Subcellular localization of tissue polypeptide antigen and cytokeratins in epithelial cells (salivary ad mammary glands). Combined use of the cryoultramicrotomy and the protein A-gold technique

Epithelial cells from various sites and at various stages of differentiation reveal distinct cytokeratin polypeptide patterns. WE have localized these heterogeneous elements at the subcellular level in human salivary glands and in a solid tumor of the breast using a monoclonal and a polyclonal antibody against cytokeratin, and an antibody against tissue polypeptide antigen (TPA) which seems to be related to some cytokeratins. Labeling by the cytokeratin antibodies was more intense in squamous and duct cells than in acinar cells. The TPA:B1 antibody reacted predominantly with duct cells and to a lesser extent with acinar and squamous cells. A precise evaluation of the labeling pattern and a well-preserved cell structure appeared to be important factors in obtaining more detailed information about intermediate filament proteins. The cryoultramicrotomy and the protein A-gold technique are suitable for these studies.     

Differential modulation by N4, 2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate of neutrophil activation.

The cyclic pyrimidine nucleotide, cCMP, is an endogenous substance in mammalian cells but little is known on its functional role. We studied the effects of cCMP, its cell-permeant analogue, N4,2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate (Bt2cCMP), and of butyrate on superoxide (O2-) formation and cytosolic Ca2+ [( Ca2+]i) in human neutrophils. Bt2cCMP inhibited O2- formation and the rise in [Ca2+]i induced by a chemotactic peptide at submaximally effective concentrations. O2- formation induced by platelet-activating factor was potentiated by Bt2cCMP, whereas the cyclic nucleotide had no effect on the rise in [Ca2+]i induced by this agonist. Bt2cCMP enhanced O2- formation induced by tau-hexachlorocyclohexane at a submaximally effective concentration. O2- formation stimulated by complement C5a, concanavalin A, NaF, A 23187, phorbol myristate acetate and arachidonic acid was not affected by Bt2cCMP. cCMP was less effective than Bt2cCMP to inhibit fMet-Leu-Phe-induced O2- formation, and butyrate was without effect on any of the functional parameters studied. Our data show that a cell-permeant analogue of cCMP differentially inhibits and potentiates activation of human neutrophils.     

Effects of cultivation techniques and media on yields and morphology of the basidiomycete Armillaria mellea

Summary The yield of cell mass and the morphology of Armillaria mellea, strain ATCC 11114, was studied using a variety of cultivation methods: solid media, standing liquid culture, shake flasks, tower reactors and impeller-stirred reactors. Two different media, malt extract broth and a glucose/asparagine/peptone-medium, and the corresponding agar media, were used. Yields were higher in the malt extract media than in the glucose media. Generally the highest yields were obtained on solid media while agitated cultures gave the lowest yields. Morphological characteristics such as pellet formation, adhesion to surfaces and pigment production were significantly affected by culture conditions.     

Reversible activation of NADPH oxidase in membranes of HL-60 human leukemic cells

NADPH oxidase in membranes of undifferentiated and dimethylsulphoxide-differentiated HL-60 cells was activated by arachidonic acid (AA) in the presence of Mg2+ and a cytosolic cofactor (CF) found in differentiated HL-60 cells. Basal superoxide (O2-) formation was enhanced several-fold by addition of the stable GTP-analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), prior to AA and was completely prevented by that of GDP. Basal and GTP gamma S-stimulated O2- formation was terminated by GDP. In the presence of Mg2+ or EDTA, basal O2- formation ceased after 25 or 10 min, respectively, and was reinitiated by GTP gamma S or GTP gamma S plus Mg2+. Albumin terminated O2- formation, which was reactivated by AA in the presence of GTP gamma S. Our results show that (1) activation of NADPH oxidase in HL-60 membranes is dependent on endogenous GTP, Mg2+, AA and CF, which is induced during myeloid differentiation, and that (2) NADPH oxidase activation is a reversible process modulated by exogenous guanine nucleotides at various stages of activity of NADPH oxidase. We suggest crucial roles of guanine nucleotide-binding proteins in the activation, deactivation and reactivation of the enzyme.     

The penetration of exogenous tracers through the enameloid organ of developing teleost fish teeth

In order to determine whether exogenous materials permeate to the forming tooth enameloid matrix, teleost species were injected intramuscularly with horseradish peroxidase (HRP) or myoglobin, or; intracardially with lanthanum nitrate or HRP, then killed a predetermined intervals post-injection. Tooth bearing bones were processed for transmission electron microscopy. At the enameloid matrix formation stage, capillaries associated with the enameloid organ were few in number and rarely fenestrated. Both organic tracers reached the matrix at cervical but not coronal, regions of the teeth in all species examined. Lanthanum was rarely observed extravascularly and never extended to the enameloid matrix at the secretion stage. At the enameloid mineralization stage, fenestrated capillaries were closely associated with the outer dental epithelial cells (ODE). All tracers were observed in the plasma membrane invaginations of the ODE. Only intracardially injected HRP compromised the apical intercellular junctions of the inner dental epithelial cells (IDE) to reach the mineralizing enameloid Lanthanum did not extend past the ODE-IDE cell junctions. It is concluded that the close association of mineralization stage fenestrated capillaries with the highly invaginated ODE cells result in increased tracer penetration compared to the secretory stage. The deeper penetration of the organic tracers, compared with lanthanum, between mineralization stage IDE cells may be due to longer in vivo circulation of the former material. The apical junctions of mineralization stage IDE cells, however, remained impermeable to the organic tracers. The absence of mineral in secretory stage enameloid mineral could not be due to specialized cell junctions preventing access of molecules to the matrix. It is suggested that controlling factors other than cellular permeability initiate enameloid mineralization.