Gene Literature Dashboard

In vitro synthesis of an antigen-specific T cell suppressor factor (TsF) has been accomplished by using partially purified poly(A)-containing RNA in a rabbit reticulocyte lysate cell-free translation system. The poly(A)-containing mRNA was isolated from a cloned T cell hybridoma that constitutively produces a TsF specific for the synthetic polypeptide antigen poly-(LGlu60LAla30LTyr10) (GAT). The RNA was fractionated by size and translated in vitro. The 16S RNA fraction stimulated synthesis of a biologically active protein that specifically suppressed both the GAT-specific antibody response by spleen cells in vitro and the proliferation response to GAT by lymph node T cells from GAT-primed mice. Further, the suppressor factor had a binding site for GAT, a determinant encoded by the I subregion of the major histocompatibility complex (MHC), and an apparent Mr 19,000 estimated by functional assays on protein separated by NadodSO4/polyacrylamide gel electrophoresis. These results indicate that virtually no posttranslational modifications (other than proteolytic cleavage) are necessary to obtain biologically active TsF. Hence, the presence of carbohydrate or other chemical groups does not contribute to either the serological properties of GAT-TsF or its biological properties.

Antithrombin III (AT III), the primary inhibitor of plasma protease coagulation proteins, was evaluated in groups of patients with different neoplastic disease. When compared with that of 25 health subjects, the mean value of AT III biologic activity was elevated in all groups, significantly in gastrointestinal (p less than 0.02) and lung (p less than 0.001) tumors, lymphoma (p less than 0.02) and in the group with various primary cancers (p less than 0.05). No difference was found between 46 patients tested under chemoradiotherapy and 216 patients who had not undergone therapy for at least 1 month. Four patients with clinical thrombosis had normal ATIII biologic activity. Of the 262 patients studied, 14 had low AT III biologic activity without clinical thrombosis and normal protein concentration. In 71 patients (27%) an excess of immunoassayable protein of 20% or more over biologic activity was found. Double immunoelectrophoresis performed in 5 of these showed for 3 patients an enlarged second are of precipitation. Moreover, no correlation was observed between the activity and protein AT III concentration (r = 0.05). The likelihood of the presence of circulating AT III-protease complexes is discussed.

T lymphocytes express a unique I-A subregion-controlled surface molecule that is not expressed on B lymphocytes. We produced antisera and monoclonal antibodies recognizing this structure. Exhaustive B cell adsorption (A.TH X B10.HTT)F antiserum, produced against activated A.TL T cells, left antibodies that bind an I-Ak specificity on some B10.A(4R) T lymphocytes (11 +/- 2%, mean +/- SEM). Similarly, exhaustive B cell adsorption of (A/J X B10.MBR)F antiserum, produced against activated B10.A(5R) cells, left antibodies specific for an I-Ab determinant on B10.A(5R)T cells (17 +/- 2%). We fused A.TL-immune (A.TH X B10.HTT)F splenocytes with NS-1 myeloma cells and identified antibody-producing hybrid cells by a fluorescent enzyme-linked immunosorbent microassay described herein. Eight monoclonal antibodies were selected; these lyse 7--26% of peripheral T cells from I-Ak strains. Thymocytes and bone marrow cells do not express the I-Ak T cells determinant. Exhaustive B cell adsorption did not remove I-Ak T cell-specific monoclonal antibodies.

The hexapeptide N-alpha-acetylalanylalanyl-lysyl-p- nitrophenylalanylalanylalanylamide has been synthesized and was found to be a good substrate for fungal aspartic proteinases that possess trypsinogen-activating activity, namely penicillopepsin, Rhizopus aspartic proteinase, Endothia aspartic proteinase and the aspartic proteinases from Aspergillus oryzae and Penicillium roqueforti. The peptide is rapidly cleaved between the lysine and p-nitrophenylalanine residues. Calf chymosin and human renin cleave the same bond, but only very slowly. The cleavage is accompanied by an absorbance decrease with a maximum at 296nm (Deltaepsilon -1800m(-1).cm(-1)). Pig pepsin and the aspartic proteinases from two Rhizomucor species cleave the peptide slowly on the carboxy side of p-nitrophenylalanine. For the five enzymes that hydrolysed the peptide rapidly, K(m) values range from 0.16 to 0.42mm and k(cat.) from 6 to 46.6s(-1) at pH 4.5 and 25 degrees C. A comparison of the kinetic parameters of the hexapeptide with those of the dipeptide N-alpha-acetyllysyl-p-nitrophenylalanylamide obtained with penicillopepsin shows that at pH 6.0 the catalytic rate constant k(cat.) is over 5000-fold greater for the hexapeptide, whereas the K(m) values are essentially the same, showing that the catalytic efficiency is strongly dependent on secondary binding. The new substrate with a p-nitrophenylalanine residue in the P'(1) position has advantages over previously used substrates for aspartic proteinases in that it offers a more sensitive spectrophotometric assay that is independent of pH up to 5.5 and can readily be used up to pH 7.0. The presence of lysine makes it very water-soluble. Stopped-flow spectrophotometric experiments with penicillopepsin gave clear evidence that the hydrolysis of the substrate by penicillopepsin is not accompanied by a ;burst' release of p-nitrophenylalanylalanylalanylamide.