Uncompetitive inhibition by adenine of the RNA-N-glycosidase activity of ribosome-inactivating proteins.

Ricin is a member of the ribosome-inactivating protein (RIP) family with RNA-N-glycosidase activity which inactivates eukaryotic ribosomes by specifically removing adenine from the first adenosine of a highly conserved GAGA loop present in 28S rRNA. Free adenine protects ribosomes in cell-free systems from inactivation by ricin. Protection by adenine is highly specific, since AMP, adenosine and modified adenines (1-methyladenine and ethenoadenine) were completely ineffective. Kinetic analysis of the behaviour of adenine as inhibitor of the RNA-N-glycosidase reaction catalysed by ricin, Shiga-like toxin I and momordin, two other members of the RIP family, established that inhibition was of the uncompetitive type, the inhibitor binding to the enzyme-substrate complex. Adenine did not protect ribosomes from alpha-sarcin, an RNAase that inactivates ribosomes by cleaving the phosphodiester bond located in the GAGA loop at one nucleotide distance from the adenosine depurinated by the RNA-N-glycosidases. Adenine at the concentration of 1 mM lowered 1.5-fold the toxicity of ricin and 3.7-fold that of Shiga-like toxin I on Vero cells in culture. The same concentration of adenine decreased 2.4-fold the inactivation of isolated ribosomes by ricin, 2.8-fold the inactivation by Shiga-like toxin I and 20-fold that by momordin.

Identification of the tRNAs which up-regulate agrostin, barley RIP and PAP-S, three ribosome-inactivating proteins of plant origin.

Ribosome-inactivating proteins (RIP) are RNA-N-glycosidases widely diffused in plants which depurinate ribosomal RNA at a specific universally conserved position, A4324 in rat ribosomes. A small group of RIPs (cofactor-dependent RIPs) require ATP and tRNA to reach maximal activity on isolated ribosomes. The tRNA which stimulates gelonin was identified as tRNA(Trp). The present paper reports the identification of three other tRNAs which stimulate agrostin (tRNA(Ala)), barley RIP (tRNA(Ala), tRNA(Val)) and PAP-S (tRNA(Gly)), while for tritin-S no particular stimulating tRNA emerged. The sequences of tRNA(Val) and tRNA(Gly) correspond to the already known ones (rabbit and man, respectively). The tRNA(Ala) (anticodon IGC) identifies a new isoacceptor. Only the stimulating activity of the tRNA(Ala) for agrostin approaches the specificity previously observed for the couple gelonin-tRNA(Trp).

Differential up-regulation by tRNAs of ribosome-inactivating proteins.

Some plant ribosome-inactivating proteins (RIPs) with RNA-N-glycosidase activity on 28S RNA require, for the inactivation of ribosomes, the presence of macromolecular cofactors present in post-ribosomal supernatants. In the case of gelonin one of the cofactors is tRNATrp lacking one or two nucleotides at the 3'-CCA end [Brigotti, M., Carnicelli, D., Alvergna, P., Pallanca, A., Lorenzetti, R., Denaro, M., Sperti, S. and Montanaro, L. (1995) Biochem. J. 310, 249-253]. In the present study it is shown that tRNAs are involved in the up-regulation of all the cofactor-requiring RIPs up to now identified (agrostin, barley RIP, PAP and tritin, besides gelonin). Polyacrylamide gel electrophoresis shows that tRNA fractions with different mobilities stimulate different RIPs. With the identification of agrostin, the cofactor-requiring RIPs (italics) add to five out of a total of thirteen investigated: barley RIP, bryodin-R, gelonin, lychnin, momordin, momorcochin-S, PAP, saporin-6, tritin [Carnicelli, D., Brigotti, M., Montanaro, L. and Sperti, S. (1992) Biochem. Biophys. Res. Commun. 182, 579-582], agrostin, luffin, trichokirin and trichosanthin (present study).

Heparin surface treatment of poly(methylmethacrylate) alters adhesion of a Staphylococcus aureus strain: utility of bacterial fatty acid analysis.

Bacterial adhesion on biomaterials is an important cause of associated infection. Many authors have studied the adhesion mechanisms of bacteria on biomaterials. These studies were useful in making materials more and more refractory to bacterial adhesion. We analysed the gas chromatographic modifications of structural fatty acids of a Staphylococcus aureus strain after adhesion on two polymers, poly(methylmethacrylate) (PMMA), whose biological compatibility is known, and heparin-surface-modified PMMA (HSM-PMMA). We noted changes to the chromatographic peaks peculiar to the fatty acids of S. aureus for each tested material and particularly for HSM-PMMA.

The RNA-N-glycosidase activity of Shiga-like toxin I: kinetic parameters of the native and activated toxin.

Shiga toxin and Shiga-like toxins are ribosome-inactivating proteins with RNA-N-glycosidase activity which remove a specific adenine from 28S RNA. The toxins are composed of an A subunit non-covalently associated to a multimer of receptor-binding B subunits. Near the COOH-terminus of the A subunit, a disulfide-bonded loop contains two trypsin-sensitive arginine residues. Proteolytic nicking at these sites, followed by reduction, removes from the A subunit the C-terminal end together with the associated B subunits. The requirement of such cleavage for biological activity of Shiga toxin and Shiga-like toxins has been recently questioned. The present paper reports the kinetic constants of the adenine release from highly purified Artemia salina ribosomes catalysed by Shiga-like toxin I and by its A subunit before and after treatment with trypsin, urea and dithiothreitol or urea and dithiothreitol alone. All reactions had approximately the same Km (1 microM). The Kcat was 0.6 min-1 for the untreated holotoxin and 6 min-1 for the isolated A subunit, respectively. The trypsin treatment increased 1000-fold the Kcat of the holotoxin (770 min-1) and 100-fold the Kcat of the A subunit (640 min-1). The same Kcat (693 min -1) was also observed when the A subunit was treated only with urea and dithiothreitol. Thus the full activity of Shiga-like toxin I required not only removal of the B subunits but also activation of the A subunit itself. Such activation could be largely induced in vitro by drastic loosening of the molecule induced by urea and dithiothreitol, but in vivo would probably require a proteolytic cleavage of the toxin. Inactivation of ribosomes by Shiga-like toxin I did not require sensitization of ribosomes by ATP and macromolecular cofactors present in postribosomal supernatants.

tRNA(Trp) as cofactor of gelonin, a ribosome-inactivating protein with RNA-N-glycosidase activity. Features required for the cofactor activity.

Purified beef and rabbit liver tRNA(Trp), but not yeast tRNA(Trp), increase the in vitro inactivation of eukaryotic ribosomes by gelonin, a ribosome-inactivating protein with RNA-N-glycosidase activity on 28S rRNA. Aminoacylation and stepwise trimming of the 3'-end of bovine tRNA(Trp) affect the cofactor activity, the most active species being that shortened by the last two nucleotides. ATP only moderately increases the activity of purified mammalian tRNA(Trp) and in this increase the cognate aminoacyl-tRNA synthetase apparently has no role. Mobility shift experiments indicate that bovine tRNA(Trp) binds both to ribosomes and to gelonin and favours the dissociation of gelonin from ribosomes.

Microstructural investigation of bone-cement interface.

We evaluated the microstructural characteristics of newly formed bone tissue at the interface with cement. The bone-cement interfaces of the femoral components of nine hip prostheses retrieved after loosening were investigated by means of X-ray diffraction on microareas and microhardness. The bone far from the interface of two stable prostheses was used as a control. The newly formed bone adjacent to cement in the loosened prostheses showed a maturity degree lower than that of bone adjacent to cement in stable prostheses. The lattice parameters of bone apatite did not show significant variations as compared to the reference values. Bone trabeculae at the interface with loosened prostheses often showed an osteoid lining characterized by a strongly demineralized lamellar and haversian structure. Radioopaque cement particles are sometimes found in the trabecular bone tissue around the prosthesis.

3'-immature tRNA(Trp) is required for ribosome inactivation by gelonin,a plant RNA N-glycosidase.

Inactivation of ribosomes by gelonin, a ribosome-inactivating protein with RNA N-glycosidase activity on 28 S rRNA, requires macromolecular cofactors present in post-ribosomal supernatants. One of these cofactors has been purified from a rat liver extract and identified as an RNA about 70 nt long which in sequence analysis shows a high level of similarity with mammalian (bovine) tRNA(Trp). The pattern of the sequencing gel is consistent with the co-existence in the preparation of two 3'-immature tRNA(Trp) species, missing only A75, or both A75 and C74. In the presence of ATP, CTP and tRNA nucleotidyltransferase, the gelonin-stimulating RNA is a good acceptor of tryptophan. An oligodeoxynucleotide complementary to positions 55 to 72 of mammalian (bovine) tRNA(Trp) hybridizes with the gelonin-stimulating RNA as demonstrated by gel mobility shift and ribonuclease H digestion. The oligodeoxynucleotide-directed ribonuclease H treatment also abolishes the gelonin-promoting activity of crude preparations of RNA, giving strong evidence that the only active RNA is a tRNA(Trp)-like molecule.