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Cyclothialidine, a new DNA gyrase inhibitor
ANHYDROTETRACYCLINE BINDING TO TET REPRESSOR INDUCES CONFORMATIONAL AND DYNAMIC CHANGES IN THE OPERATOR BINDING DOMAIN Chabbert M.1, Peviani C.2, Ettner N. 3, Lami H. 1, Doglia S.2, & Hillen
PROTECTIVE EFFECTS OF MELATONIN IN MICE INFECTED WITH ENCEPHALITISVIRUSES. A. Conti 1, G.J.M. Maestroni 1, S. Lustig2 and D.Ben -Nathan2. llstituto Cantonale di Patologia, 6604 Locarno Switzerland, 2 Israel Institute for Biological Research, Ness Ziona, Israel. In the past several years it has been recognized that the pineal neurohormone melatonin (MLT) can augment the immune response and correct immunodeficiency states which may follow acute stress, viral diseases, aging or drug treatment. The immunostimulating properties of MLT seem to depend on activated T helper cells which upon MLT stimulation show an enhanced synthesis and/or release of opioid peptides, IL-2, IL-4 and IFN-7. It is known that viral infections cause an increase in glycocorticoids and involution of thymus and spleen as well as generalized immunosuppression.The question which arise from the above data is Wether MLT may act as an ativiral agent.In the present study we examined the effect MLT on protection from viral encephalitis.The antiviral activity of MLT was evaluated in normal mice inoculated with Semliki Forest Virus (SFV) and in stressed mice injected with the attenuated non-invasive West Nile Virus (WN-25). Daily subcutaneous administration of MLT from 3 days before through 10 days after virus inoculation, reduced viremla and significantly post-poned the onset of disease and death by 7 to 10 daysl Moreover, MLT injection reduced mortality of SFV (10 PFU) inoculated mice from 100%to 44%. In mice inoculated with high dose of SFV (100 PFU), MLT postponed death and reduced mortality by 20%. In all of the surviving mice anti-SFV antibodies were detected 22 days after virus inoculation. Stress exposure (isolation or dexamethasone injection) of WN-25 infected mice induced mortality of 75% and 50% respectively, which was reduced by MLT administration to 31% and 25%. The efficiency of MLT in protecting from lethal, viral infections warrats further investigations on its mechanisms of action and possible clinical application.
1Laboratoire de Physique, Facult~ de Pharmacie de Strasbourg, CNRS URA 491, F-67401 lllkirch; 2Consorzio Biodcerche, Via Emanueli t5, 1-20126 Milano; ~Lehrstul far Mikrobiologie, Institut far Mikrobiologie und Biochemie der Frieddch-Alexander Universit~t Edangen-N~3mberg,D-8520 Edangen. Tet repressor (TetR) controls transcription of the TnlO encoded tet genes conferring resistance to tetracycline in Gram-negative bacteria. This control is negatively regulated by the antibiotic tetracycline that functions as an inducer. The engineered F75 TetR contains a single Trp residue located at position 43 in the operator recognition o~-helix.This Trp can be used as a fluorescent probe to investigate changes in the operator binding domain of TetR upon induction. Binding of the tetracycline derivative anhydrotetracycline (AnTc) to F75 TetR induced a 35% decrease in Trp-43 fluorescence intensity without shift of the emission maximum. Either in the presence or absence of AnTc, the fluorescence decay of Trp-43 could be described by the sum of three exponential components. In both cases, the lifetimes were independent of the emission wavelength, suggesting that these components might be related to three conformations of Trp-43. AnTc binding induced a marked decrease in the amplitude of the longest component. The Ti'p-43 anisotropy decay showed the existence of motion with two distinct timescales. The shortest rotational correlation time, in the picosecond-subnanosecond range, was related to fast wobbling of Trp-43. The longest one, in the nanosecond range, was related to segmental motion of the operator binding domain. Upon AnTc binding, this rotational correlation time decreased from 3.5 ns to 3.0 ns, indicating an increase in segmental mobility. Our results clearly indicate that AnTc binding induces conformational and dynamic changes in the operator binding domain of TetR which might be crucial for the allostedc regulation mechanism.
DIFFERENT CONVULSANT PROPERTIES OF IMIPENEM AND M E R O P E N E M A. De Sarro, C. Imperatore, F. Nava and G.B. De Sarro*. Institute of Pharmacology, School of Medicine, University of Messina, *Department of Experimental and Clinical Medicine, School of Medicine, University of Reggio Calabria (Italy).
CYCLOTHIALIDINE, A NEW DNA GYRASE INHIBITOR H.GmQnder1, P,Angehrn 1, E.G6tschi 1, N.Nakada2, M. Stieger1 F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland1 Nippon Roche Research Center, Kamakura, 247, Japan 2 Cyctothialidine is a new DNA gyrase inhibitor isolated from Streptomyces filipinensis. Structurally it belongs to a new class of natural products containing a unique 12-membered lactone ring that is partly integrated into a pentapeptide chain. Cyclothialidine was found to be one of the most active inhibitors of E. coil DNA gyrase and cyclothialidine congeners exhibit also very good activity against Gram.-positive bacteria. No substantial difference in suscept=bility to cyclothialidine congeners was seen among multiple resistant (methicillin, erythremycin, vancomycin, quinolones) and susceptible strains, indicating no cross-resistance between this type of compounds and other antibacterials. Cyclothialidine binds to the B subunit of DNA gyrase and inhibits the ATPase activity of this subunit. Binding affinity increased depending on the assembly of the DNA gyrase-DNA complex (affinity for B < A2B 2 < A2B2-DNA ). Binding of C-14 labelled cyclothialidine to the B subunit was strongly competed by cyclothialidine, novobiocin and coumermycin, only weakly by ADPNP and ATPyS, but not by ofloxacin. The gyrB genes of S. aureus mutants resistant against novobiocin showed a single point mutation leading to the replacement of Arg-144 by lie. Arg-144 in S. aureus is homologous to Arg-136 in E. coil, which when mutated lead also to novobiocin resistance, suggesting that the mechanism leading to coumadn resistance is similar for these two DNA gyrases. This Arg-144 mutation affected considerably the activity of both novobiocin and coumermycin but did not change the susceptibility to cyclothialidine congeners. However, single point mutations at other sites (causing alterations of Ser-128 to Leu, Thr-173 to Asn, and Ile-175 to Thr) decreased the susceptibility to cyclothialidine congeners but affected also the susceptibility to the coumarins. These results suggest an ovedapping binding site for the cyclothialidine congeners and for the coumarins.
Imipenem-cilastatin has been reported to induce sometimes a seizure-like activity. We studied the possible convulsant effects of imipenem in DBA/2 mice, a strain genetically susceptible to sound-induced seizures and in C57 mice, a no seizure prone strain. In addition, we compared the neurological effects of imipenem and meropenem, a new carbapenem derivative, in both strains of mice. Groups of DBA/2 and CsLmice (18-28 g, 42-48 days old) were injected (i.p.) with several doses of imipenem-cilastatin, imipenem alone and meropenem and the occurenee of seizures was recorded for 2 hr. Our experiments showed that no significant differences were observed between imipenem-cilastatin and imipenem treated animals. Thus, we exclude a probenecid-like effect of cilastatin with reference to imipenem. An increased excitation of the central nervous system (CNS) by inhibition of GABA binding to receptors and a slow clearance of imipenem from the CNS were postulated as causes of convulsant properties of the diag. Meropenem showed weak or no convulsant effects in the same experimental conditions. We emphasize that several factors deserve to be considered in explaining the different convtflsant properties ofimipenem and meropenem.
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PROTECTIVE EFFECTS OF MELATONIN IN MICE INFECTED WITH ENCEPHALITISVIRUSES. A. Conti 1, G.J.M. Maestroni 1, S. Lustig2 and D.Ben -Nathan2. llstituto Cantonale di Patologia, 6604 Locarno Switzerland, 2 Israel Institute for Biological Research, Ness Ziona, Israel. In the past several years it has been recognized that the pineal neurohormone melatonin (MLT) can augment the immune response and correct immunodeficiency states which may follow acute stress, viral diseases, aging or drug treatment. The immunostimulating properties of MLT seem to depend on activated T helper cells which upon MLT stimulation show an enhanced synthesis and/or release of opioid peptides, IL-2, IL-4 and IFN-7. It is known that viral infections cause an increase in glycocorticoids and involution of thymus and spleen as well as generalized immunosuppression.The question which arise from the above data is Wether MLT may act as an ativiral agent.In the present study we examined the effect MLT on protection from viral encephalitis.The antiviral activity of MLT was evaluated in normal mice inoculated with Semliki Forest Virus (SFV) and in stressed mice injected with the attenuated non-invasive West Nile Virus (WN-25). Daily subcutaneous administration of MLT from 3 days before through 10 days after virus inoculation, reduced viremla and significantly post-poned the onset of disease and death by 7 to 10 daysl Moreover, MLT injection reduced mortality of SFV (10 PFU) inoculated mice from 100%to 44%. In mice inoculated with high dose of SFV (100 PFU), MLT postponed death and reduced mortality by 20%. In all of the surviving mice anti-SFV antibodies were detected 22 days after virus inoculation. Stress exposure (isolation or dexamethasone injection) of WN-25 infected mice induced mortality of 75% and 50% respectively, which was reduced by MLT administration to 31% and 25%. The efficiency of MLT in protecting from lethal, viral infections warrats further investigations on its mechanisms of action and possible clinical application.
1Laboratoire de Physique, Facult~ de Pharmacie de Strasbourg, CNRS URA 491, F-67401 lllkirch; 2Consorzio Biodcerche, Via Emanueli t5, 1-20126 Milano; ~Lehrstul far Mikrobiologie, Institut far Mikrobiologie und Biochemie der Frieddch-Alexander Universit~t Edangen-N~3mberg,D-8520 Edangen. Tet repressor (TetR) controls transcription of the TnlO encoded tet genes conferring resistance to tetracycline in Gram-negative bacteria. This control is negatively regulated by the antibiotic tetracycline that functions as an inducer. The engineered F75 TetR contains a single Trp residue located at position 43 in the operator recognition o~-helix.This Trp can be used as a fluorescent probe to investigate changes in the operator binding domain of TetR upon induction. Binding of the tetracycline derivative anhydrotetracycline (AnTc) to F75 TetR induced a 35% decrease in Trp-43 fluorescence intensity without shift of the emission maximum. Either in the presence or absence of AnTc, the fluorescence decay of Trp-43 could be described by the sum of three exponential components. In both cases, the lifetimes were independent of the emission wavelength, suggesting that these components might be related to three conformations of Trp-43. AnTc binding induced a marked decrease in the amplitude of the longest component. The Ti'p-43 anisotropy decay showed the existence of motion with two distinct timescales. The shortest rotational correlation time, in the picosecond-subnanosecond range, was related to fast wobbling of Trp-43. The longest one, in the nanosecond range, was related to segmental motion of the operator binding domain. Upon AnTc binding, this rotational correlation time decreased from 3.5 ns to 3.0 ns, indicating an increase in segmental mobility. Our results clearly indicate that AnTc binding induces conformational and dynamic changes in the operator binding domain of TetR which might be crucial for the allostedc regulation mechanism.
DIFFERENT CONVULSANT PROPERTIES OF IMIPENEM AND M E R O P E N E M A. De Sarro, C. Imperatore, F. Nava and G.B. De Sarro*. Institute of Pharmacology, School of Medicine, University of Messina, *Department of Experimental and Clinical Medicine, School of Medicine, University of Reggio Calabria (Italy).
CYCLOTHIALIDINE, A NEW DNA GYRASE INHIBITOR H.GmQnder1, P,Angehrn 1, E.G6tschi 1, N.Nakada2, M. Stieger1 F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland1 Nippon Roche Research Center, Kamakura, 247, Japan 2 Cyctothialidine is a new DNA gyrase inhibitor isolated from Streptomyces filipinensis. Structurally it belongs to a new class of natural products containing a unique 12-membered lactone ring that is partly integrated into a pentapeptide chain. Cyclothialidine was found to be one of the most active inhibitors of E. coil DNA gyrase and cyclothialidine congeners exhibit also very good activity against Gram.-positive bacteria. No substantial difference in suscept=bility to cyclothialidine congeners was seen among multiple resistant (methicillin, erythremycin, vancomycin, quinolones) and susceptible strains, indicating no cross-resistance between this type of compounds and other antibacterials. Cyclothialidine binds to the B subunit of DNA gyrase and inhibits the ATPase activity of this subunit. Binding affinity increased depending on the assembly of the DNA gyrase-DNA complex (affinity for B < A2B 2 < A2B2-DNA ). Binding of C-14 labelled cyclothialidine to the B subunit was strongly competed by cyclothialidine, novobiocin and coumermycin, only weakly by ADPNP and ATPyS, but not by ofloxacin. The gyrB genes of S. aureus mutants resistant against novobiocin showed a single point mutation leading to the replacement of Arg-144 by lie. Arg-144 in S. aureus is homologous to Arg-136 in E. coil, which when mutated lead also to novobiocin resistance, suggesting that the mechanism leading to coumadn resistance is similar for these two DNA gyrases. This Arg-144 mutation affected considerably the activity of both novobiocin and coumermycin but did not change the susceptibility to cyclothialidine congeners. However, single point mutations at other sites (causing alterations of Ser-128 to Leu, Thr-173 to Asn, and Ile-175 to Thr) decreased the susceptibility to cyclothialidine congeners but affected also the susceptibility to the coumarins. These results suggest an ovedapping binding site for the cyclothialidine congeners and for the coumarins.
Imipenem-cilastatin has been reported to induce sometimes a seizure-like activity. We studied the possible convulsant effects of imipenem in DBA/2 mice, a strain genetically susceptible to sound-induced seizures and in C57 mice, a no seizure prone strain. In addition, we compared the neurological effects of imipenem and meropenem, a new carbapenem derivative, in both strains of mice. Groups of DBA/2 and CsLmice (18-28 g, 42-48 days old) were injected (i.p.) with several doses of imipenem-cilastatin, imipenem alone and meropenem and the occurenee of seizures was recorded for 2 hr. Our experiments showed that no significant differences were observed between imipenem-cilastatin and imipenem treated animals. Thus, we exclude a probenecid-like effect of cilastatin with reference to imipenem. An increased excitation of the central nervous system (CNS) by inhibition of GABA binding to receptors and a slow clearance of imipenem from the CNS were postulated as causes of convulsant properties of the diag. Meropenem showed weak or no convulsant effects in the same experimental conditions. We emphasize that several factors deserve to be considered in explaining the different convtflsant properties ofimipenem and meropenem.
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