Inhibitory effects of various synthetic substrates for aminopeptidases on phagocytosis of immune complexes by macrophages

The inhibitory effects of various 7-(aminoacyl)-4-methylcoumarinylamides (AA-MCA's), synthetic substrates for aminopeptidases, on phagocytosis of immune complexes by guinea pig peritoneal macrophages were investigated by measuring the intracellular uptake of sensitized 51Cr-sheep erythrocytes and 125I-alpha-amylase complexed with homologous IgG2 antibody. Among the AA-MCA's examined, MCA compounds of hydrophobic amino acids (Phe, Tyr, Leu, and Pro) were found to inhibit the intracellular uptake and digestion of immune complexes. Sucrose density gradient centrifugation of the homogenates of macrophages treated with the inhibitors for 1 h at 37 degrees C showed that they modulated the lysosomes, resulting in a decrease in buoyant density of the organella. These effects of the inhibitors on the buoyant density of the lysosomes as well as on the phagocytic activity of macrophages disappeared upon removal of the inhibitors from the cells by washing. Since none of 7-amino-4-methylcoumarin, L-phenylalanine, and bestatin methyl ester could significantly inhibit the phagocytosis of immune complexes by macrophages, the MCA compounds of hydrophobic amino acids appear to inhibit the phagocytosis as a consequence of their lysosomotropic nature.     

Histochemical staining of exogenous chromium and iron with the catalytic oxidation of phenylamines

Summary A highly sensitive and specific method for staining exogenous chromium and iron in tissues is described. This method is superior to conventional complex-forming methods with regard to its sensitivity and specificity for these metals. The staining reaction is based on the metalcatalysed oxidation of phenylamines. Tissue sections were incubated in a medium containing hydrogen peroxide and phenylamines, p-phenylenediamine or phenylhydrazine. Results obtained from test-tube experiments concerning the catalytic activities of metals indicated that the staining reactions depends on the activities of metals in tissues.     

Escherichia coli K-12 tolZ mutants tolerant to colicins E2, E3, D, Ia, and Ib: defect in generation of the electrochemical proton gradient

Spontaneous Escherichia coli K-12 mutants tolerant to colicin E3 were isolated, and on the basis of their tolerance patterns to 19 kinds of colicins, a new phenotypic class of tolZ mutants was found. The tolZ gene was located between min 77 and 78 on the E. coli K-12 genetic map. The tolZ mutants were tolerant to colicins E2, E3, D, Ia, and Ib, and showed an increased sensitivity to ampicillin, neomycin, and EDTA, but not to deoxycholate; they were able to grow on glucose minimal medium, but not on nonfermentable carbon sources (succinate, acetate, pyruvate, lactate, malate, etc.). The pleiotropic phenotype of the tolZ mutant was due to a single mutation. Both respiration and membrane ATPase activity of the tolZ mutant were normal. The tolZ mutant had a defect in the uptake of proline, glutamine, thiomethyl-beta-D-galactoside, and triphenylmethylphosphonium ion; these uptake systems are driven by an electrochemical proton gradient (delta-mu H+) or a membrane potential (delta psi). In contrast, the uptake of methionine and alpha-methyl-D-glucoside, which is not dependent on delta-mu H+ and delta psi, was normal in the tolZ mutant. Glucose 6-phosphate uptake at pH 5.5, which is driven by a transmembrane pH gradient, in the tolZ mutant was similar to the parent level. These results indicate that the tolZ mutant has a defect in the generation of delta-mu H+ and delta psi.     

Nucleosidediphosphate kinase from Ehrlich ascites tumor cells

A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.     

Isolation of functional cDNA clones for human thymidylate synthase

Thymidine auxotrophic mutants of mouse FM3A cells due to thymidylate synthase deficiency can be transformed into prototrophs by DNA-mediated gene transfer using total human DNA (Ayusawa, D., Shimizu, K., Koyama, H., Takeishi, K., and Seno, T. (1983) J. Biol. Chem. 258, 48-53). From one such transformed cell clone, cloned recombinant lambda phages containing DNA fragments were obtained recently that were concluded by circumstantial genetic evidence to have been derived from the human thymidylate synthase gene (Takeishi, K., Ayusawa, D., Kaneda, S., Shimizu, K., and Seno, T. (1984) J. Biochem. (Tokyo) 95, 1477-1483). Using a DNA segment derived from the cloned genomic DNA fragment and free of repetitive sequences as a probe, functional cDNA corresponding to thymidylate synthase mRNA could be cloned from a cDNA library of SV40 transformed human fibroblasts constructed by Okayama and Berg (Okayama, H. and Berg, P. (1983) Mol. Cell. Biol. 3, 280-289). The cloned cDNA plasmid containing an insert of approximately 1.7-kilobase transformed mouse thymidine auxotrophic mutant cells to thymidine prototrophic cells at a frequency of 2-3 transformants/micrograms of DNA/10(5) cells, a value almost comparable to the highest so far reported. The resultant transformants retained the introduced cDNA and expressed human thymidylate synthase protein sufficient for supporting normal growth of otherwise auxotrophic mouse cells.     

Folic acid as a dietary factor affecting the wing morph of the planthopper, Laodelphax striatellus (hemiptera, delphacidae)

A factor effecting the production of brachypterous adults in Laodelphax striatellus was studied by comparing a diet on which brachypterous adults sometimes developed and a diet on which they never developed. By substituting components distinctive of the latter diet into the former, or by varying the concentration of certain components of the latter diet, the production of brachypterous adults was found to vary with the concentration of a vitamin, folic acid. Brachypterous adults were produced only when the diet contained folic acid at concentrations between 0.5 mg/l and 7.5 mg/l.

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Résumé Un facteur efficace pour la production d'adultes brachyptères chez Laodelphax striatellus a été recherché en comparant une nourriture sur laquelle des adultes brachyptères se développent parfois et une nourriture sur laquelle ils ne se développent jamais. En substituant les constituants spécifiques de cette dernière par ceux de la première ou en variant les concentrations de certains des constituants de la dernière on a montré que la production d'adultes brachyptères varie avec la concentration d'une vitamine, l'acide folique. Ceux-ci sont produits seulement lorsque l'alimentation contient de l'acide folique à des concentrations comprises entre 0,5 mg/l et 7,5 mg/l.

     

Structure of the murine mb-1 gene encoding a putative sIgM-associated molecule

Genomic DNA clones containing the B cell-specific murine mb-1 gene were isolated and a 5.6-kb BamH I fragment was characterized. It is 5629 bp long and contains five exons: an exon containing the 5' untranslated and the coding sequence of the signal peptide, an exon of 294 bp, which contains most of the extracellular sequence of the MB-1 protein, a 119-bp long exon coding mainly for the transmembrane portion, and two exons of 69 bp and 427 bp encoding the cytoplasmic domain and the 3'-untranslated region, respectively. The mb-1 gene does not contain a "TATA box" found in many eukaryotic promoters. The 5'-flanking region has sequence stretches homologous to IgVH 5'-promoter regions and a bcl 2 intron sequence. It contains the decanucleotide sequence (ATGGCAAATA) almost identical to the octamer motif of IgVH promoters. A B cell-specific DNase I-hypersensitive site was found in the 3'-flanking region indicating that this region might be involved in B cell-specific expression of mb-1. Southern blot analysis of genomic liver DNA with the cloned mb-1 cDNA suggests the existence of another mb-1-related gene segment.     

Necessity of IgE antibodies and mast cells for manifestation of resistance against larval Haemaphysalis longicornis ticks in mice

Genetically mast cell-deficient WBB6F1-W/Wv mice showed an apparent defect in manifestation of the resistance against larval Haemaphysalis longicornis ticks, but their serum IgE levels increased more than 100-fold after the second tick infestation. Immune sera obtained from the WBB6F1-W/Wv mice were adoptively transferred to the other WBB6F1-W/Wv mice which had received intracutaneous injections of WBB6F1-+/+ mouse-derived cultured mast cells. Because the resistance against ticks was detectable only when both mast cells and IgE antibodies were available, immediate hypersensitivity reaction appeared to have a physiologic role in the manifestation of the resistance against H. longicornis ticks.