Kirromycin, a new inhibitor of protein synthesis, is shown to interfere with the peptide transfer reaction by acting on elongation factor Tu (EF-Tu). All the reactions associated with this elongation factor are affected. Formation of the EF-Tu·GTP complex is strongly stimulated. Peptide bond formation is prevented only when Phe-tRNA Phe is bound enzymatically to ribosomes, presumably because GTP hydrolysis associated with enzymatic binding of Phe-tRNA Phe is not followed by release of EF-Tu·GDP from the ribosome. This antibiotic also enables EF-Tu to catalyze the binding of Phe-tRNA Phe to the poly(U)·ribosome complex even in the absence of GTP. EF-Tu activity in the GTPase reaction is dramatically affected by kirromycin: GTP hydrolysis, which normally requires ribosomes and aminoacyl-tRNA, takes place with the elongation factor alone. This GTPase shows the same K m for GTP as the one dependent on Phe-tRNA Phe and ribosomes in the absence of the antibiotic. Ribosomes and Phe-tRNA Phe , but not tRNA Phe or Ac-Phe-tRNA Phe , stimulate the kirromycin-induced EF-Tu GTPase. These results indicate that the catalytic center of EF-Tu GTPase that is dependent upon aminoacyl-tRNA and ribosomes is primarily located on the elongation factor. In conclusion, kirromycin can substitute for GTP, aminoacyl-tRNA, or ribosomes in various reactions involving EF-Tu, apparently by affecting the allosteric controls between the sites on the EF-Tu molecule interacting with these components.

We have investigated the mechanism which allows the antibiotic kirromycin to inhibit peptide bond formation by acting on elongation factor Tu (EF-Tu). Gel filtration experiments show that with kirromycin after enzymic binding of aminoacyl-tRNA to ribosomes and subsequent GTP hydrolysis EF-Tu is not released from the ribosome. However, ability of the ribosomal peptidyl transferase center to support puromycin reactivity of peptidyl-tRNA bound to the ribosomal donor site is still preserved. This shows that inhibition of peptide bond formation by kirromycin is the consequence of the failure of EF-Tu to leave the ribosome, which blocks interaction of the C-C-A end of aminoacyl-tRNA bound to the acceptor site with the peptityl-transferase center.In line with these observations, kirromycin inhibits ribosome . EF-Tu GTPase activity down to one round of GTP hydrolysis when poly(U) and Phe-tRNAPhe are simultaneously present. The intrinsic inability of EF-Tu bound to ribosomes to interact in a productive way with free GTP appears to be a primary reason for inhibition of the turnover of GTPase activity in this condition.In the absence of poly(U), ribosomes are very active in stimulating the kirromycin-induced EF-Tu GTPase either with or without Phe-tRNAPhe, which increases their affinity for the reaction. When Phe-tRNAPhe is present, kirromycin enables either ribosomal subunit to stimulate GTP hydrolysis of EF-Tu, showing that each subunit harbours an active site for the regulation of the catalytic center of EF-Tu. Addition of poly(U) plus Phe-tRNA'"' inhibits the turnover of the kirromycin-induced GTP hydrolysis in the presence of the 30-S subunits, but not of that in the presence of the 50-S subunit, which is inhibited when the 30-S subunit is also added. The 30-S subunit alone appears therefore to be sufficient for formation of a stable complex with EF-Tu and kirromycin. Our results show that expression of EF-Tu GTPase activity coupled with polypeptide synthesis is the result of an highly specific and coordinated mechanism, in which both ribosomal subunits participate and act on the catalytic center of the elongation factor.No kirromycin-like activity was found in the two fragments of the antibiotic molecule, obtained by acid hydrolysis. None of the partial reactions of polypeptide synthesis tested, dependent on elongation factor G, initiation factors or aminoacyl-tRNA-synthetases, appeared to be affected by kirromycin.In the preceding paper [I] we have studied the effect of the antibiotic kirromycin on the EF-Tudependent reactions in the absence of ribosomes ; in this communication we try to explain the mechanism which allows kirromycin to block peptide bond formation ~ in polypeptide synthesis by acting on the Abhreviarions. EF-Tu, elongation factor Tu; EF-Ts, elongation factor Ts; EF-T, the complex formed by the elongation factors Tu and Ts; EF-G, elongation factor G ; IF-2, initiation factor 2; AcPhe, M-acetylphenylalanine; tRNAPhe, phenylalanine acceptor, tRNA; Guo-5'-P2-CH2-P, guanosine 5'-(~,y-methylene)triphosph...

The antigen-presenting cell system in the gastrointestinal tract, one of three main sites (skin and lung being the others) of primary antigen contact, is poorly understood. Our study focused on dendritic cells (DC) as possible candidates for antigen uptake, processing and presentation in mucosal inductive sites, such as Peyer's patches (PP). To investigate the morphology, immunophenotype and stimulatory activity of intestinal DC, a procedure was developed to obtain a cell population by using collagenase digestion of PP, density centrifugation and cell sorting on the basis of CD11c expression. The resultant low-density cell fraction consisted of a nonadherent cell population expressing different intensities of CD11c that could at least be characterized by typical DC morphology (e.g. abundant cytoplasma with veil-like cytoplasmatic dendrites, irregularly shaped nuclei, multivesicular and multilamellar bodies), constitutive levels of surface MHC class II, the presence of macrophage-specific markers, such as F4/80, Mac-I and Fc receptors, respectively, on subpopulations of CD11c+ sorted cells and expression of adhesion and co-stimulatory receptors like ICAM-1 and CD44. The capability of this low-density CD11c+ fraction to stimulate T cell responses was demonstrated in primary allogeneic mixed-lymphocyte reactions (MLR). Herein, we show that the freshly isolated CD11c+ cells showed weak accessory function, but develop this capacity following short-term culture in vitro in the presence of granulocyte/macrophage colony-stimulating factor. Although the nature and functional capacity of the isolated CD11c+ needs further clarification, these preliminary results describing phenotype and accessory function provide some evidence that these cells isolated from the PP may be immature forms of DC and play a crucial role as antigen-presenting cells with important implications for understanding the complex network regulating intestinal antigen uptake, processing and presentation.

SummaryThe genetic basis for aging is being intensely investigated in a variety of model systems. Much of the focus in Drosophila has been on the molecular-genetic determinants of lifespan, whereas the molecular-genetic basis for age-related functional declines has been less vigorously explored. We evaluated behavioural aging and lifespan in flies harbouring loss-of-function mutations in myospheroid , the gene that encodes β β β β PS, a β β β β integrin. Integrins are adhesion molecules that regulate a number of cellular processes and developmental events. Their role in aging, however, has received limited attention. We report here that age-related declines in locomotor activity are ameliorated and that mean lifespan is increased in myospheroid mutants. The delayed functional senescence and altered mortality in myospheroid flies are independent of changes in body size, reproduction or stress resistance. Our data indicate that functional senescence and age-dependent mortality are influenced by β β β β integrins in Drosophila .