Inhibition of ATPase Activity in Pea Plasma Membranes In Situ by a Suppressor from a Pea Pathogen, Mycosphaerella pinodes

A pathogenic fungus of pea, Mycosphaerella pinodes, secretesa so-called "suppressor" in its pycnospore germination fluid.The suppressor blocks the defense responses and induces localsusceptibility (accessibility) in pea plants to agents thatare not pathogenic in pea. The suppressor nonspecifically inhibitsthe ATPase activity in plasma membranes prepared from pea, soybean,kidney bean, cowpea and barley plants. However, cytochemicalstudies by electron microscopy indicate that the suppressorspecifically inhibits the ATPase in pea cell membranes, butnot in those of four other plant species tested. That is, thespecificity of the suppressor appears at the cell and/or tissuelevel, but is not evident in vitro. Furthermore, the inhibitoryeffect of the suppressor is temporary because the ATPase activityrecovers 9 h after the treatment. A similar effect was observedafter inoculation with M. pinodes but not with a nonpathogenof pea, M. ligulicola. The role of the suppressor in host-parasitespecificity is discussed. (Received April 9, 1991; Accepted August 6, 1991)     

Kinetic properties of aromatase mutants Pro308Phe, Asp309Asn, and Asp309Ala and their interactions with aromatase inhibitors.

Mutant forms of aromatase cytochrome P-450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for androstenedione for expressed wild type (11.0 +/- 0.3 nM SEM, n = 3) increased 4-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. 4-Hydroxyandrostenedione (4-OHA) a strong inhibitor of wild type aromatase activity (IC50 = 21 nM and Ki = 10 nM), was even more effective against mutant Pro308Phe (IC50 = 13 nM and Ki = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC50 = 345 and 330 nM and Ki = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 16949A (IC50 = 4.0 and 4.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC50 = 140 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced Ki = 0.4 nM for CGS 16949A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for androstenedione aromatization. The substitution of Pro308 with Phe produces weaker affinity for androstenedione in the substrate pocket, but this alteration favors 4-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase.     

Levels of tRNAs in bacterial cells as affected by amino acid usage in proteins.

Transfer RNAs of Mycoplasma capricolum were separated by two-dimensional polyacrylamide gel electrophoresis, and the relative abundance of each of the 28 known tRNA species was measured. There existed a correlation between the relative amount of isoacceptor tRNAs and the frequency in choosing synonymous codons that could be translated by the isoacceptors. Furthermore, it was observed that the total amount of tRNAs for a particular amino acid was paralleled by the composition of the amino acid in ribosomal proteins. A similar relationship was obtained from reexamination of the previous data on Escherichia coli tRNAs, suggesting that the amount of tRNAs for an amino acid is affected by the usage of the amino acid in proteins.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.     

Cloning and expression of human lymphotoxin mRNA derived from a human T cell hybridoma

We cloned human lymphotoxin (LT) cDNA from a cDNA library prepared from phorbol myristate acetate (PMA) and Con A-stimulated human T cell hybridoma (AC5-8) cells, The nucleotide sequence of the cDNA insert in the plasmid, pLT13, was determined and compared with those of peripheral blood mononuclear cell derived LT cDNA and the LT gene. Four stretches, containing 14 nucleotides in total, were different among the three sequences. The differences included one base change from C to A in the coding region. Because this change was expected to result in a change in the amino acid at position 26 from Thr to Asn, we constructed an E. coli expression plasmid (pLT13tac6-8.2) and a mammalian cell expression plasmid (pSV2-LT) in order to confirm that pLT13 contains the coding sequence of human LT. Both plasmids were found to synthesize active LT molecules after transfection into JM105 and COS-1 cells, respectively, and their lytic activities were found to be completely neutralized by anti human LT serum. Using an insertion mutant and a deletion mutant, we examined the role of the C terminal domain in the LT activity. The results obtained strongly suggested that the intactness of the C terminal less than 10 residues is required for the LT activity.     

Postnatal development of the anulospiral endings of Ia fibers in muscle spindles of mice

The formation of the anulospiral ending of Ia fibers in muscle spindles was investigated in the masseter muscle of developing mice. Before 15 days after birth, the complete anulospiral ending was not observed in almost all of the muscle spindles examined. With the growth of mice, the Ia fiber began to construct the spiral ending, and by the 40th postnatal day after weaning, almost all of the Ia fibers of the muscle spindles had complete coiled endings, though the formation still continued in some spindles. The continuous formation of anulospiral endings for a long period after weaning indicates that muscle spindle morphogenesis may be affected by muscle tension in the masseter muscle due to the movement activated after weaning.     

Inhibition of IL 2-dependent proliferation of rat T lymphoblasts by the monoclonal antibody ART62 which reacts with MHC class 1 antigens

During the course of studies designed to obtain monoclonal antibodies (mAb) that recognize the rat interleukin 2 receptor, a mouse IgG1 mAb (ART62) was identified which inhibits the interleukin 2 (IL 2)-dependent proliferation of rat T lymphoblasts without affecting the binding of IL 2 to such cells. In order to characterize the cell surface components that react with the mAb ART62, T lymphoblasts were surface-labeled with 125I, and the radioactive molecules were immunoprecipitated by the antibody analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The mAb ART62 precipitated two major components of 48,000 m.w. and 12,000 m.w., respectively, which were different from those which react with the anti-IL 2-receptor antibody ART18, a molecule of 50,000 to 55,000 m.w. Sequential immunoprecipitation studies revealed that the mAb ART62 reacts with the MHC class 1 antigen that reacts with the classical anti-rat MHC class 1 mAb OX18, and vice versa. In contrast to the mAb ART62, OX18 that does not affect and several other mAbs known to inhibit the rat MLR failed to inhibit IL 2-dependent proliferation of rat T lymphoblasts. In contrast to the anti-IL 2 receptor antibody ART18, ART62 effectively inhibited IL 2-driven proliferation even when added to cells already committed to proliferate by IL 2-IL 2 receptor interaction. These data raise the possibility that MHC class 1 antigens could be involved in the chain of reactions mediating the signals required for cell proliferation.     

Establishment of a nucleoside-specific suppressor T cell line from SLE prone (NZB/NZW)F1 mice

A long-term cultured suppressor T cell line (GTS-124) was established from an autoimmune mouse strain, (NZB X NZW)F1, by a two-part procedure: a) B/W F1 mice were made tolerant to guanosine (G) by administration of a tolerogen, the G-modified copolymer of D-glutamic acid and D-lysine (G-D-GL); and b) the spleen cells obtained from tolerant mice were repeatedly stimulated with mitomycin C-treated G-modified syngeneic spleen cells. The GTS-124 cells suppressed the secondary in vitro response to G-keyhole limpet hemocyanin (G-KLH) but did not suppress the response to unrelated antigens, sheep erythrocytes (SRBC), or trinitrophenyl-KLH (TNP-KLH). The expression of Thy-1 antigen on the cell surface of GTS-124 was demonstrated by flow cytometry. Growth of GTS-124 cells was dependent on IL 2. To determine whether GTS-124 cells could suppress the response to nucleosides other than G, KLH coupled with four nucleosides (adenosine [A], G, cytidine [C], and thymine riboside [T]) collectively (AGCT-KLH) was first used as the antigen in the assay system. The PFC response to the individual nucleosides (anti-A, -G, -C, and -T PFC) were effectively inhibited by GTS-124 cells, suggesting that the GTS-124 cells mediated cross-suppression toward all four nucleosides. A more stringent cross-suppression test was conducted by using only the T moiety bound to KLH (T-KLH) as antigen. The results showed that GTS-124 cells were capable of suppressing the T-specific response. The cross-suppression could be seen after repeated selection on a G-BSA-coated dish. These results provide direct evidence that the suppressor T cells induced by in vitro stimulation with G-modified self can indeed suppress the response to nucleosides other than G.     

Mechanism of phorbol myristate acetate-induced lymphotoxin production by a human T cell hybridoma

Various hydroxyl radical scavengers markedly inhibited phorbol myristate acetate (PMA)-induced lymphotoxin (LT) production by a human T cell hybridoma, AC5-8. Among those we tested, tetramethylurea (TMU) was the most potent scavenger, and it was revealed that TMU must be added before 2 h have elapsed after PMA addition in order for LT production to be inhibited. In concordance with this fact, soluble NADPH dependent O2- forming enzyme(s) were activated several fold by PMA. PMA also induced DNA strand breaks, a process markedly inhibited by TMU. As expected, ADP-ribosyl transferase (ADPRT), which is well known to require DNA strand breaks for its enzymatic activity, was activated by PMA treatment. In addition, specific inhibitors for ADPRT, namely 3-amino-benzamide and nicotinamide, inhibited PMA-induced LT production. Taken together, these three successive events, activation of soluble NADPH dependent O2- forming enzyme(s), DNA strand breaks and activation of ADPRT, may be required for PMA-induced LT production by AC5-8.