Estimation of symbiotically fixed nitrogen in field grown soybeans: An application of natural15N/14N abundance and a low level15N-tracer technique

Summary Plants from agricultural and natural upland ecosystem were investigated for¹⁵N content to evaluate the role of symbiotic N₂-fixation in the nitrogen nutrition of soybean. Increased yields and lower δ¹⁵N values of nodulating soybeansvs, non-nodulating isolines gave semi-quantitative estimates of N₂ fixation. A fairly large discrepancy was found between estimations by δ¹⁵N and by N yield at 0 kg N/ha of fertilizer. More precise estimates were made by following changes in plant δ¹⁵N when fertilizer δ¹⁵N was varied near¹⁵N natural abundance level. Clearcut linear relationships between δ¹⁵N values of whole plants and of fertilizer were obtained at 30 kg N/ha of fertilizer for three kinds of soils. In experimental field plots, nodulating soybeans obtained 13±1% of their nitrogen from fertilizer, 66±8% from N₂ fixation and 21±10% from soil nitrogen in Andosol brown soil; 30%, 16% and 54% in Andosol black soil; 7%, 77% and 16% in Alluvial soil, respectively. These values for N₂ fixation coincided with each corresponding estimation by N yield method. Other results include: 1)¹⁵N content in upland soils and plants was variable, and may reflect differences in the mode of mineralization of soil organics, and 2) nitrogen isotopic discrimination during fertilizer uptake (δ¹⁵N of plant minus fertilizer) ranged from −2.2 to +4.9‰ at 0–30 kg N/ha of fertilizer, depending on soil type and plant species. The proposed method can accurately and relatively simply establish the importance of symbiotic nitrogen fixation for soybeans growing in agricultural settings.     

Caffeine contracture in the cultured chick myotube

A possible function of Ca store site in cultured chick myotubes was examined by recording contraction of the myotube with special reference to the effect of caffeine. Caffeine at low concentrations (below 1 mM), applied focally on the myotube through a micropipette with a pressure pulse, elicited focal contraction without membrane potential changes. Procaine inhibited the caffeine contracture. Deuterium oxide also inhibited the caffeine contracture at low concentrations, but enhanced the maximal contracture. These observations are similar to those in the mature frog muscle fiber in which the sarcoplasmic reticulum (SR) is a main site of caffeine action. On the basis of these similarities, it was considered that caffeine acts on SR to elicit contracture in the myotube. The ability of SR to accumulate and release Ca ion seemed to be low, because caffeine contracture decreased or disappeared in a Ca-free solution in many myotubes.     

Influence of monovalent cation transport on anabolism of glycosphingolipids in cultured human fibroblasts

We have reported [Saito, M., Saito, M., & Rosenberg, A. (1984) Biochemistry 23, 1043-1046] that the monovalent cationic ionophore monensin reduced the incorporation of labeled galactose into oligosaccharidyl glycosphingolipids (globotriaosylceramide, globotetraosylceramide, and gangliosides) and induced a cellular accumulation of glucosyl- and lactosylceramide in cultured diploid human fibroblasts. We have undertaken further studies on the effects of monensin and made comparison with the effects of related monovalent cation transporters on plasma membrane glycosphingolipid anabolism in human fibroblasts. Our results demonstrate that ionic flux can markedly influence glycosphingolipid synthesis, and they indicate that, like glycoprotein, the sites of glycosylation of the initial, precursor glycosphingolipids are different from the sites of higher glycosylation. At a concentration of 10(-7) M, monensin induced the maximum inhibition of incorporation of labeled galactose into polyglycosyl sphingolipids: globotriaosylceramide, globotetraosylceramide, and gangliosides; increased incorporation of labeled galactose into glucosyl- and lactosylceramide was clearly evident, and their content rose measurably in the cell at concentrations of monensin as low as 10(-8) M. These effects of monensin were reversible. Incorporation of labeled galactose into higher glycosylated neutral glycosphingolipids and gangliosides slowly resumed, and the accumulated glycosylceramide diminished after removal of monensin from the culture medium. Ouabain (plasma membrane Na+,K+-ATPase inhibitor) and A23187 (Ca2+ ionophore) also caused a rapid increase in incorporation of labeled hexose into glucosylceramide and decreased its incorporation into higher neutral glycosphingolipids and into gangliosides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atrial natriuretic factor increases cyclic GMP and inhibits cyclic AMP in rat renal papillary collecting tubule cells in culture

The present study was undertaken to determine whether human atrial natriuretic factor (hANF) produces guanosine-3', 5'-monophosphate (cGMP) and alters arginine vasopressin (AVP)- and forskolin (F)- induced adenosine-3', 5'-monophosphate (cAMP) production in the cultured rat renal papillary collecting tubule cells. hANF increased cellular cGMP levels in a dose dependent manner. AVP and F, however, did not affect cGMP production. hANF significantly inhibited AVP- and F-stimulated cAMP levels, but hANF by itself did not affect cellular cAMP production. Since F activates adenylate cyclase at a step of catalytic unit and the cellular action of AVP to activate adenylate cyclase is mediated through receptor-catalytic units, the present results indicate that hANF may directly inhibit the AVP- and F-stimulated adenylate cyclase in renal papillary collecting tubules.     

A wasp venom mastoparan-induced polyphosphoinositide breakdown in rat peritoneal mast cells

The phospholipid metabolism of rat peritoneal mast cells stimulated with mastoparan, a secretagogue purified from wasp venom, was investigated. Mastoparan at 20 micrograms/ml caused a rapid secretion of histamine. Mastoparan induced a transient decrease of phosphatidylinositol 4,5-biphosphate on 32P labeling and generation of a water-soluble degradation product, inositol trisphosphate on [3H]inositol labeling, suggesting the activation of phospholipase C upon stimulation.     

Activation and stabilization of asparaginase by anti-asparaginase IgG and its Fab

Modified asparaginase, in which 4 tryptophan residues were modified with 2-hydroxy-5-nitrobenzyl bromide, had little enzymic activity and retained immunoreactivity [(1976) FEBS Lett. 65, 11-15]. Addition of IgG or its Fab towards asparaginase to the modified asparaginase gave rise to marked enhancement of the enzymic activity. Native asparaginase (4 subunits) lost the enzymic activity due to dissociation into subunits by dilution of the enzyme solution. However, in the presence of Fab, asparaginase did not lose enzymic activity on dilution, probably due to no dissociation into subunits occurring.     

Murinoglobulin, a novel protease inhibitor from murine plasma. Isolation, characterization, and comparison with murine alpha-macroglobulin and human alpha-2-macroglobulin

Two glycoproteins having trypsin-protein esterase activity were purified to apparent homogeneity from murine plasma. One was alpha-macroglobulin, a homologue of human alpha-2-macroglobulin, while the other, tentatively named murinoglobulin, did not correspond to any of the known plasma protease inhibitors that have been well characterized in men or other mammals. Murinoglobulin contained about 7.6% carbohydrate and was composed of a single-polypeptide chain of Mr = 180,000 as judged by the equilibrium sedimentation analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Murinoglobulin did not cross-react immunologically with mouse alpha-macroglobulin nor with human alpha-2-macroglobulin. Protease-inhibiting properties of murinoglobulin were compared with those of mouse alpha-macroglobulin and human alpha-2-macroglobulin. All the three proteins inhibited trypsin, papain, and thermolysin, although they differed considerably in both the degree of inhibition and the binding stoichiometry of protease-inhibitor complexes. The two macroglobulins inhibited pepsin at pH 5.5, whereas murinoglobulin was inactivated at this pH. Murinoglobulin was more sensitive to methylamine than the two macroglobulins. No protein corresponding to murinoglobulin was detected in human plasma.     

Molecular species of platelet-activating factor generated by human neutrophils challenged with ionophore A23187

Two species of platelet-activating factor (PAF), 1-hexadecyl- and 1-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16 = 0 AGEPC and C18 = 0 AGEPC) were detected in ionophore A23187-stimulated human neutrophils. The amount of AGEPC in 1 x 10(7) neutrophil cells was 80 +/- 26 pmol (mean +/- standard error) with a range of 14 to 223 pmol (n = 8), and it consisted of 80% of the C16 = 0 species and 20% of the C18 = 0 species. Most of the AGEPC derived from ionophore-treated neutrophils remained cell associated rather than being secreted into the medium, even when the medium contained ample albumin protein, which can trap AGEPC. These results were obtained by a technique of gas chromatography-mass spectrometry coupled with selected ion monitoring.     

The target minor H antigen for F1 cytotoxic T lymphocytes induced by Igh-congenic parental spleen cells is coded for by gene linked to H-2

Graft-vs-host reaction (GvHR) induced in (B10.BR X CWB)F1 (BWF1; H-2k/b, Ighb/b) by i.v. injection with CWB (H-2b, Ighb) spleen cells resulted in complete suppression of cytotoxic T lymphocyte (CTL) responsiveness of the F1 host spleen cells (GvHR-associated immunosuppression). In contrast, GvHR induced in BWF1 mice with CSW (H-2b, Ighj; Igh-congenic to CWB) spleen cells did not affect CTL responsiveness of the F1 host spleen cells at all. The BWF1 hosts undergoing the CSW-induced GvHR generated anti-CSW CTL in their spleens, and the subsequent culture of such BWF1 spleen cells with CSW stimulator cells, augmented the CTL activity. The BWF1 anti-CSW CTL lysed both Con A- and LPS-induced splenic blasts from mouse strains carrying the Ighj allele in the context of self H-2Kb. However, determination of the Igh haplotype in the serum IgG and of the susceptibility of the splenic lymphocytes to the BWF1 anti-CSW CTL on backcross mice, which carry either Ighb/j or Ighb/b in the context of H-2b/b or H-2b/k, showed clearly that Ighj and the gene coding for the target antigen for the BWF1 anti-CSW CTL segregated at ratios close to 1:1. The study in which linkage between the gene(s) coding for the target antigen for the BWF1 anti-CSW CTL and H-2 was examined on CWB X (C3H X CWB)F1 backcross mice and (B10.BR X CSW)F1 X B10 mice demonstrated that the gene, most likely a single gene, coding for the target antigen for the BWF1 anti-CSW CTL is located at 8.5 +/- 4.3 cross-over units to the right or left of the H-2 complex. We designated the minor H antigen to be recognized by the BWF1 anti-CSW CTL as H-X+, and we discuss the distinction between the H-X+ locus and the other minor H loci on chromosome 17.     

In vivo priming of mouse CTL precursors directed to product of a newly defined minor H-42 locus is under a novel control of class II MHC gene

In a previous study, we discovered a new mouse minor histocompatibility antigen encoded by a locus at 8.5 cM apart from the H-2 complex, and we have since named the locus H-42. One allele of H-42, which is named H-42a, had been elucidated, but the other alleles, which we tentatively named H-42b, have not been elucidated. In the present study, we explored MHC control on the anti-H-42a cytotoxic T lymphocyte (CTL) responsiveness in H-42b mice. In vivo immunization (i.v. injection) of H-42b mice with 5 to 30 X 10(6) spleen cells (SC) bearing allogeneic H-42a antigen but carrying H-2 complex (mouse MHC) matched with the H-42b mice failed to prime anti-H-42a CTL but induced stable and specific anti-H-42a CTL unresponsiveness, i.e., tolerance, in the H-42b recipient mice. In contrast, H-2 heterozygous H-42b F1 mice injected with SC bearing H-42a alloantigen on either of the parental H-2 haplotypes were effectively primed to generate anti-H-42a CTL. Exploration of the region or subregion in the H-2 complex of H-42a donor SC that should be compatible with H-42b recipient mice for the induction of their anti-H-42a CTL tolerance demonstrated that the compatibility at I region, most probably I-A subregion, but not at K, S, or D region, determined the induction of the tolerance. MHC class II compatible H-42a skin graft (SG) to H-42b mice, however, consistently primed the anti-H-42a CTL in the H-42b recipients. These results were discussed in several aspects, including uniqueness of MHC class II control on the CTL response to minor H-42a antigen, possibility of inactivation of responding anti-H-42a precursor CTL or helper T cells in H-42b mice by encountering the veto cells present in MHC class II-matched H-42a SC population, and significance of the present observations as a mechanism of CTL tolerance to self-components.