Effect of Bordetella pertussis leukocytosis (lymphocytosis)-promoting factor (LPF) on the physical lymphoepithelial-cell association studied with the use of an in vitro model of mouse thymus

The effect of highly purified leukocytosis (lymphocytosis)-promoting factor (LPF) of Bordetella pertussis on physical lymphocyte and reticuloepithelial (RE) cell association was studied in an in vitro thymus model. First, a simplified in vitro system to assess the lympho-RE-cell association was developed. A completely confluent layer of thymic RE cells was formed by cultivating trypsinized thymus cell suspensions from 2- to 7-day-old mice. When thymic lymphoid cells were seeded on this cell layer and cultivated overnight, a significant proportion of them were found underneath the RE cell layer. This physical lympho-RE-cell association was quantitated by counting the lymphoid cells underneath the RE cell layers. Second, the effect of LPF on this physical lympho-RE-cell association phenomenon was investigated. Addition of LPF to the culture markedly inhibited the formation of the lympho-RE-cell complex; that is, it inhibited the infiltration of lymphoid cells under the RE cell layer. LPF rendered a nearly maximal level of inhibitory effect at a dose of 0.1 ng/ml. Furthermore, LPF enhanced the liberation of lymphoid cells from preformed lympho-RE-cell complexes. On the other hand, LPF had no direct cytotoxic effect on lymphoid cells at doses below 1 microgram/ml. In order to investigate whether LPF produced the effect by acting on lymphoid cells, RE cells, or both, the following experiments were performed. When lymphoid cells were pretreated with LPF and added to normal RE cell layers, the lympho-RE-cell association was maximally inhibited above the dose of 1 ng/ml. Treatment of these LPF-treated lymphoid cells with anti-LPF antibodies failed to abrogate the effect of LPF. When RE cell layers were similarly pretreated with LPF and were cultivated with normal lymphoid cells, however, much higher doses of LPF, above 100 ng/ml, were required for maximal inhibition. Furthermore, treatment of these LPF-treated RE cells with anti-LPF antibodies abrogated the effect of LPF. Therefore, the apparent effect of LPF on RE cells was considered to be due to the carry-over by RE cells of LPF, which should directly act on lymphoid cells at extremely low doses. On the basis of these results, it was concluded that LPF acted directly on lymphoid cells without mediation of RE cells. These in vitro results appear to parallel the effects of LPF in vivo, where it induces a depletion of cells in the thymus. The model may be useful to study this phenomenon and the concomitant accumulation of blood lymphocytes.     

Extracellular matrix during laminar pattern formation of neocortex in normal and reeler mutant mice

The spatial and temporal distribution of extracellular matrix, which occupied the large extracellular spaces in the developing cerebral cortex, was studied during pre- and perinatal ontogenesis of normal and reeler mutant mice. Colloidal iron-staining material was localized principally in the marginal zone and subplate of normal mice, whereas in reeler mutants, most of the material was found in the outer layers of the cortex. Patterns of extracellular matrix localization in both genotypes followed the laminar pattern formation of cerebral cortex architecture. Histochemical ultrastructural visualization of this extracellular matrix and its susceptibility to enzymatic treatment suggested that the major components are glycosaminoglycans. Their possible role in relation to afferent axon targeting is discussed.     

Molybdic and tungstic heteropolyanions for "ionic fixation" of acetylcholine in cholinergic motor nerve terminals

The treatment of neuromuscular junctions with phosphomolybdic acid (PMA) and silicotungstic acid (STA) heteropolyanions permits the visualization of electron dense precipitates in the synaptic vesicles of the cholinergic motor nerve terminals. At the light microscopic level, the uncolored molybdenum salt is visualized after reduction to molybdenum blue. The blue coloration is confined to the nerve terminals. Since PMA and STA are known as strong precipitating agents of quaternary ammonium compounds (cations) it is supposed that they have insolubilized in situ the acetylcholine (Ach) of the synaptic vesicles by means of a rapid ionic interaction. Furthermore, in spite of the strong acidity of PMA and STA solutions, the ultrastructure of the treated tissue is not significantly altered but on the contrary seems to be well preserved. The ionic insolubilization of Ach, added to the good preservation of the ultrastructure prompted us to use the term "ionic fixation".     

Production of neutral protease from Aspergillus oryzae by a novel cultivation method on a microporous membrane

Summary A novel cultivation method using a microporous membrane (membrane-surface liquid culture) was developed, in which moulds are grown on the membrane surface with its opposite side being in contact with a liquid medium. The amount of neutral protease from Aspergillus oryzae produced was more than 10 times higher than that produced by the conventional liquid culture.     

Receptor-mediated calcium signal playing a nuclear third messenger in the activation of antigen-specific B cells.

We have studied receptor-mediated calcium signals in antigen-specific B cells (trinitrophenol-specific B cell clone, TP67.21) using a confocal fluorescence microscope with an argon ion laser (488 nm) and a He-Cd laser (325 nm). Confocal fluorescence images of fluo-3 loaded B cells, excited by an argon ion laser, became much brighter and more nonhomogeneous than those before antigen stimulation. Time-dependent fluorescence changes in intensities were abrupt and quite similar to the patterns of the intracellular calcium ion concentration [Ca2+]i observed by a conventional fluorescence microscope using fura-2. From the morphological patterns of the calcium images, the parts of the bright fluorescence seemed to belong to the nucleus in B cells. To confirm the above events we measured the confocal fluorescence images of the nucleus. From the fluorescence images of co-loaded Hoechst 33342 (a DNA-specific fluorescent probe), which excited by a He-Cd laser, the brighter parts of the fluo-3 fluorescence intensities were identified to the nucleus in B cells. This suggested the possibility that the increased intranuclear calcium ions may play a nuclear third messenger in B cells.     

Irradiation of cells in tissue culture

Summary Cultures of human amnion were employed to check the hypothesis that cell strains with heteroploid chromosome counts regularly produce giant cells within 12 days following treatment with 2000 r and 4000 r of gamma irradiation from a cobalt source, while this response has not been obtained from primary cultures whose cells were presumed to be diploid.The giant cell reaction not only was obtained from two transfer passage lines of a well-established amnion strain developed at Berkeley (No A 185-21C-26 and No A 185-21C-45) but was also found for a 20-day second passage culture of amnion. Since this line has continued to reproduce at a rapid rate, it is presumed to have assumed the features of a typical strain within the period of observation. This impression was reinforced by the finding that the chromosome number for 32 cells fixed on the 35th day had a modal value of 67.In contrast, both untrypsinized and trypsinized spindle cells in primary cultures as well as unaltered epithelial elements which had not been subcultured gave no evidence of giant cell formation 12 days after exposure to 2000 r and 4000 r from a Cobalt⁶⁰ source.These data lend evidence that giant cell formation is related to the chromosomal constitution of the irradiated elements.This research was supported by funds provided under Contract AF 18(600)-1263 with the School of Aviation Medicine, USAF, Randolph Air Force Base, Texas.Fellow of the Instituto de Alta Cultura and the Fundacão Calouste Gulbenkian of Lisbon, Portugal.Tobacco Industry Research Committee Fellow.     

Antithrombotic effect of topically applied 3-Oxa-Methano-prostaglandin I1 in the microcirculation and antiplatelet functions of its active form

The antithrombotic effect of topical application of the 3-oxamethano-prostaglandin (PG) I₁ analog, SM-10902 in the microcirculation and in vitro antiplatelet functions of its active form SM-10906 were estimated in comparison with PGI₂ and PGE₁. In rat platelets, SM-10906 evoked accumulation of intracellular cyclic adenosine 3′,5′-monophosphate, and exhibited antiaggregatory and disaggregatory activities, which were all enhanced by the phosphodiesterase inhibitor theophylline. Additionally, SM-10906 was shown to inhibit platelet adhesion to collagen in human platelet-rich plasma. PGI₂ and PGE₁ also showed in vitro antiplatelet effects in the order of PGI₂ > SM-10906 ≥ PGE₁. SM-10902 exhibited a dose-dependent antithrombotic effect in the guinea pig mesenteric arteriole by a topical application, and this activity might be exerted by the antiplatelet functions of SM-10906. Although SM-10906, PGI₂ and PGE₁ also showed the antithrombotic effects, SM-10902 was the most potent. In conclusion, the present studies indicate that an external topical preparation of SM-10902 may be useful for the therapy of peripheral circulatory insufficiency.     

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H2O at pH 2.5 or 5.5 were examined by H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to D-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the beta-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.