Sucrose modifies ribosomal stability and conformation

High concentrations of sucrose have a strong protective effect on heat-induced modifications of rat liver ribosomal subunits. They prevent to a large extent subunit inactivation, measured by poly (U)-dependent [14C] Phe tRNA binding (40S subunits) and puromycin reaction (60S subunits), subunit unfolding into light forms, and the release of both free and protein-complexed 5S RNA. They also increase the temperature at which subunits start to melt. Our data indicate that sucrose affects subunit conformation.     

tRNA binding modifies the properties of the small ribosomal subunit of rat liver

The binding of a specific tRNA (acylated or not) to the 40S subunits in the presence of the proper codon was shown to produce two striking effects on the subunits. First, the subunits were no longer able to dimerize at low ionic strength. Second, they became fully resistant to 1.25 M LiCl treatment: bound tRNA prevented subunit inactivation as measured by polyphenylalanine synthesis; it also prevented large sedimentation changes of subunits and ribosomal protein release induced by LiCl. The number of protected proteins far exceeded that of the proteins crosslinked with tRNA after irradiation at 254 nm (A.M. REBOUD, S. DUBOST and J.P. REBOUD (1983) FEBS Lett. 158, 285-288). These results strongly suggest that tRNA binding induces modifications of rRNA-protein interactions in large domains of the subunits. A weak interaction of tRNA with the 40S subunit was demonstrated in the absence of the codon.     

tRNA binding stabilizes rat liver 60 S ribosomal subunits during treatment with LiCl

We have shown recently that, in the absence of mRNA, 1 molecule of nonacylated tRNA binds to the large ribosomal subunit of rat liver with a high affinity constant (Buisson, M., Reboud, A.M., Dubost, S., and Reboud, J. P. (1979) Biochem. Biophys. Res. Commun. 90,634-640). In this paper, free and tRNA-bound 60 S subunits were treated with increasing concentrations of LiCl to obtain information on tRNA binding site. The rationale for using deacylated tRNA was that it is assumed to bind to the peptidyl donor site. We observed that tRNA has a strong protective effect on subunit modifications produced by LiCl: tRNA prevents subunit inactivation as measured by puromycin reaction and polyphenylalanine synthesis and it shifts the Li+/Mg2+ ratio value needed to reach 50% inactivation, from 60 to 250; it also prevents ribosomal protein and 5 S RNA release and large sedimentation changes of subunits, induced by LiCl. To explain the mechanism of 60 S subunit stabilization by tRNA, two hypotheses are considered: stabilization can be consequent on direct interaction of tRNA with specific proteins, or on maintenance on subunits of essential cations which are otherwise displaced by Li+, or both.     

Non-invasive assessment of reproductive status in Chinese water deer (Hydropotes inermis): Correlation with sexual behaviour

The annual reproductive cycle of Chinese water deer (Hydropotes inermis), a nearly threatened small cervid species, was studied by means of fecal steroid analysis coupled with behavioural observations. Data showed a clearly seasonal reproductive pattern. In adult males, the onset of androgen secretion, in October, was concomitant with the first manifestations of territoriality. Androgen metabolites concentrations reached significant peak values in December, when matings occurred. In mature females, there was a close synchrony in reproductive states: lactational/seasonal anoestrus from June to November, pregnancy from December to May. Fecal progesterone metabolites profiles suggested that silent ovulations occurred at the onset of breeding season and that females conceived at their first ovulation with behavioural estrus. The female sexual receptivity state might last only a few hours. High levels of sniffing, parades and pursuits, concomitant of the highest concentrations of androgen, could allow the males to detect the furtive estrus in the females present in their territory. We concluded that the non-invasive method applied for the first time in this species was useful for the evaluation of the endocrine status and its relation with behaviour.     

Prolyl oligopeptidase of Trypanosoma brucei hydrolyzes native collagen,peptide hormones and is active in the plasma of infected mice

Proteases play important roles in many biological processes of parasites, including their host interactions. In sleeping sickness, Trypanosoma brucei proteases released into the host bloodstream could hydrolyze host factors, such as hormones, contributing to the development of the disease's symptoms. In this study, we present the identification of the T. brucei prolyl oligopeptidase gene (poptb) and the characterization of its corresponding enzyme, POP Tb. Secondary structure predictions of POP Tb show a structural composition highly similar to other POPs. Recombinant POP Tb produced in E. coli was active and highly sensitive to inhibitors of Trypanosoma cruzi POP Tc80. These inhibitors, which prevent T. cruzi entry into non-phagocytic cells, arrested growth of the T. brucei bloodstream form in a dose-dependent manner. POP Tb hydrolyzes peptide hormones containing Pro or Ala at the P1 position at a slightly alkaline pH, and also cleaves type I collagen in vitro and native collagen present in rat mesentery. Furthermore, POP Tb is released into the bloodstream of T. brucei infected mice where it remains active. These data suggest that POP Tb might contribute to the pathogenesis of sleeping sickness.     

Unexpected inhibition of peptidoglycan LD-transpeptidase from Enterococcus faecium by the beta-lactam imipenem

The beta-lactam antibiotics mimic the D-alanyl(4)-D-alanine(5) extremity of peptidoglycan precursors and act as "suicide" substrates of the DD-transpeptidases that catalyze the last cross-linking step of peptidoglycan synthesis. We have previously shown that bypass of the dd-transpeptidases by the LD-transpeptidase of Enterococcus faecium (Ldt(fm)) leads to high level resistance to ampicillin. Ldt(fm) is specific for the L-lysyl(3)-D-alanine(4) bond of peptidoglycan precursors containing a tetrapeptide stem lacking D-alanine(5). This specificity was proposed to account for resistance, because the substrate of Ldt(fm) does not mimic beta-lactams in contrast to the D-alanyl(4)-D-alanine(5) extremity of pentapeptide stems used by the DD-transpeptidases. Here, we unexpectedly show that imipenem, a beta-lactam of the carbapenem class, totally inhibited Ldt(fm) at a low drug concentration that was sufficient to inhibit growth of the bacteria. Peptidoglycan cross-linking was also inhibited, indicating that Ldt(fm) is the in vivo target of imipenem. Stoichiometric and covalent modification of Ldt(fm) by imipenem was detected by mass spectrometry. The modification was mapped into the trypsin fragment of Ldt(fm) containing the catalytic Cys residue, and the Cys to Ala substitution prevented imipenem binding. The mass increment matched the mass of imipenem, indicating that inactivation of Ldt(fm) is likely to involve rupture of the beta-lactam ring and acylation of the catalytic Cys residue. Thus, the spectrum of activity of beta-lactams is not restricted to transpeptidases of the DD-specificity, as previously thought. Combination therapy with imipenem and ampicillin could therefore be active against E. faecium strains having the dual capacity to manufacture peptidoglycan with transpeptidases of the LD- and DD-specificities.     

Identification of the L,D-transpeptidases responsible for attachment of the Braun lipoprotein to Escherichia coli peptidoglycan

The L,D-transpeptidase Ldt(fm) catalyzes peptidoglycan cross-linking in beta-lactam-resistant mutant strains of Enterococcus faecium. Here, we show that in Escherichia coli Ldt(fm) homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the L,D-transpeptidation reaction.     

Role of class A penicillin-binding proteins in PBP5-mediated beta-lactam resistance in Enterococcus faecalis

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis, class B PBP5 mediates intrinsic resistance to the cephalosporin class of beta-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 (ponA, pbpF, and pbpZ). Among mutants with single or double deletions, only JH2-2 DeltaponA DeltapbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus. Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of beta-lactams formed a subset of the class A PBPs of E. faecalis, and heterospecific complementation was observed with an ortholog from E. faecium. Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.     

Acute oxidative stress is associated with cell proliferation in the mouse liver

Oxidative stress is known to produce tissue injury and to activate various signaling pathways. To investigate the molecular events linked to acute oxidative stress in mouse liver, we injected a toxic dose of paraquat. Liver necrosis was first observed, followed by histological marks of cell proliferation. Concomitantly, activation of the MAP kinase pathway and increased levels of the anti-apoptotic protein Bcl-XL were observed. Gene expression profiles revealed that the differentially expressed genes were potentially involved in cell proliferation. These data suggest that paraquat-induced acute oxidative stress triggers the activation of regeneration-related events in the liver.