A mode of formation of tubular myelin from lamellar bodies in the lung.

A mechanism is suggested by which the membranes of lamellar bodies are converted to tubular myelin (TM) in the lung. It is argued that a simple corrugation of the membranous sheets can produce the TM formation. Such corrugation would occur in response to simple stresses acting on the lamellar body membranes. The intersections of the tubular figures are formed by fusion of adjacent corners in the corrugations. This results in a more stable hydrophobic bonding of phospholipid molecules. Strong supportive evidence for the mechanism is given by electron micrographs of TM formations.

Enzymes of the lung. I. Detection of esterase with a new cytochemical method.

The esterases of rabbit lung have been investigated from two viewpoints, the cytochemical and the biochemical. To accomplish this objective, we designed and synthesized a series of ester substrates which provide both a cytochemical indicator of the location of the enzyme and a means of following the enzymatic activity in tissue homogenates and subfractions. The substrates are p-nitrophenylthiol esters which yield, upon hydrolysis, carboxylic acid and p-nitrothiophenol. The latter can react with aurous ions to give an electron-opaque deposit; in addition, the strong absorption of p-nitrothiophenol at 410 mmicro permits continuous kinetic measurements. Thus, it is possible to correlate the intracellular site of action and the biochemical behavior of the esterases. The new substrates are the thiol analogues of the p-nitrophenyl esters frequently employed as esterase substrates. The rates of hydrolysis of the two series of esters are compared in vitro. During tissue fractionation, most of the esterase activity sediments with a particulate fraction. The effects of a number of common esterase inhibitors, such as diisopropyl phosphorofluoridate and eserine sulfate, are examined, and the effects of enzyme concentration and heat inactivation are shown with the use of the partially purified preparations. The cytochemical work shows that the esterase activity is most prominent in the lamellar bodies of the giant alveolar (type II, septal, or granular pneumatocyte) cells of the lung and to a lesser extent in squamous (type I, or membranous pneumatocyte) epithelial and endothelial cells. In both the cytochemical and biochemical studies, the enzymes are inhibited by diisopropyl phosphorofluoridate and phenyl methylsulfonyl fluoride but are insensitive to eserine sulfate.

Polyanionic agents as inhibitors of phagosome-lysosome fusion in cultured macrophages: evolution of an alternative interpretation.

Various natural and synthetic substances classified as polyanionics have been implicated in antagonizing phagosome-lysosome fusion in cultured macrophages. The phenomenon has been judged by comparing the transfer of selected markers from secondary lysosomes to phagosomes in control and in "polyanion" cells. Our earlier studies showed that use of one of the markers, the membrane-permeating acridine orange, was plagued with artifacts that were especially misleading in the presence of polyanionic agents. We now question the validity of data obtained by the alternative technique, electron microscopy. Our present evidence shows that nonionic hydrocolloids of sufficiently high molecular weight prevent the transfer of various colloidal electron-opaque markers from lysosomes to phagosomes in the same manner as does the powerful polyanionic "fusion inhibitor" dextran sulfate. Both kinds of hydrocolloids, however, allow delivery of lysosomal, low-molecular-weight highly charged non-permeant fluorescent markers to phagosomes, probably by a fusion process. We propose that neither type of hydrocolloid inhibits fusion; instead, when sufficiently concentrated, they trap particulate electron-opaque markers in a gelatinous matrix, which may move only slowly out of lysosomes. The polyanionics trap the electron-opaque markers physically and acridine orange ionically. Hence, the semblance of "fusion inhibition."

Polyanionic agents do not inhibit phagosome-lysosome fusion in cultured macrophages.

The survival of some intracellular pathogens within macrophages may be aided by an ability of the organism to antagonize, from within the entrapping phagosome, its fusion with lysosomes. On the other hand, certain polyanionic agents have been implicated in imposing a similar block to fusion from the lysosomal domain--because the transfer of various foreign markers from lysosomes to newly formed phagosomes is remarkably inhibited in these polyanion-containing cells. Based on an analysis of various observations and our own recent data, we propose that the polyanionics do not, in fact, prevent phagosome-lysosome fusion but, instead, physically entrap the usual markers in a gelatinous matrix within the lysosomes. This view accounts for many paradoxical consequences of polyanionic accumulation and for the curiously normal behavior of macrophages that are presumed to be suffering from such a crucial intracellular dysfunction.

Macrophage effector function in pulmonary oxygen toxicity: hyperoxia damages and stimulates alveolar macrophages to make and release chemotaxins for polymorphonuclear leukocytes.

Macrophages synthesize many secretory products in vitro but the stimuli for their production and their pathophysiologic significance in vivo are largely unknown. In the present investigation, we found that hyperoxia damaged rabbit alveolar macrophages (AM) in vitro as manifested by decreased cell numbers, increased lactate dehydrogenase (LDH) release, and the development of ultrastructural abnormalities that resembled those seen in AM in situ or lavaged from lungs of rabbits exposed to hyperoxia in vivo. Hyperoxia also stimulated cultured rabbit AM to release chemotaxins for polymorphonuclear leukocytes (PMN) that were similar in molecular weight to chemotaxins obtained from lung lavages of rabbits exposed to hyperoxia in vivo. Our results suggest that alveolar macrophage secretory products may play a physiologically relevant role in recruitment of PMN to the lungs in pulmonary oxygen toxicity.

Adenine nucleotides in the secretory granule fraction of rat islets.

Concomitant with glucose-induced insulin release, there occurred an increase of ATP from 4.40 plus or minus 0.21 to 23.16 plus or minus 0.52 pmol/100 islets/min (P less than 0.001) in the effluent from perifused rat islets. There is a linear relationship between circulating ATP and insulin levels both in the stimulated and basal state (r = 0.689, P less than 0.01). Islets incubated with labeled adenine for a short period of time (37.5 min) showed no release of radioactivity upon subsequent glucose-induced insulin release. Islets incubated for a prolonged interval with labeled adenine (150 min) showed an increase in acid soluble radioactivity in the effluent during glucose-induced insulin release. Following incubation of the islets with labeled adenine for 150 min, approximately 5% of the homogenate radioactivity was found in the secretory granules. Using column chromatography to separate the adenine nucleotides, the distribution of radioactivity among the various nucleotides in the secretory granule fraction was found to be: AMP 54.42 plus or minus 4.96%, ADP 14.20 plus or minus 1.63%, ATP 15.39 plus or minus 3.84%, and cAMP 16.07 plus or minus 2.11%. The distribution of radioactivity in the effluent adenine nucleotides after glucose-induced insulin release was: AMP 32.83 plus or minus 4.62%, ADP 24.52 plus or minus 2.77%, ATP 28.13 plus or minus 5.45%, and cAMP 26.01 plus or minus 3.34%. The absolute levels of adenine nucleotides in the secretory granules were ATP 4.19 plus or minus 0.88, ATP madp 7.94 plus or minus 2.20 and cAMP 4.46 plus or minus 1.74 pmol/ug prot. The levels in the islet effluent were ATP, 15.30 plus or minus 2.70, ATP qDP, 29.43 plus or minus 3.49 and cAMP 7.66 plus or minus 1.93 pmol/100 islets/min for the first ten min of glucose-stimulated insulin release. Thereafter there was a rapid decline in effluent cAMP while ATP and ADP remained in essentially equivalent amounts. The distribution of radioactivity and absolute levels of the adenine nucleotides in the effluent reflects that found in the secretory granules, confirming previous observations that insulin release is occurring by exocytosis.

Fimbriae of Actinomyces viscosus t14v: their relationship to the virulence-associated antigen and to coaggregation with Streptococcus sanguis 34.

1) Fimbriae from A. viscosus T14V may be similar to those found on other bacteria. 2) The antigenic difference between virulent and avirulent A. viscosus T14 appears to be of a quantitative rather than a qualitative nature and is related to fimbriae and not to the cell wall polysaccharide. 3) Coaggregation between A. viscosus T14V and S. sanguis 34 is mediated by fimbriae on the former which have specificity for beta-linked galactosyl residues.

Surface fibrils (fimbriae) of Actinomyces viscosus T14V.

Surface antigens of Actinomyces viscosus T14V were released from cell walls by digestion with lysozyme. These were separated by ion-exchange and gel filtration chromatography into fractions rich in carbohydrate or protein. The former contained a polysaccharide high in 6-deoxytalose, along with a peptide fragment from the cell wall. In the protein-rich fractions, material of high molecular weight was present, which contained some carbohydrate and up to 14.3% nitrogen. Aspartic acid, threonine, glutamic acid, lysine, alanine, and glycine were detected in these fractions, along with smaller amounts of 10 other amino acids. Most of the alanine was present as the L isomer and thus was not from peptidoglycan. Electron microscopy of the high-molecular-weight material revealed long fibrils, 3.5 to 4.5 nm in diameter, which resembled those seen on bacterial cells. V-specific antiserum, prepared by absorbing anti-A. viscosus T14V serum with cell walls of the avirulent strain (A. viscosus T14AV), did not react with the 6-deoxytalose polysaccharide but reacted well with isolated fibrils, and this was not inhibited by 6-deoxytalose.

Synaptogenesis in reaggregating brain cell culture.

Dissociated cells from embryonic mouse brain reassociate in rotation culture to form highly organized aggregates. Electron microscopic observations of aggregates show the presence of synapses that mature and increase in number during culture. Apparent myelination of axons is observed in the aggregates after 5 weeks of culture. Thus, brain cell aggregates perform two highly specialized morphological events that are characteristic of mouse brain differentiation.

Identification of the virulence-associated antigen on the surface fibrils of Actinomyces viscosus T14.

Actinomyces viscosus T14V is virulent (V) for monoinfected rats, causing periodontal disease and bone loss, whereas, A. viscosus T14AV, a mutant strain, is avirulent (AV). Surface antigens from the T14V and T14AV strains were prepared by lysozyme digestion of cell walls and were compared by immunodiffusion against antisera to T14V and T14AV whole cells. The V-associated antigen (V-antigen) was detected readily in the T14V, but not readily in the T14AV cell wall extract. Antiserum specific for the V-antigen was prepared by absorbing anti-A. viscosus T14V serum with cell walls from the T14AV strain. This antiserum was used in the indirect peroxidase-labeled antibody technique to localize the V-antigen on the bacterial cell surface at the ultrastructural level. With whole bacterial cells, the V-antigen was found on fine fibrils and was detected in both the T14V and T14AV strains. The presence of V-antigen on the AV strain was supported by the demonstration of antibodies against the V-antigen in anti-A. viscosus T14AV serum. Examination of isolated bacterial cell walls revealed a greater amount of fibrils and V-antigen on the T14V cell wall than on the T14AV cell wall. The data suggest that the presence of V-antigen represents a quantitative rather than a qualitative difference between the V and the AV strains of A. viscosus T14. Samples of human plaque were examined, and the V-antigen was found to be a specific marker for the fibril-containing layer of certain plaque bacteria, which are probably strains of A. viscosus or A. naeslundii.

Inhibitors of coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34: beta-galactosides, related sugars, and anionic amphipathic compounds.

Coaggregation between Actinomyces viscosus T14V (T14V) and Streptococcus sanguis 34 (Ss34) depends upon specific reaction between lectin on T14V and carbohydrate on Ss34. Studies on coaggregation inhibition by sugars related to D-galactose, beta-galactosides, and amphipathic molecules revealed: (i) D-fucose, D-talose approximately equal to D-galactose, which was 0.2 potency of lactose. No other hexoses or pentoses inhibited at 0.1 M. (ii) Gal beta (1 leads to 3)GalNAc alpha OCH2C6H5 was the most potent beta-galactoside inhibitor; it had 20 times the potency of lactose. (iii) Anionic nonaromatic amphipathic compounds were good inhibitors; sodium deoxycholate (I) was equal to lactose; sodium dodecyl sulfate (II) had 15 times the potency of lactose; there was 90 to 100% irreversible inhibition when T14V was treated with 0.005 M (II). Treatment of Ss34 with II had no effect. (iv) Synergism of inhibition was observed between lactose and I or lactose and II, e.g., inhibition by 0.01 M lactose = 5%; inhibition by 0.01 M I = 9%; inhibition by 0.01 M lactose + 0.01 M I = 87%. (v) The irreversible inhibition by II was prevented when 0.25 M lactose or 0.25 M I was present during treatment of T14V with 0.005 M II. (vi) Synergism and prevention by lactose or by I of irreversible inhibition by II suggest that all three react at the same site on T14V lectin. We hypothesize that the T14V lectin combining site for Ss34 carbohydrate has specific affinity for beta-galactosides and for anionic nonaromatic amphipathic molecules. This site can be saturated by either kind of reagent to exclude the other reagent or to inhibit coaggregation.