UVB radiation induced effects on cells studied by FTIR spectroscopy

We have made a preliminary analysis of the results about the effects on tumoral cell line (lymphoid T cell line Jurkat) induced by UVB radiation (dose of 310 mJ/cm²) with and without a vegetable mixture. In the present study, we have used two techniques: Fourier transform infrared spectroscopy (FTIR) and flow cytometry. FTIR spectroscopy has the potential to provide the identification of the vibrational modes of some of the major compounds (lipid, proteins and nucleic acids) without being invasive in the biomaterials. The second technique has allowed us to perform measurements of cytotoxicity and to assess the percentage of apoptosis. We already studied the induction of apoptotic process in the same cell line by UVB radiation; in particular, we looked for correspondences and correlations between FTIR spetroscopy and flow cytometry data finding three highly probable spectroscopic markers of apoptosis (Pozzi et al. in Radiat Res 168:698–705, 2007). In the present work, the results have shown significant changes in the absorbance and spectral pattern in the wavenumber protein and nucleic acids regions after the treatments.     

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Kinetics of binding of macrolides, lincosamides, and synergimycins to ribosomes

The synergistic effect of type A (virginiamycin M (VM)) and type B (virginiamycin S (VS)) synergimycins and their antagonistic effect against erythromycin (a 14-membered macrolide) for binding to the large ribosomal subunit (50 S) have been related. This investigation has now been extended to 16-membered macrolides (leucomycin A3 and spiramycin) and to lincosamides (lincomycin). A dissociation of VS-ribosome complexes was induced as well by 16-membered macrolides as by lincosamides. The observed dissociation rate constant of VS-ribosome complexes was identified with the kappa-vs in the case of 16-membered macrolides, but linearly related to lincomycin concentration, suggesting a direct binding of the latter antibiotic to VS-ribosome complexes and the triggering of a conformational change of particles entailing VS release. Two different mechanisms were also involved in the VM-promoted reassociation to ribosomes of VS previously displaced by either macrolides or lincosamides. By binding to lincosamide-ribosome complexes, VM induced a conformational change of ribosomes resulting in higher affinity for VS and lower affinity for lincosamides. On the contrary, an incompatibility for a simultaneous binding of VM and 16-membered macrolides to ribosomes was observed. These results have been interpreted by postulating specific (nonoverlapping) and aspecific (overlapping) antibiotic binding sites at the peptidyltransferase domain. All the kinetic constants of five antibiotic families (type A and B synergimycins, 14- and 16-membered macrolides, and lincosamides) and a topological model of peptidyltransferase are presently available.     

Analysis of the reversible binding of virginiamycin M to ribosome and particle functions after removal of the antibiotic

Type A synergimycins (VM) were shown to act catalytically and to induce two ribosomal alterations: (a) inability to promote polypeptide synthesis; (b) high-affinity binding of type B synergimycins (VS). A claim for irreversible binding of type A synergimycins to ribosomes has promoted the present reinvestigation. Submission of ribosomes from VM-treated bacteria to a purification procedure (supposed to remove the drug, according to a low association constant previously reported) yielded particles still holding residual VM. The formation of VM.ribosome complexes, more stable than previously inferred but without covalent linkage, was deduced from the extractability of complexed VM by organic solvents. Moreover, incubation of these complexes with increasing amounts of anti-VM immunoglobulins progressively restored ribosome activity in protein synthesis. Binding of VS to ribosomes, by fluorimetric titrations in the presence of substoichiometric concentrations of VM, was incompatible with catalytic action of type A synergimycins. Ribosomes from VM-treated bacteria displayed also a higher affinity for VS than did control ribosomes. This property did not disappear when ribosome.VM complexes were incubated with anti-VM IgG, nor when VM-IgG complexes were withdrawn from the reaction mixture by protein A-agarose binding. We can conclude that VM binding produces: (1) an inhibition of ribosome-promoted peptide bond formation, which occurs only in the presence of the drug; and (2) an increase of ribosome affinity for VS, which lasts after VM removal. The linkage of this drug with ribosomes is tight but reversible and its action is stoichiometric.     

Analysis of fluorescence quenching of ribosome-bound virginiamycin S

The two virginiamycin components VM and VS interact synergistically with bacterial ribosomes in vitro and in vivo. Ribosome affinity for virginiamycin S increases about 10-fold upon incubation with virginiamycin M. This effect has been previously traced by spectrofluorimetric measurement based on the enhancement of virginiamycin S fluorescence upon its binding to the 50 S ribosomal subunit. In the present work the action of two virginiamycin S fluorescence quenchers, acrylamide and iodide, has been explored to gather information about the accessibility of ribosome-bound virginiamycin S and the variation of the accessibility level in the presence of virginiamycin M. Both acrylamide (non-ionized quencher) and iodide (ionized quencher) proved powerful quenchers of free virginiamycin S solutions. Since a comparable effect was obtained on 3- hydroxypicolinamide , the latter was indicated as the part of the molecule involved in the fluorescence effect. Fluorescence quenching by either agent was of the dynamic, i.e. collisional, type. Such an inference was based on the fact that these quenchers merely modified the emission spectrum (not the absorption spectrum), the bimolecular rate constant for the quenching process decreased linearly with the viscosity of the medium (static-type quenching is viscosity-independent), and that linear Stern-Volmer plots were obtained. The quenching ability of both agents underwent a sharp decrease in the presence of ribosomes; however, the Stern-Volmer equation was followed only in the case of acrylamide, whereas Lehrer 's relationship had to be applied in the case of iodide. When ribosomes were incubated with virginiamycin M, the fluorescence quenching ability of acrylamide and iodide was significantly reduced. Conclusions are as follows: a) the 3- hydroxypicolinyl residue of virginiamycin S is buried within an open well on the ribosome surface and is likely to be involved in the interaction with the binding site; b) the accessibility to the well is partly controlled by electrostatic forces; c) interaction of ribosomes with virginiamycin M entails a conformational change whereby the access to the well is reduced. These findings provide a molecular explanation for the previously observed increase of the association constant of virginiamycin S to ribosomes incubated with virginiamycin M which was found to be due to the decrease of the dissociation rate constant (the association rate constant remains practically the same).     

Fenfluramine Releases Serotonin from Human Brain Nerve Endings by a Dual Mechanism

Abstract: Fenfluramine is the most widely used anorexigenic drug in humans. In animal experiments d -fenfluramine has been shown to act as a potent releaser of brain serotonin [5-hydroxytryptamine (5-HT)]. Here we have investigated the effects of d -fenfluramine on the release of [³H]5-HT from isolated nerve endings of human neocortex. The drug elicited release of unmetabolized [³H]5-HT, and this effect was concentration dependent. However, the mechanism of release seems to differ profoundly depending on the concentrations of d -fenfluramine used. At 5 µ M , the release of [³H]5-HT was blocked by the 5-HT transporter inhibitor fluoxetine and was Ca²⁺ independent and insensitive to the human autoreceptor 5-HT_1D agonist sumatriptan. The release of [³H]5-HT elicited by 0.5 µ M d -fenfluramine was similarly blocked by fluoxetine, but it was strongly Ca²⁺ dependent and sensitive to sumatriptan. It is suggested that, at relatively high concentrations, d -fenfluramine largely diffuses into serotonergic terminals and causes release of 5-HT through the 5-HT carrier working in the inside-outside direction; at relatively low concentrations d -fenfluramine enters the terminals through the 5-HT transporter but elicits release of 5-HT by an exocytotic-like mechanism.     

Identification of a single base change in ribosomal RNA leading to erythromycin resistance.

The molecular basis of a mutation conferring an erythromycin-resistance phenotype was explored, as an approach to the role of 23 S rRNA in the peptidyl-transferase activity of 50 S ribosomal subunits. Mutagenization of an Escherichia coli strain, which carried the multicopy plasmid pLC7-21 containing the rrnH operon, led to the production of an erythromycin-resistant strain. Plasmid pBFL1 isolated from this mutant was able to transform the sensitive RecA- strain EM4 and to induce a "dissociated" type of antibiotic resistance. Two ribosome populations occurred in EM4/pBFL1: normal particles coded for by the seven rrn chromosomal genes and mutated particles containing rRNA of plasmid origin. The latter particles displayed in vitro lower affinity and susceptibility to erythromycin than wild type particles. The mutation within plasmid pBFL1 was mapped by a multiple primer extension technique. Three synthetic primers were used to sequence the central loop in domain V of 23 S rRNA, leading to identification of a C to U transition at position 2611. This base change was proved to be responsible for the erythromycin-resistance phenotype by the plasmid-plasmid marker rescue technique. A molecular explanation for the rrn mutations leading, respectively, to undissociated and to dissociated types of resistance to the MLSb (macrolide-lincosamide-synergimycin B) group of antibiotics is proposed. These results and some literature data support the notion that rRNA bases involved in antibiotic resistance play a conformational role in the ribosomal binding sites for the MLSb antibiotics.     

The role of rRNA bases in the interaction of peptidyltransferase inhibitors with bacterial ribosomes.

Synergism of streptogramins A (virginiamycin M, VM) and B (virginiamycin S, VS), peptidyltransferase inhibitors, was explored in EM4/pLC7-21 (wild type) and EM4/pERY (VS-resistant). These bacterial strains contained multicopy plasmids carrying an rrnH operon with wild type (pLC7-21) or mutated (A2058----U transversion) 23 S rRNA gene. Ribosomes with wild type and mutated rRNA were both present in EM4/pERY. The latter particles did not bind VS; in the presence of VM, however, high affinity VS binding occurred. As shown previously, VS protected against chemical reagents certain bases in domain V rRNA and VM in the stems flanking this loop. Differences between wild type and mutant ribosomes were observed: A2058, A2059, A2062, and G2505, protected by VS and ERY in EM4/pLC7-21, were unshielded in EM4/pERY. A2062 was shielded by VM in EM4/pERY, not in EM4/pLC7-21, and G2505 of mutant ribosomes became protected by VS when VM was simultaneously present. Induction by VM of a high affinity VS binding site in VS-sensitive and -resistant ribosomes indicates A2058 mutation to entail a conformational change of this site, which is counteracted by VM fixation. Accessibility of A2062 to chemical reagents (unlike behavior of EM4/pERY and EM4/pLC7-21 in the presence of VM) implies different conformations for wild type and mutant ribosomes.     

The sensitivity of catecholamine release to botulinum toxin C1 and E suggests selective targeting of vesicles set into the readily releasable pool

The impact of syntaxin and SNAP-25 cleavage on [3H]noradrenaline ([3H]NA) and [3H]dopamine ([3H]DA) exocytotic release evoked by different stimuli was studied in superfused rat synaptosomes. The external Ca2+-dependent K+-induced [3H]catecholamine overflows were almost totally abolished by botulinum toxin C1 (BoNT/C1), which hydrolyses syntaxin and SNAP-25, or by botulinum toxin E (BoNT/E), selective for SNAP-25. BoNT/C1 cleaved 25% of total syntaxin and 40% of SNAP-25; BoNT/E cleaved 40% of SNAP-25 but left syntaxin intact. The GABA uptake-induced releases of [3H]NA and [3H]DA were differentially affected: both toxins blocked the former, dependent on external Ca2+, but not the latter, internal Ca2+-dependent. BoNT/C1 or BoNT/E only slightly reduced the ionomycin-evoked [3H]catecholamine release. More precisely, [3H]NA exocytosis induced by ionomycin was sensitive to toxins in the early phase of release but not later. The Ca2+-independent [3H]NA exocytosis evoked by hypertonic sucrose, thought to release from the readily releasable pool (RRP) of vesicles, was significantly reduced by BoNT/C1. Pre-treating synaptosomes with phorbol-12-myristate-13-acetate, to increase the RRP, enhanced the sensitivity to BoNT/C1 of [3H]NA release elicited by sucrose or ionomycin. Accordingly, cleavage of syntaxin was augmented by the phorbol-ester. To conclude, our results suggest that clostridial toxins selectively target exocytosis involving vesicles set into the RRP.     

The chitinolytic activity of Penicillium janthinellum P9: purification, partial characterization and potential applications

AIMS: To purify and characterize the chitinolytic activity of Penicillium janthinellum P9 and to evaluate possible uses of the purified enzymes in the control of fungal growth and spore germination. METHODS AND RESULTS: The chitinolytic activity of P. janthinellum P9 was associated to two beta-N-acetyl-hexosaminidases (CHI1 and CHI2) that were purified by preparative isoelectric focusing and preparative electrophoresis and partially characterized. Treatment of test fungi with purified enzyme solutions caused reduced spore germination, reduction of hyphal length and mycelial damage. The combined action of the two enzymes and a systemic fungicide completely inactivated pests and food-spoiling moulds such as Fusarium solanii, P. canescens and Cladosporium cladosporioides. Treatment with the two enzymes increased germination of freeze-dried fungal spores. CONCLUSION: The chitinolytic activity of P. janthinellum P9 is associated with two extracellular beta-N-acetyl-hexosaminidases that can cause damage to the cell walls of other fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: This appears to be the first report on the characterization of extracellular chitinolytic enzymes produced by a Penicillium strain. The results of this study might have some impact in the applied research field.