Role of glutamic acid 177 of the ricin toxin A chain in enzymatic inactivation of ribosomes.

The gene for the A chain of ricin toxin was fused to a beta-galactosidase marker cistron via a DNA sequence encoding a short collagen linker, and the tripartite fusion protein was expressed in Escherichia coli. Site-specific mutagenesis was used to change glutamic acid residue 177 to aspartic acid or alanine. When the mutant proteins were expressed, purified, and tested quantitatively for enzymatic activity, the carboxylate function at position 177 was found not to be absolutely essential for ricin toxin A-chain catalysis.     

Immune response gene control of antibody specificity

The expression of the histocompatibility-linked PLL Ir gene was investigated in guinea pig B cells. Strain 2 and F₁ (2 × 13) guinea pigs, immunized with the αDnp-Lys₉, produce both T cells and antibody which are equally discriminatory for αDnp-Lys₉. In contrast strain 13 (PLL Ir gene negative) guinea pigs immunized with αDnp-Lys₉ do not develop specific T-cell responses and the antibody produced while restricted in heterogeneity cannot differentiate the immunizing antigen from Dnp-OH. However, if in a F₁ (2 × 13) environment, PLL Ir gene-negative B cells are provided with F₁ (2 × 13) T cells they express the ability to make antibody as specific and discriminatory as the antibody produced by PLL Ir gene-positive B cells. These findings strongly suggest that in the guinea pigs the PLL Ir gene defect is localized to the T cells and that the repertoire of specificity of B cells is similar if not identical in both responder and nonresponder animals. In addition these observations support the notion that the cellular locus for the PLL Ir gene expression in the guinea pigs is limited to T cells and not to macrophages and B lymphocytes.     

Microsomal sn-glycerol 3-phosphate and dihydroxyacetone phosphate acyltransferase activities from liver and other tissues. Evidence for a single enzyme catalizing both reactions.

Liver microsomal cytochrome P-448 purified from 3-methylcholanthrene-treated rats or rabbits contained seven free sulfhydryl groups per mole of enzyme as determined by amino acid analysis or by spectrophotometric titrations with 5,5′-dithiobis(2-nitroben-zoic acid), 4,4′-dipyridinedisulfide, 2-nitro-5-thiocyanobenzoic acid, and p-mercuribenzoate. The rat cytochrome P-448-catalyzed hydroxylation of benzo[a]pyrene was inhibited 70% after modification of the enzyme with 5,5′-dithiobis(2-nitrobenzoic acid) but was unaffected after titration of the enzyme with other sulfhydryl reagents, suggesting that the sulfhydryl groups may not be essential for catalysis. On the other hand, the rabbit cytochrome P-448-catalyzed hydroxylation of benzo[a]pyrene was inhibited following the modification of this enzyme with all of the sulfhydryl reagents listed above. Whether the loss in catalytic activity in this case is due to the essential role of the sulfhydryl groups in catalysis or to the steric hindrance or conformational change due to the substituent is uncertain.     

Natural killer-like activity mediated by activated T lymphocytes

Diverse types of lymphocytes mediate in vitro cytotoxic activity. In addition to CTLs (cytotoxic T lymphocytes) and NK (natural killer) cells which differ in their activation requirements, target specificities, and lytic mechanisms, a natural killer-like activity of activated cells (A-NK) has recently been described. The data presented here suggest that an activated T lymphocyte can mediate A-NK activity. A-NK activity can be separated from resting NK activity by its requirement for activation and an effector phenotype (T12+,Ia+,Mol-) which includes the presence of the T12 and Ia antigens and the absence of the Mol antigen. In contrast, resting NK activity is mediated by T12-,Ia-,Mol+ cells. Cells that mediate A-NK activity can be differentiated from CTLs by their differing kinetics of activation and susceptibility to inhibition by monoclonal antibodies. An additional distinguishing feature is the fact that A-NK cells are predominantly Ia+ and are derived from either the T4+ or T8+ T-cell subsets whereas CTLs generated under similar conditions are predominantly T8+,T4-,Ia-. The in vivo relevance of this newly defined T-cell cytolytic activity remains to be defined.     

CD45 isoforms associated with distinct functions of CD4 cells derived from unusual healthy donors lacking CD45RA- T lymphocytes.

We now report two healthy individuals whose T lymphocytes were over 95% positive for CD45RA antigen expression. However, these donors normally expressed both the CD29 high (CD29+) and CD45RO high (CD45RO+) antigens on approximately 40 and 50% of their CD4 cells, respectively. Despite the strong CD45RA expression on the surface of almost all CD4 cells, the CD29 marker allowed T cells from these donors to be divided phenotypically into subsets having distinct in vitro function. CD4+CD29+ cells from these donors responded maximally to recall antigens such as TT and provided strong helper function for B cell Ig synthesis. In contrast, CD4+CD29- cells responded poorly to recall antigens and had poor helper function for B cell Ig synthesis, but had strong suppressor activity. Thus, CD29 antigen expression was still predictive of the in vitro functional activity as previously described for normal donors. Furthermore, biochemical analysis of the distribution of individual CD45 isoforms on the surface of these subsets of CD4 cells revealed distinct differences. The CD4+CD29 high (CD4+CD29+) subset of cells primarily expressed the 180-, 190-, and 205-kDa CD45 isoforms, while the CD4+CD29 low (CD4+CD29-) cells primarily expressed the 190-, 205-, and 220-kDa CD45 isoforms. These results suggest that despite the superficial phenotypic similarity of CD4 cells in these donors, distinctions in the distribution of both CD29 and the 180- and 220-kDa CD45 isoforms exist and might play a role in the different functions of freshly isolated CD4 lymphocytes.     

B5, a new B cell-restricted activation antigen

The characterization of a new human B cell-restricted activation antigen (B5) is described in this report. With the use of a monoclonal antibody to B5, we show that B5 can be detected on peripheral blood or splenic B cells after 1 day of stimulation with either anti-immunoglobulin, protein A, Epstein Barr virus, or pokeweed mitogen. In contrast, B5 was not expressed on resting B, T, or myeloid cells. More important, B5 could not be detected on activated T cells or monocytes. The B5 antigen was expressed on some lymphoblastoid B cell lines and B cell neoplasms but was not expressed on leukemias or lymphomas of T or myeloid origin. The B5 antigen is distinct from previously reported B cell activation antigens by its m.w. and pattern of cellular expression. These studies suggest that B5 is a novel B cell-restricted activation antigen, which may be useful to study the events of early human B cell activation.     

Common tree shrews and primates share leukocyte membrane antigens

Monoclonal antibodies reactive with human peripheral blood lymphocyte and myeloid cell surface antigens were utilized to study the phylogeny of the common tree shrew. Blood cells from the common tree shrew, but not the bat or short-tailed shrew, react with certain of these antibodies. These data strengthen the argument that the Tupaiidae are primitive primates rather than insectivores. They also indicate that this approach should be useful for further work in taxonomic systemization.     

Phenotypic and functional distinctions between the TH2+ and JRA+ T cell subsets in man.

Prior work has demonstrated the existence of distinct human peripheral blood T cell subsets by utilizing heterologous as well as autoimmune antisera. In the present study, the relationship between the TH2+ and JRA+ T cell subsets was examined. T cells were purified with Sephadex G-200 anti-F(ab)2' affinity chromatography and E-rosetting technique, and subsequently fractionated into TH2+ and TH2- subsets by utilizing indirect immunofluorescence on FACS. Approximately 40 to 45% of the TH2- subset was shown to be JRA+, whereas less than 5% of the TH2+ subset was JRA+. In reciprocal studies, T cells were fractionated into JRA+ and JRA- subsets and reacted with heterologous antisera with anti-TH2+ specificity and indirect immunofluorescence. FACS analysis demonstrated that the JRA+ population contained no TH2+ T cells. In contrast, the JRA- population contained TH2+ T cells and accounted for the entire TH2+ subset found in the unfractionated T cell population. Functional studies showed that the TH2+ subset, and not the JRA+ subset, contain the effector population for cell-mediated lympholysis. It is concluded that the TH2+ and JRA+ T cell subsets define distinct and different T cell populations in man.