Human B cell lines can be triggered to secrete an interleukin 2-like molecule

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt's and non-Burkitt's types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 ng/ml) triggered IL-2 secretion in 7/8 (87%) EBV-negative lymphoma cell lines of American origin and in 6/6 (100%) normal lymphoblastoid cell lines, but in only 1/6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5/5 (0%) African Burkitt's cell lines. IL-2 secretion was not detected in control supernatants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS-1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tac monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti-tac monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation.     

Integration of phage Mu into the RP4::D3112A-plasmid in Escherichia coli cells

Hybrid plasmids obtained as a result of Mu phage insertions into the RP4::D3112 plasmid in Escherichia coli cells were studied. Stable maintenance of RP4::D3112 plasmid in E. coli cells was provided by using the D3112 phage genome with a point polar mutation in the A gene which prevented early genes' expression. The presence of D3112A- in the RP4 plasmid has been shown to have no effect on efficiency of phage Mu transposition into this plasmid. Moreover, RP4 and D3112 genomes were equivalent targets for Mu integration. The integration of transposable phage into genome of nonrelated phage can be used as one of the approaches to construct recombinant phage genomes in vivo in the absence of DNA homology.     

An evolutionary comparison of Acinetobacter calcoaceticus trpF with trpF genes of several organisms

The deduced amino acid sequence of Acinetobacter calcoaceticus N-(5'-phosphoribosyl) anthranilate isomerase (PRAI), which is coded by trpF, was compared with TrpF of Caulobacter crescentus, Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Neurospora crassa, and Aspergillus nidulans. Sixty percent of identical or similar amino acids were located in alpha/beta TIM (triose-phosphate isomerase) barrels and in residues important in substrate binding and catalysis. In addition, the analysis of trpF genes presented here supports a model by which fusion between separate trpC and trpF genes arose in some cases by in-frame deletions.     

The circadian rhythm of the N-terminus and C-terminus of the atrial natriuretic factor prohormone

Circadian variation in the circulating concentrations of the N-terminal and C-terminal portions of the atrial natriuretic factor prohormone (pro ANF) was evaluated in 8 men, ages 41-47, who have been followed for 19 years with respect to circadian variation in physiological variables including blood pressure and clinical chemistries. The N-terminus of the ANF prohormone contains two peptides consisting of amino acids 1-30 and 31-67 while the C-terminus contains 1 peptide (amino acids 99-126) of this 126 amino acid prohormone which lower blood pressure and have natriuretic properties. To determine if either the N-terminus and/or the C-terminus of the prohormone have a circadian variation in their circulating plasma concentrations these 8 men had blood samples obtained for radiommunoassay every 3 hr during a 24-hr period. Three radiommunoassays which immunologically recognize (1) the whole N-terminus (i.e. amino acids 1-98), (2) the midportion of the N-terminus (amino acids 31-67) and (3) the C-terminus (amino acids 99-126) of the ANF prohormone were utilized. The whole N-terminus, the midportion of the N-terminus which circulates after being proteolytically cleaved from the rest of the N-terminus, and the C-terminus each had a peak circulating concentration between 0400 and 0700 which were significantly (P less than 0.001) higher than their concentrations at any other time throughout the 24-hr period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of lymphocyte nuclear matrix organization in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole: an autoradiographic and immunofluorescence study

Assembly of active nuclei in lymphocytes stimulated by mitogen is paralleled by the elaboration of a structurally and biochemically complex nuclear matrix (NM). To examine the dynamics of individual NM polypeptide components during blastogenesis, we have applied immunofluorescence labelling with anti-NM antibodies to concanavalin A-stimulated mouse splenocytes. Whereas peripherin and PI2 antigens did not reorganize during stimulation, labelling of PI1 and small nuclear ribonucleoprotein (snRNP) antigens increased markedly in intensity and redistributed in concert with the previously reported NM restructuring. Double-labelling showed, furthermore, that snRNPs and the internal staining component of PI1 were largely co-localized. As an approach to studying the role of RNA and RNA synthesis in NM organization, we have further examined the effects of the inhibitor of RNA synthesis, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), on NM antigen distribution. The rapid inhibition of 3H-uridine incorporation by DRB was accompanied by coordinate aggregation of snRNPs and of the internal PI1 component into large, brightly stained patches. Both 3H-uridine incorporation levels and antigen localization were readily reversed upon removal of DRB. We conclude that NM antigens behave independently during nuclear and NM assembly and that NM organization, as reflected by NM antigen distribution, is modulated by con A- and DRB-induced alterations in RNA synthesis. We propose, furthermore, that the PI1 antigen plays a role in RNA metabolism, and is possibly involved in RNA transport to the nuclear periphery.     

Cellular proteins that associate with the middle and small T antigens of polyomavirus.

We have used two-dimensional gel electrophoresis to analyze in more detail the cellular proteins which associate with the middle and small tumor antigens (MT and ST, respectively) of polyomavirus. Proteins with molecular masses of 27, 29, 36, 51, 61, 63, and 85 kilodaltons (kDa) that specifically coimmunoprecipitated with MT were identified on these gels. The 36-, 51-, 61-, 63-, and 85-kDa proteins are probably the same as the proteins of similar sizes previously reported by a number of groups, whereas the 27- and 29-kDa proteins represent proteins that are heretofore undescribed. The 27- and 29-kDa proteins were abundant cellular proteins, whereas the others were minor cellular constituents. The association of each of these proteins with MT was sensitive to one or more mutations in MT that rendered it transformation defective. The association of the 85-kDa protein was the most sensitive indicator of the transformation competence of MT mutants. In addition, the 85-kDa protein was the only associated protein whose association with MT changed consistently in parallel with MT-associated phosphatidylinositol kinase activity. Furthermore, the fraction of the 85-kDa protein which was found associated with the MT complex contained 15 to 20% of its phosphate content on tyrosine. The 36- and 63-kDa proteins complexed with both polyomavirus MT and ST and comigrated on two-dimensional gels with two simian virus 40 ST-associated proteins originally described by Rundell and coworkers (K. Rundell, E. O. Major, and M. Lampert, J. Virol. 37:1090-1093, 1981). None of the other MT-associated proteins associated significantly with ST.     

Mutational analysis of upstream AUG codons of poliovirus RNA.

The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.     

Characterization of rat skeletal muscle sarcolemmal insulin receptors and a sarcolemmal insulin binding inhibitor

When insulin receptors of rat skeletal muscle sarcolemmal vesicles were solubilized with Triton X-100, the specific binding of 125I-labeled insulin increased by more than 10-fold over that seen in the intact vesicles. Partial purification of the skeletal muscle insulin receptors on wheat germ agglutinin affinity columns increased the total insulin binding activity by 7-fold and reduced the Kd for insulin binding from 1.92 to 0.20 nM, suggesting that an inhibitor of insulin binding was removed by this purification step. This was confirmed when the unbound fractions of the affinity column were dialyzed and reconstituted with the insulin receptors. The inhibitory activity in the sarcolemmal extract could not be accounted for by the presence of Triton X-100. The skeletal muscle inhibitor was more potent in inhibiting insulin binding to skeletal muscle insulin receptors than to liver or adipose receptors. The inhibitor was very effective in inhibiting insulin binding to wheat germ agglutinin-purified IM-9 receptors, but had negligible effects on insulin binding to intact IM-9 cells. The properties of the alpha and beta subunits of the skeletal muscle insulin receptors appear to be the same as those of insulin receptors of other tissues: cross-linking of 125I-labeled insulin to the receptor revealed a band of 130,000 daltons, and insulin stimulated the phosphorylation of bands of 90,000 and 95,000 daltons in the receptor preparation. The skeletal muscle insulin binding inhibitor elutes from molecular sieves in a major 160,000-dalton peak and minor 75,000-dalton peak. The binding inhibitor is not inactivated by heat, by mercaptoethanol, or by trypsin, pepsin, or proteinase K. Collectively, these data suggest that the inhibitor may be a small molecule that aggregates with itself, with larger proteins, or with detergent micelles.