Generation of specific antibodies against the rap1A, rap1B and rap2 small GTP-binding proteins. Analysis of rap and ras proteins in membranes from mammalian cells.

Specific antibodies against rap1A and rap1B small GTP-binding proteins were generated by immunization of rabbits with peptides derived from the C-terminus of the processed proteins. Immunoblot analysis of membranes from several mammalian cell lines and human thrombocytes with affinity-purified antibodies against rap1A or rap1B demonstrated the presence of multiple immunoreactive proteins in the 22-23 kDa range, although at strongly varying levels. Whereas both proteins were present in substantial amounts in membranes from myelocytic HL-60, K-562 and HEL cells, they were hardly detectable in membranes from lymphoma U-937 and S49.1 cyc- cells. Membranes from human thrombocytes and 3T3-Swiss Albino fibroblasts showed strong rap1B immunoreactivity, whereas rap1A protein was present in much lower amounts. In the cytosol of HL-60 cells, only small amounts of rap1A and rap1B proteins were detected, unless the cells were treated with lovastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A reductase, suggesting that both proteins are isoprenylated. By comparison with recombinant proteins, the ratio of rap1A/ras proteins in membranes from HL-60 cells was estimated to be about 4:1. An antiserum directed against the C-terminus of rap2 reacted strongly with recombinant rap2, but not with membranes from tested mammalian cells. In conclusion, rap1A and rap1B proteins are distributed differentially among membranes from various mammalian cell types and are isoprenylated in HL-60 cells.     

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.     

Fatty-acid-induced activation of NADPH oxidase in plasma membranes of human neutrophils depends on neutrophil cytosol and is potentiated by stable guanine nucleotides

Both cis and trans unsaturated fatty acids and sodium dodecyl sulfate activated NADPH oxidase in plasma membranes of human neutrophils in the presence of neutrophil cytosol. In contrast, 5,8,11,14-icosatetraynoic acid, saturated fatty acids, esters, peroxides and 4 beta-phorbol 12-myristate 13-acetate, a potent activator of protein kinase C, were inactive. 5,8,11,14-icosatetraynoic acid inhibited superoxide formation elicited by fatty acids. Guanosine 5'[gamma-thio]triphosphate (GTP[gamma S]), a potent activator of guanine-nucleotide-binding proteins (N-proteins) enhanced superoxide formation elicited by fatty acids up to fourfold, supporting our previous suggestion that NADPH oxidase is regulated by an N-protein [Seifert, R. et al. (1986) FEBS Lett. 205, 161-165]. Cytosols from various tissues, soybean lipoxygenase and protein kinase C, purified from chicken stomach, did not substitute neutrophil cytosol. The activity of neutrophil cytosol was destroyed by heating at 95 degrees C. Superoxide formation was not affected by the inhibitor of protein kinase C 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). Removal of cytosolic ATP by preincubation with hexokinase and glucose, dialysis of neutrophil cytosol or chelation of calcium with EGTA did not abolish the stimulatory effect of arachidonic acid and GTP[gamma S]. Thus, the cytosolic cofactor appears to be a neutrophil-specific and heat-labile protein, which is neither a lipoxygenase nor protein kinase C.     

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.     

Ultrastructural changes in maturing pollen grains ofApium nodiflorum L. (Apiaceae), with special reference to the endoplasmic reticulum

Summary The ultrastructural events in 3-cellular pollen grains ofApium nodiflorum L. are investigated during pollen maturation. Three distinct developmental stages are distinguished from the formation of sperm cells up to anthesis, whereby the rough endoplasmic reticulum (RER) is mainly involved. The most conspicious form is the highly dilated RER in the vegetative cytoplasm of the youngest pollen grains, which changes to vesicular RER in the following stage. In mature pollen grains the RER has a narrow cisternal configuration and often forms stacks. Pollen activation is preceded by the accumulation of polysaccharide particles.     

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.