The epithelium overlying rabbit bronchus-associated lymphoid tissue does not express the secretory component of immunoglobulin A

Summary The epithelium associated with lymphoid aggregates in the bronchial tract (BALT) was studied in rabbits by immunohistochemistry using monoclonal antibodies against the secretory component (SC) of IgA. The normal bronchus epithelium was intensely labelled. In contrast, epithelium overlying the central parts of the follicles was negative. This specialized epithelium cannot participate in the SC-mediated transport of IgA, which might be a basis for the adherence and transport of microorganisms into the lymphoid tissue, thus initiating immune responses of the BALT.     

O-ribosyl-phosphate purine as a constant modified nucleotide located at position 64 in cytoplasmic initiator tRNAs(Met) of yeasts.

The unknown modified nucleotide G*, isolated from both Schizosaccharomyces pombe and Torulopsis utilis initiator tRNAs(Met), has been identified as an O-ribosyl-(1"----2')-guanosine-5"-phosphate, called Gr(p), by means of HPLC, UV-absorption, mass spectrometry and periodate oxidation procedures. By comparison with the previously published structure of Ar(p) isolated from Saccharomyces cerevisiae initiator tRNA(Met), the (1"----2')-glycosidic bond in Gr(p) has been postulated to have a beta-spatial conformation. The modified nucleotide Gr(p) is located at position 64 in the tRNA(Met) molecules, i.e. at the same position as Ar(p). Since we have also characterized Gr(p) in Candida albicans initiator tRNA(Met), the phosphoribosylation of purine 64 can be considered as a constant nucleotide modification in the cytoplasmic initiator tRNAs(Met) of all yeast species so far sequenced. Precise evidence for the presence of Gr(p) in initiator tRNAs(Met) of several plants is also reported.     

Evidence that the 5'-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4G

The efficiency of translation of alfalfa mosaic virus (AMV) RNA 4, barley alpha-amylase (B alpha A) mRNA, and two chimeric mRNAs, AMV 4-B alpha A and B alpha A-AMV 4 (in which the 5' leader sequences of the two mRNAs were interchanged), was measured in an S30 extract from wheat germ and a fractionated system from wheat germ in which translation could be made dependent upon initiation factor (eIF) 3, 4A, 4F, or 4G. In the S30 system, AMV RNA 4 and the chimeric mRNA AMV 4-B alpha A are translated much more efficiently than B alpha A mRNA and the chimeric mRNA B alpha A-AMV 4. When the S30 system was supplemented with high amounts of purified eIF-3, eIF-4A, eIF-4F, and eIF-4G, B alpha A and B alpha A-AMV 4 mRNAs were translated as efficiently as AMV RNA 4 and AMV 4-B alpha A mRNA. These findings indicated that the mRNAs containing the B alpha A leader sequence required higher amounts of one or more of the initiation factors (eIF-3, eIF-4A, eIF-4F, and eIF-4G) for efficient translation. Determination of the amounts of the initiation factors required for translation in the fractionated system showed that AMV RNA 4 required 2-4-fold lower amounts of eIF-3, eIF-4A, eIF-4F, and eIF-4G than did B alpha A mRNA. Replacement of the B alpha A leader sequence with that of AMV RNA 4 decreased the amounts of eIF-4A, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4F required. Replacement of the AMV RNA 4 leader sequence with that of B alpha A mRNA increased the amounts of eIF-4F, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4A required. These data strongly suggest that the amounts of the factors required are affected not only by the 5' leader itself but also by interactions between the 5' leader and a region(s) of the mRNA 3' to the initiation codon.     

Biological activity and receptor binding of human prointerleukin-1 beta and subpeptides

We report here that the human interleukin-1 beta precursor (proIL-1 beta) protein as well as several interleukin-1 beta (IL-1 beta) subpeptides bind cellular receptors specifically and exhibit biological activity by stimulating proliferation of helper T-cells. IL-1 beta polypeptides have been synthesized by in vitro translation of mRNAs transcribed from plasmid vectors containing the bacteriophage SP6 promoter joined to the complete IL-1 beta cDNA or to deletion constructs. The quantity of IL-1 beta in vitro translation products was increased significantly by replacing the cognate IL-1 beta untranslated leader sequence with a 37-nucleotide plant viral untranslated leader. Translation of chimeric mRNAs followed by direct bioactivity assay demonstrated that mature IL-1 beta-(117-269), proIL-1 beta-(1-269), and peptide IL-1-(71-269) were all biologically active. Specific binding to cellular receptors was observed with these three IL-1 beta molecules; moreover, several peptides with minimal biological activity also bound receptor specifically. The biological activity and receptor binding properties of the IL-1 beta proteins reported here contrast with those described by Mosley et al. (Mosley, B., Urdal, D. L., Prickett, K. S., Larsen, A., Cosman, D., Conlon, P. J., Gillis, S., and Dower, S. K. (1987) J. Biol. Chem. 262, 2941-2944; Mosley, B., Dower, S. K., Gillis, S., and Cosman, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4572-4576), who reported that proIL-1 beta-(1-269) had no biological activity and does not bind receptor. Our results indicate that proIL-1 beta is active at a relatively high concentration, and analysis of the proIL-1 beta-(1-269) and IL-1-(71-269) bioactivity data suggests a possible relationship with membrane-bound IL-1.     

DNA methylation is not increased in mouse-human somatic cell hybrids

The level of DNA methylation in three mouse-human cell lines that retained different human chromosomes and in the parental mouse and human lines has been determined by high-pressure liquid chromatography (HPLC). The level of methylation is similar in the hybrid and parental cells, indicating that interspecific somatic cell hybridization followed by preferential chromosome segregation can occur without an increase in overall DNA methylation.     

Protein isolation by solution-controlled gel sorption.

Illustrated are the principles for isolating proteins from solution by sorption into a polymer gel phase, driven by the addition of a water-soluble polymer to the protein solution. The separation is shown to be analogous to conventional two-phase aqueous extraction. However, the use of a gel phase rather than a solution for absorbing the protein makes separation of the protein from the polymer and the recycling of the gel phase much simpler. The model system used was linear poly(ethylene glycol) (PEG) and dextran gel. Increasing the molecular weight and concentration of the PEG favored sorption by the gel of ovalbumin, bovine serum albumin, cytochrome c, and hemoglobin. The proteins could be quantitatively recovered by immersing the gel in PEG-free solution.     

Multiple amino acid substitutions suggest a structural basis for the separation of biological activity and receptor binding in a mutant interleukin-1 beta protein.

Receptor binding and biological activity properties of human interleukin-1 beta can be dissociated by mutating a single amino acid, arginine 127, to glycine (IL-1 beta R----G) [Gehrke et al. (1990) J. Biol. Chem. 265, 5922-5925]. The mechanism underlying the reduced biological activity has been examined by replacing arginine 127 with several other amino acids, followed by determination of biological activity using a T-helper cell proliferation assay. Mutant IL-1 beta proteins containing lysine, glutamic acid, tryptophan, or alanine in place of arginine 127 maintain biological activity. These data strongly suggest that IL-1 beta biological activity is not directly dependent upon the specific properties of charge, hydrophobicity/hydrophilicity, or side-chain group presented by the residue at position 127. Molecular modeling analyses indicate that the structural integrity of the antiparallel beta-strand 1/12 pair is disturbed in the glycine 127 mutant protein. Collapse of beta-strand 1 into a hydrated space between strands 1, 2, and 4 could structurally alter a cleft in IL-1 beta that contains a cluster of highly conserved amino acids, including a key aspartic acid residue [Ju et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2658-2662]. Mutagenesis data and the differential activities of the IL-1 beta R----G and IL-1 receptor antagonist proteins in stimulating early and late gene expression [Conca et al. (1991) J. Biol. Chem. 266, 16265-16268] suggest that multiple receptor-ligand contacts, exclusive of those required for receptor binding, are required for the stimulation of full IL-1 biological activity.     

Diaminopimelic Acid Synthesis in Cultures of an Escherichia coli Double Auxotroph: Effects of Cultural Conditions

The metabolic response to L-lysine of Escherichia coli ATCC 13002, a lysine-histidine double auxotroph, has been examined in a synthetic medium containing sucrose. In shaken cultures largest amounts of extracellular DAP were produced with an initial lysine concentration of 7·5 mg/1 and in static cultures of 2·5 mg/1. Considerably smaller amounts of DAP accumulated under stationary conditions. In cultures shaken for 20 and 43 h there was an overall decrease in the yields of DAP, expressed in terms of cell biomass and of sucrose consumed, as the initial concentration of lysine was increased from 0·75 mg/1 in steps up to 25 mg/1. The regulatory effect of lysine on DAP production was also observed when lysine was supplied to cultures at a constant rate employing diffusion capsules.