Lyb-8.2: A new B cell antigen defined and characterized with a monoclonal antibody

A DBA/1 B10.D2-specific monoclonal antibody (CY34) is described which defines a new murine B lymphocyte differentiation antigen designated Lyb-8.2. The ontogeny, strain distribution, and cell-surface density of the antigen were studied by radioimmunoassay and by fluorescence-activated cell sorter (FACS) analysis. Lyb-8.2 appears to be expressed on pre-B cells and on all mature B lymphocytes. Lyb-8.2 molecules immunoprecipitated from surface labeled B10.D2 spleen cells migrated in polyacrylamide gels with an apparent mol. wt. of 95000–105000 daltons and were bound by lentil lectin. The expression of Lyb-8.2 is controlled by a locus on chromosome 7 that is closely linked to Gpi-1 and RP-2. Added Lyb-8.2-specific antibody did not measurably impair B lymphocyte function in several in vitro systems studied.     

Induction of B lymphocyte proliferation by monoclonal anti-Lyb 2 antibody

Monoclonal antibody to Lyb 2, a differentiation antigen present on all B cells, has been used to study the role of Lyb 2 molecules in B cell activation. Monoclonal anti-Lyb 2 antibody (m-anti-Lyb 2) transforms resting B cells into blast cells and induces proliferation in these activated B cells. The proliferative response to anti-Lyb 2 is a property of the Lyb 5+ subset of B cells, since the antibody fails to stimulate B cells from mice expressing the CBA/N immune defect. B cells activated by anti-Lyb 2 mature into antibody-secreting cells in the added presence of T cell-replacing factors contained in the supernatants from concanavalin A-activated T cells. Thus, Lyb 2 molecules may participate in the delivery of stimulatory signals during the early phases of B lymphocyte activation.     

A specific defect in the proliferative capacity of B cells from old mice stimulated with autoreactive T cells

B lymphocytes from aged mice were found to be defective in their ability to proliferate in response to stimulation with an autoreactive T cell clone D1.4. The differentiative response leading to antibody secretion was also impaired in the auto D1.4 T cell-stimulated B cells from old mice in comparison to similarly stimulated B cells from young mice. The B cells from old mice were competent in activating the autoreactive T cells such that the T cells were induced to proliferate. The B cell defect appears to be restricted to a certain phase of B cell activation, since old mouse B cells responded to the auto D1.4 T cells by increasing cell surface Ia as well as size, but failed to incorporate tritiated thymidine. The responsiveness to interleukin-4 was found to be similar between B cells from young and old mice. It appeared that the B cells from old mice are specifically defective in progressing from the G0 phase of cell cycle into the S phase when stimulated with the auto D1.4 T cells.     

Influence of cyclic AMP on DNA synthesis in slices of regenerating rat liver.

DNA synthesis in slices of regenerating rat liver is inhibited by adenosine cyclic 3',5'-monophosphate [cAMP]. The number of cells synthesizing DNA as assayed by 2-14C-thymidine incorporation is reduced by 65% in the presence of 10(-3) M cAMP. The inhibition of cAMP is not specific; other adenosine compounds, N6,O2,-dibutyryl adenosine 3',5'-monophosphate, 5'AMP and adenosine have the same effect. Moreover, the concentration of cAMP in the cell required for this inhibition is much higher than the normal levels of cAMP in liver cells.     

Biological nitrification inhibition (BNI) activity in sorghum and its characterization

Aims

The ability to suppress soil nitrification through the release of nitrification inhibitors from plant roots is termed ‘biological nitrification inhibition’ (BNI). Here, we aimed at the quantification and characterization of the BNI function in sorghum that includes inhibitor production, their chemical identity, functionality and factors regulating their release.

Methods

Sorghum was grown in solution culture and root exudate was collected using aerated NH₄Cl solutions. A bioluminescence assay using recombinant Nitrosomonas europaea was employed to determine the BNI activity. Activity-guided chromatographic fractionation was used to isolate biological nitrification inhibitors (BNIs). The chemical structure was analyzed using NMR and mass spectrometry; pH-stat systems were deployed to analyze the role of rhizosphere pH on BNIs release.

Results

Sorghum roots released two categories of BNIs: hydrophilic- and hydrophobic-BNIs. The release rates for hydrophilic- and hydrophobic- BNIs ranged from 10 to 25 ATU?g^?1 root dwt. d^?1. Addition of hydrophilic BNIs (10 ATU?g^?1 soil) significantly inhibited soil nitrification (40 % inhibition) during a 30-d incubation test. Two BNI compounds isolated are: sakuranetin (ED₈₀ 0.6 μM; isolated from hydrophilic-BNIs fraction) and sorgoleone (ED₈₀ 13.0 μM; isolated from hydrophobic-BNIs fraction), which inhibited Nitrosomonas by blocking AMO and HAO enzymatic pathways. The BNIs release required the presence of NH ₄ ⁺ in the root environment and the stimulatory effect of NH ₄ ⁺ lasted 24 h. Unlike the hydrophobic-BNIs, the release of hydrophilic-BNIs declined at a rhizosphere pH >5.0; nearly 80 % of hydrophilic-BNI release was suppressed at pH ≥7.0. The released hydrophilic-BNIs were functionally stable within a pH range of 5.0 to 9.0. Sakuranetin showed a stronger inhibitory activity (ED₅₀ 0.2 μM) than methyl 3-(4-hydroxyphenyl) propionate (MHPP) (ED₅₀ 100 μM) (isolated from hydrophilic-BNIs fraction) in the in vitro culture-bioassay, but the activity was non-functional and ineffective in the soil-assay.

Conclusions

There is an urgent need to identify sorghum genetic stocks with high potential to release functional-BNIs for suppressing nitrification and to improve nitrogen use efficiency in sorghum-based production systems.