Empty Envelopes Research Articles

FATE OF NEUROHYPOPHYSIAL GRANULE MEMBRANES AFTER SECRETION IN THE RAT: GENERATION OF A NEW APPARENT ORGANELLE BY FREEZING AND THAWING SECRETORY GRANULES

The contents and membranes of the secretory granules in the rat neurohypophysis were labelled in vivo with [35S]cysteine and [3H]choline respectively. Density-gradient centrifugation of the labelled granules showed the membrane label to be distributed largely between two peaks: one associated with intact granules and one with the characteristics of empty granule envelopes. Stimulation of hormones release in vitro led to the movement of membrane label from the intact granule fraction to the other one, consistent with the recapture of membrane as large vesicles. Freezing and thawing the crude granule fraction, ostensibly to aid osmotic fracture, produced a single membrane component with a density intermediate between the two original fractions.

A negative staining study of human erythrocyte ghosts and rat liver nuclear membranes

An electron microscopic study of membrane features has been performed using negative staining. Hemoglobin-free human erythrocyte ghosts and membranes from rat liver nuclei have been observed in the electron microscope after negative staining with ammonium molybdate and the uranyl acetate-oxalic acid complex, both at pH 7.0. The surfaces of partially empty nuclear envelopes have been shown to be covered with pores approximately 500 A in diameter and surrounded by annuli approximately 250 A wide. Many of the annuli appeared to be composed of eight subunits. Individual annuli have been found to separate themselves from the overall membrane sheet in regions where the membranes have undergone disruption. Hemoglobin-free human erythrocyte ghosts have been shown to possess membrane holes or pits, approximately 140 A in diameter. These pits are very similar to those formed by complement hemolysis, but are fewer in number and may be slightly larger. The erythrocyte pit structures have much smoother edges than the nuclear pores and do not appear to be composed of subunits, though this cannot be ruled out. Apart from the pit structures the surface of the erythrocyte ghost has an overall fine structure. Within this fine structure individual protein molecules have been located, though it has very often been difficult to obtain sufficient contrast around the molecules owing to the double layer of membrane beneath them on the specimen grid. The uranyl complex provided a superior contrast to ammonium molybdate at this order of resolution, though it tended to impart a more granular texture to the electron images than did ammonium molybdate. The results presented are discussed in relation to the possibilities and limitations of the negative staining method when applied to membrane systems.

Separation and characterization of products of two measles virus variants.

Products released from HeLa cells infected with the DP (Diluted Passage) and UP (Undiluted Passage) variants of measles virus were characterized by fractionation in discontinuous CsCl gradients. DP virus materials contained non-soluble products, mainly represented by infectious hemagglutinin (HA), density 1.24–1.25 g/ml. UP virus materials contained some particles of this kind, but to a major extent the products present were non-infectious HA — both non-soluble and soluble. Of particular prominent occurrence among non-infectious products was a population of non-soluble particles with a density of 1.20–1.21 g/ml, which carried both HA and hemolysing activity. Electron microscopy demonstrated these particles to be empty envelopes, contrasting with the infectious HA, density 1.24–1.25 g/ml, which was represented by intact particles or envelopes containing, or associated with nucleocapsids.

Comparison of the Action of Vantocil, Cetrimide and Chlorhexidine on Escherichia coli and its Spheroplasts and the Protoplasts of Gram Positive Bacteria

SUMMARYThe actions of chlorhexidine, cetrimide and Vantocil (the hydrochloride of a polymeric biguanide) on bacteria and spheroplasts or protoplasts derived therefrom have been compared. Lysis of Escherichia coli spheroplasts by chlorhexidine or cetrimide is due to rupture of the cytoplasmic membrane. Membranes ruptured with cetrimide can reform to give small, empty envelopes. With increasing concentrations of cetrimide or chlorhexidine more and more granules appear in the cytoplasm, increasing in size with increasing concentration, until the structure becomes transformed to a granular body similar in size to the original spheroplast. With chlorhexidine or Vantocil this now electron‐dense body is rigid, but with cetrimide it collapses to a flat disc which does not lyse. The granules appear to consist of coagulated cytoplasm. This effect of excess of these antiseptics not only prevents osmotic lysis of the spheroplast or the collapsed disc but renders them immune to lysis by a variety of chemical agents. In isolated cytoplasm, chlorhexidine or cetrimide causes precipitation over a narrow range of concentrations but greater than those required for lysis. With Vantocil precipitation occurs gradually over a wide range of concentrations, starting at concentrations equal to those which cause a limited amount of lysis of the spheroplast or a relatively small increase in permeability of intact bacteria. Lanthanous and uranyl ions also precipitate cell contents but do not lyse spheroplasts; spheroplasts treated with these ions are protected from lysis by other agents.

The sarcolemma and fibrous envelope of striated muscles in beef

A Waring blendor with dulled knife and reduced speed was used for isolating muscle fibers and connective tissues from small samples of beef muscles. Significantly, the sarcolemma proper, a thin, homogeneous, non-fibrous membrane about 0.1 μ thick was found linking the ends of broken muscle fibers from raw unfixed tissue only. From cooked or/and chemically fixed samples, the isolated muscle fibers were “bare”, i.e. deprived of their membranes, the latter being found in the form of empty tubular envelopes either singly or in groups. The separation between the muscle fiber substance (actomyosin) and the surrounding membranes, so observed, is the result of a differential protein denaturation brought about by the cooking and fixing processes as evidenced by the structures of cooked muscle tissues seen in 10 μ celloidin sections. No structural details were obtained from the muscle fiber envelopes on account of the limited resolving power of the light microscope, but judging from their characteristic appearance of having numerous tiny interstices, they are probably fibrous, though not likely of the collagenous type in the light of the recent results obtained with electron microscopy. The results of this work have confirmed the bipartite nature of the muscle fiber membrane held by many investigators in this field, and have advanced reasons for believing that the true sarcolemma and the outer fibrous envelope are truly components of an intact muscle fiber membrane. It is suggested on these grounds that the term sarcolemma, when used alone, may refer to the composite membrane of a muscle fiber as a whole, whereas the two components should be known, respectively, as the sarcolemma proper (or true sarcolemma) and the sarcolemmous envelope.