Monoclonal antibodies inhibiting RNA polymerase from Escherichia coli

Monoclonal hybridoma antibodies directed against RNA polymerase from E. coli have been obtained. Only a few have been found to inhibit the enzyme activity. Antibodies produced by two clones, PYN-1 and PYN-2, inhibit RNA polymerase at the stage of RNA chain elongation. The PYN-1 antibodies react with the beta'-subunit of the enzyme. The PYN-2 antibodies react with the beta-subunit and with its 130 kDa amber fragment.     

Antigenic determinants of the constant region of human immunoglobin kappa-chain detected by monoclonal antibodies

Ten different monoclonal antibody (monAB) preparations reacting with human IgL chains of the kappa type have been obtained. Nine of the monAB interacted with the kappa-chain C domain, whereas only one monAB reacted with the V domain. It has been determined that monAB against the C domain react with three different epitopes. One epitope is expressed on intact Ig molecules as well as on isolated kappa-chains, whereas the other two epitopes are found only on isolated kappa-chains. The expression of these epitopes in 40 different myeloma kappa-chain preparations belonging to four various subgroups was studied. The level of this C domain epitope expression has been shown to depend on the variable subgroups of kappa-chains indicating a close association between V and C domains. This association leads to the alteration of antigenic activity of some C domain epitopes. The alterations are thought to be local because, as a rule, they involve only one of the three epitopes.     

Analysis of the frequency of allelic exclusion of immunoglobulin light chain genes and molecular characteristics of immunoglobulins secreted by hybridomas with expression of both allelic genes

The investigation of 750 B-lymphocyte hybridoma clones obtained by fusion of mouse myeloma and newborn heterozygous Igk-la/Igk-1b rat splenocytes has revealed that 9,8% of Ig kappa-chain genes are rearranged productively. Seventeen hybridomas secrete kappa-chains of both allelic variants. The analysis of IgM molecules of nine such clones demonstrated that in six cases only one L-chain allotype is present in IgM. Thus for the first time the high frequency of selective association of H and L chains was shown. Evidently this selectively may function as one of the allelic exclusion mechanisms at the Ig assembly stage.     

Seasonal variation in foliar carbon exchange in Pinus radiata and Populus deltoides: respiration acclimates fully to changes in temperature but photosynthesis does not

We investigated seasonal variation in dark respiration and photosynthesis by measuring gas exchange characteristics on Pinus radiata and Populus deltoides under field conditions each month for 1 year. The field site in the South Island of New Zealand is characterized by large day-to-day and seasonal changes in air temperature. The rate of foliar respiration at a base temperature of 10 °C ( R ₁₀) in both pine and poplar was found to be greater during autumn and winter and displayed a strong downward adjustment in warmer months. The sensitivity of instantaneous leaf respiration to a 10 °C increase in temperature ( Q ₁₀) was also greater during the winter period. The net effect of this strong acclimation was that the long-term temperature response of respiration was essentially flat over a wide range of ambient temperatures. Seasonal changes in photosynthesis were sensitive to temperature but largely independent of leaf nitrogen concentration or stomatal conductance. Over the range of day time growth temperatures (5–32 °C), we did not observe strong evidence of photosynthetic acclimation to temperature, and the long-term responses of photosynthetic parameters to ambient temperature were similar to previously published instantaneous responses. The ratio of foliar respiration to photosynthetic capacity ( R _d/ A _sat) was significantly greater in winter than in spring/summer. This indicates that there is little likelihood that respiration would be stimulated significantly in either of these species with moderate increases in temperature – in fact net carbon uptake was favoured at moderately higher temperatures. Model calculations demonstrate that failing to account for strong thermal acclimation of leaf respiration influences determinations of leaf carbon exchange significantly, especially for the evergreen conifer.     

Study of the antigenic structure of human immunoglobulin lambda-chain using monoclonal antibodies]

Nine monoclonals against human Ig lambda chains were produced, 4 antibodies react with C-domain, 5--with V-domain of the lambda chain. Anti-C lambda domain antibodies recognize not less than 3 epitopes and one of them is expressed only on the isolated chain. Anti-V lambda antibodies bind both isolated lambda chain and intact IgG, IgM, IgA. Four epitopes are expressed by few lambda Bence Jones proteins of the III subgroup, the immunogen possessing the same isotype. The 4 mentioned epitopes represent private idiotypic determinants. The epitope 3E10 is characteristic of 50% Bence Jones proteins of the II and III V lambda-subgroups thus representing a common idiotypic determinant. Using anti-V lambda antibodies germ line variability of V lambda III proteins was analysed and the similarity of antigenic structure of normal and myeloma human Ig lambda chains was demonstrated.     

Overexpression of BAD potentiates sensitivity to tumor necrosis factor-related apoptosis-inducing ligand treatment in the prostatic carcinoma cell line LNCaP

Here we show that LNCaP, which is resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, becomes sensitive to TRAIL after overexpression of full-length, wild-type BAD (BAD WT). TRAIL induces caspase-dependent cleavage of BAD WT that results in generation of a M(r) 15,000 protein. LNCaP stably expressing truncated BAD (tBAD) and cells expressing mutated BAD at the caspase cleavage site were less sensitive to TRAIL treatment when compared to LNCaP expressing BAD WT. Cytochrome c and Smac/DIABLO release from mitochondria into cytosol was found after TRAIL treatment only in cells overexpressing BAD WT. Furthermore, differences in phosphorylation of serine residues for BAD WT and tBAD were identified. BAD WT was phosphorylated at positions S136 and S155, whereas tBAD was phosphorylated at positions S112, S136, and S155. LNCaP stably expressing BAD mutated at serine 112 to alanine was less sensitive to TRAIL treatment when compared to LNCaP expressing BAD WT. Lastly, recombinant BAD cleaved by caspase-3 is a more potent inducer of cytochrome c and Smac/DIABLO release than BAD WT. In summary, BAD-mediated sensitivity of LNCaP to TRAIL depends on the phosphorylation status of BAD WT and tBAD.     

Appearance of Neoantigen in Mouse IgG1 Upon Reduction of Interchain Disulfide Bridges: Assessment of Local and General Conformational Rearrangements Using Monoclonal Antibody and Small-angle X-ray Scattering

Abstract

Mouse hybridoma antibody E5D2 reacting with murine mono- and polyclonal IgG1 has been produced. MonAb E5D2 recognizes the antigenic determinant (epitope) buried in intact IgG1 and expressed upon mild reduction of interchain S-S bridges. Neither H nor L chains alone maintain epitope E5D2. Reassociation of gamma 1 chains (H chains of IgG1) with L chains results in complete restoration of this antigenic determinant. The data strongly suggest that epitope E5D2 depends on the quaternary structure of IgG1. The epitope is also expressed by reduced F(ab)₂ fragment of IgG1 but is not connected with its antigen binding site. The likely localization of the epitope E5D2 is the interface between C_H and C_L domains. The second produced monAb F6C2 reacts with C_H1-C_L region of reduced mouse IgG2.

Small-angle X-ray scattering experiments have demonstrated pronounced decrease of the radius of gyration of reduced IgG1 as compared to the intact one. This indicates general conformational changes of IgG1 molecule following mild reduction of Fab region S-S groups. Epitope E5D2 is the first quaternary antigenic subclass specific determinant described for C the region of mouse IgG. Thus, serologic expression of epitope E5D2 reveals precise conformational perturbations of small area near reduced S-S bridges while small-angle scattering demonstrates accompanying general transformation of IgG structure.