of atrophy, somewhat overlapping in time, were observed. In the first, a degenerative autolytic process takes place in areas of the fiber, with loss of striation. It can be detected as early as the 7th day, but the maximum is observed at the 14th day, and accounts for a gross weight loss of 50 per cent. The first alteration appears in the Z lines; disorder in the disposition of filaments then follows. The process occurs very rapidly, leaving large areas in the cell in which one can detect only ground substance, glycogen, rare randomly disposed vesicular elements, and some mitochondria. Several lysosomes and masses of lipoproteins, which assume the configuration of concentric lamellae, show up in these fibers. Subsequently large parts of the waste sarcoplasm are discarded into the intercellular spaces. In the second major phase the so called "simple" atrophy takes place. The process starts early, but its effects are more detectable after 1 month. In this period, single myofibrils undergo different degrees of reduction in diameter, while the spatial disposition of primary and secondary filaments inside the fibrils remains normal. The appearance of the fibrils in longitudinal sections suggests that the process takes place by the detachment of filaments from the periphery of the fibrils and by their subsequent breakdown in the interfibrillary spaces. The sarcoplasmic reticulum is still well preserved, and relatively overdeveloped. Mitochondria disappear in parallel with the contractile material.     

Fluorescence of eosinophil leucocyte granules induced by 1-hydroxy-3,6,8-pyrenetrisulfonate. Visualization of differences in protein isoelectric points

After treatment of horse, rat and human blood smears with alkaline solutions of 1-hydroxy-3,6,8-pyrenetrisulfonate (HPTS), eosinophil leucocyte granules were the unique cell components which showed a bright green fluorescence. When stained with HPTS at pH 10, the whole granule of horse eosinophils showed high emission which strongly diminished after washing or staining in salt solutions or by using blocking methods for amino groups. Using HPTS at pH 12, the fluorescence reaction of house granules was specifically located in the peripheral region, appearing as fluorescent rings. These microscopic observations, which indicate differences in the isoelectric point of proteins within the eosinophil granule, were also confirmed by HPTS staining of protein blots as model substrates. Spectral analysis of HPTS at pH 10 and 12 showed practically identical absorption and emission spectra with peaks at 450 nm and 510 nm, respectively. Our results indicate that mainly ionic binding occurs between cationic proteins and HPTS in alkaline solution, and that the most cationic proteins (with isoelectric points at pH higher than 12) are located in the peripheral annular region of horse eosinophil granules.     

Structural simplicity of theZonula Occludens in the electrolyte secreting epithelium of the avian salt gland

Summary The structure of thezonula occludens in the secretory epithelium of the salt gland of the domestic duck was determined by thin section and freeze-fracture electron microscopy. These glands secrete an effluent with a NaCl concentration four times that of plasma, and thus maintain a steep ionic gradient across their secretory epithelium. Freezefracture replicas from salt stressed ducks demonstrate that thezonula occludens is surprisingly shallow in depth (20–25 nm) and generally consists of two parallel junctional strands which are juxaposed along their entire length. In addition to the simplicity of the junction separating mucosal and serosal compartments, the ratio of junctional length to apical surface area is large since luminal surfaces of secretory cells are narrow and intermesh with one another. Thezonula occludens in nonsecreting fresh water-adapted birds is similar to the salt stressed group except that two sets of double strand junctions are seen in addition to junctions consisting of a single set. Based on previous ultrastructural, cytochemical and physiological studies in salt glands and in other epithelia, a model for salt secretion was suggested in which intercellular space Na⁺, generated by basolateral ouabain-sensitive Na⁺ pumps, reaches the lumen via a paracellular route (Ernst & Mills, 1977,J. Cell Biol. 75:74). The simplicity of the morphological appearance of thezonula occludens in the salt gland, which resembles that described for several epithelia known to be leaky to ions, is consistent with this hypothesis.