The role of the carboxyl and amino groups of polyene macrolides in their interactions with sterols and their selective toxicity. A P-NMR study

The permeability induced by amphotericin B and vacidin A derivatives in large unilamellar lipidic vesicles containing various sterols has been studied using the proton-cation exchange method and ³¹P-NMR spectroscopy. Derivatives which have a free ionizable carboxyl group induce biphasic ‘all or none’ permeability typical of channel-forming ionophores, whatever the sterol present. In sterol-free membranes, they have no significant activity. Derivatives which lack a free ionizable carboxyl group exhibit this channel-like mode of action only in membranes containing ergosterol or sterols with an alkyl side like that of ergosterol. In membranes containing cholesterol or sterol whose side-chain is alike, a slow and progressive permeability is observed at high concentrations. This activity is observed in sterol-free membranes as well. Derivatives containing sugars with substituted amino groups always have lower ionophoric activity than those which are unsubstituted. The greatest decrease in activity was observed for N-acetyl derivatives. Substitution of the amino groups has no effect on the mode of action. A model of interaction of polyenes with sterols is presented accounting for the data obtained on vesicles and the observed selective toxicity of polyene derivatives in biological membranes.     

Aspects réglementaires et organisationnels de l’activité d’autoconservation. Le point de vue du juriste

At the present time, legal texts in application of bioethics laws only briefly mention cryopreservation. The conditions of cryopreservation differ according to the type of tissue stored: while cryopreservation of oocytes and ovarian tissue corresponds to the field of biomedical research, semen cryopreservation corresponds to medically assisted procreation. Cryopreservation activity is more clearly defined in the draft revision of the bioethics law. Concerning the cryoconservation of ovarian tissue and oocytes, these difficulties result in particular in this activity’s mixed nature. It is located indeed, halfway between research and care. These two spheres of medical activity are subjected to their own distinct and exclusive, and their application is conditioned by the qualification of the implemented act. However, this qualification is dubious here, because of, in particular, the impossibility of determining which acts of sampling, cryoconservation and use might be concerned with a research protocol. Concerning the cryoconservation of sperm, the texts, first of all, seem to assimilate the activity of cryoconservation within an activity of assisted medical procreation. However, such assimilation would be equivalent to the impossibility of its implementation, because of the difficulty of respecting all of the legal conditions of assisted medical procreation. However, another more favourable interpretation of the provisions seems to be possible. Taking into consideration these uncertainties, contradictions and difficulties, the legislator intervened and devoted, by the widening of the indications of assisted medical procreation, the activity of cryoconservation in the project of revision of the laws of bioethics. He however did not solve all the difficulties. These cryoconserved elements can be restored only to the depositor. Indeed, the texts make obstacle to a delivery for a third party of the cryoconserved sperm, whatever the moment. They can also be used. This use can consist of assisted medical procreation, which is the first finality of cryoconservation — but it could only be implemented in respect of the whole of the legal conditions. It seems that the cryoconserved elements could also be used within the framework of research, whatever its nature (biomedical or not) and the moment of its implementation (while the person is alive or after his death). Its implementation should however be subjected to prior agreement and expressed while the person, whose elements were preserved, is alive. Such a use is expressly made possible in the project of revision of the laws of bioethics. Following a partial use or a lack of such a use, the destruction of the cryoconserved elements can be considered, as well as the continuation of the cryoconservation. However, these hypotheses raise difficulties that have not yet been resolved in the draft revision of the bioethics law.     

Phospholipid requirement for expression of ice nuclei in Pseudomonas syringae and in vitro

Delipidation of partially purified outer membranes of Pseudomonas syringae by various delipidating agents resulted in a significant loss of ice nucleation activity associated with the cell envelopes of this and other ice nucleation active bacteria. Lipopolysaccharide depletion of such membranes caused no reduction in ice nucleation activity. Both phospholipid content and ice nucleation activity of membranes were decreased by a similar fractional amount with time after treatment with phospholipase A2. A proportional quantitative relationship between loss of ice nucleation activity and lipid removal with increasing concentrations of sodium cholate and sodium dodecyl sulfate (SDS) was also observed. Significant linear relationships between the amount of lipid removed by phospholipase A2, sodium cholate, and SDS and the loss of ice nucleation activity in P. syringae outer membranes were observed. However, the slopes of these linear relationships for membranes treated with phospholipase A2 (m = 0.80), SDS (m = 0.94), and sodium cholate (m = 0.53) differed. The lower slope value for cholate-treated membranes indicated a partial substitution of sodium cholate for the phospholipids removed. The ice nucleation activity of delipidated outer membranes was restored by reconstitution with various phospholipids in a cholate dialysis procedure. Lipid classes differed in their ability to restore ice nucleation activity to sodium cholate-treated outer membranes. These results suggest that a hydrophobic environment provided either by lipids or certain detergent micelles is required for proper assembly and structural organization of an oligomeric ice protein complex enabling its expression as an ice nucleus.     

Scanning electron microscopic surface analysis of cryoconserved skull bone after decompressive craniectomy

Bone flaps removed during decompressive craniectomy are commonly frozen at ?80 °C and stored until cranioplasty. Histological integrity and regenerative capacity have been shown for cryoconserved bone. The effects of cryoconservation on the surface structure are unknown, although these might cause mechanical instability or facilitate bacterial adhesion. This study evaluates the surface structure of cryoconserved bone by scanning electron microscopy. Five patients were identified who could not receive their autologous bone flaps after decompressive craniectomy. These redundant bone specimens were obtained after cryoconservation for 6–8 months and the outer surface was analyzed by scanning electron microscopy. We found varying surface structures which did not correlate with any variables, such as patient age, gender or duration of freezing, and probably reflect physiological interindividual variation. Pathological findings, such as microscopic crack formation, were not observed. Cryoconservation for up to 8 months does not appear to alter the surface structure of skull bone on scanning electronic microscopy.     

Modulation of natural killer activity by muramyl peptides: relationship with adjuvant and anti-infectious properties

Natural killer (NK) activity of spleen cells was studied in DBA/2 mice, 24 and 72 h after intravenous injection of various muramyl peptides: muramyl dipeptide (MDP) and derivatives which are both adjuvant-active and able to increase resistance against Klebsiella pneumoniae; derivatives which are adjuvant-active but devoid of anti-infectious properties; derivatives which are anti-infectious but devoid of adjuvant activity, and derivatives which are devoid of both activities such as the stereoisomer MDP[D-Ala]1. An early increase in NK activity was observed 24 h after injection of all nonadjuvant derivatives, whatever their effect on infection. A stimulation of natural cytotoxicity was always induced 72 h after injection of MDP and derivatives able to protect mice against Klebsiella pneumoniae infection. So, even if the reverse was not true, there seems to exist some correlation between the anti-infectious effect of muramyl peptides and the late increase in NK activity. The modulation of NK activity by muramyl peptides appeared to be independent of interferon production. Moreover, inhibition of the stimulatory effect by a cell cycle-specific drug, hydroxyurea, observed 72 h after MDP suggests a requirement for proliferation.     

Mechanism of stabilization of synaptosomes with alpha-tocopherol during exposure to phospholipase A2

Changes in potential-dependent fluorescence were studied, using fluorescent probe di-S-C3-(5), in synaptosome suspensions exposed to phospholipase A2, alpha-tocopherol and its derivatives. Phospholipase A2 increased potential-dependent fluorescence, i.e. depolarization of synaptosome membranes. The damaging phospholipase A2 effect was prevented and/or abolished by alpha-tocopherol added to synaptosome suspensions before and after phospholipase A2. Alpha-tocopherol derivatives (2,2,5,7,8-pentamethyl-6-hydroxychromane and alpha-tocopheryl-acetate as well as 4-methyl-2,6-di-tert-butylphenol) failed to exert a protective effect on synaptosome membranes modified by phospholipase A2. It is suggested that alpha-tocopherol effect is determined by its interaction with fatty acids, with 6-hydroxy groups of chromanol nucleus and phytol chain being essential for the complex formation.     

In vitro studies of the influence of ELF electromagnetic fields on the activity of soluble and insoluble peroxidase

The influence of an extremely low frequency (ELF) magnetic field (50 Hz and 1 mT, EMF) on the activity of a soluble and insoluble horseradish peroxidase (E.C. 1.11.17) has been studied as a function of time. Insoluble derivatives were obtained by enzyme entrapment into two different gelatin membranes or by covalent attachment of the enzyme on two nylon membranes, differently preactivated. Results have shown that the field affects the inactivation rate of the soluble enzyme, while no effects are observed with insoluble derivatives. Since in vivo enzymes are immobilised into the biomembrane bilayer or entrapped into the cytoplasmic mixture, one might speculate that our experimental conditions do not reflect the catalytic activity of the enzymes in vivo.     

Hydrogen fluoride effects on plasma membrane composition and ATPase activity in needles of white pine (Pinus strobus) seedlings pretreated with 12 h photoperiod

 Eastern white pine (Pinus strobus L.) seedlings were pretreated with 12 h photoperiod to induce dormancy. Dormant plants were fumigated with 0.5 ppb (0.4 μg m^–3) or 2.0 ppb (1.6 μg m^–3) hydrogen fluoride (HF) for 2 – 28 days. Plasma membranes were isolated from needles of treated and control seedlings to determine their chemical composition and ATPase activity. For all analyses, only those plants which did not show needle necrosis were selected. The amount of plasma membrane phospholipid expressed on a plasma membrane protein basis was higher after 2 days in the 0.5 ppb HF treatment as compared to controls. After 2 days of 2.0 ppb HF treatment as well as after 8 and 28 days of both HF treatments phospholipid to protein ratios in fluoride treated seedlings were lower as compared to control levels. A decrease in sterol levels could be observed after 2 days in both HF treatments. A large increase in the ratio of sterols to proteins was observed in plasma membranes of eastern white pine seedlings treated with 0.5 ppb HF for 28 days. Increased sterol to phospholipid ratios were observed after 8 and 28 days in 0.5 ppb and after 2 and 8 days of 2.0 ppb HF treatment. A decrease in ATPase activity was observed after 8 days with both fluoride treatments. Drastic increase of ATPase activity was observed after 28 days of HF treated plants. Observed changes of sterol and phospholipid levels after only 2 days of fumigation suggest early fluoride effects on plasma membrane composition during plant dormancy. Received: 25 October 1995 / Accepted: 24 May 1996     

Polyene--sterol interaction and selective toxicity

From permeability experiments carried out with series of amphotericin B derivatives in both biological and model membranes, it was concluded that derivatives, whose carboxyl group at the C18 position is blocked by substitution, are much more efficient at inducing permeability in ergosterol-containing than in cholesterol-containing membranes, whereas derivatives whose carboxyl group is free and ionizable are equally efficient in both membranes types. Binding measurements on erythrocyte membranes showed that all amphotericin B derivatives simply partition between membrane lipids and aqueous medium, according to their lipid solubility. There is no relationship between binding and efficiency in inducing permeability. Permeability studies carried out on lipidic vesicles containing various sterols showed that: 1) derivatives having their carboxyl free induced permeability of the 'channel' type, regardless of the sterol present, and no detectable permeability in sterol-free membranes; 2) derivatives whose carboxyl group is blocked induce channels only in membranes containing ergosterol or sterols having an alkyl side chain identical to that of ergosterol. In the presence of other sterols or in sterol-free membranes, their ionophoric activity is poor and always of the 'mobile-carrier' type. A model of polyene-sterol interaction is proposed, accounting for the data obtained with both biological and model membranes.     

A spin label study of rat brain membranes. Effects of temperature and divalent cations.

Rat brain myelin, synaptosomal plasma membranes and synaptic vesicles were spin labelled with stearic acid nitroxide derivatives. Their electron spin resonance spectra were studied as a function of temperature and devalent ions (Ca2+ and Mg2+) concentrations. (1) Synaptosomal plasma membranes and synaptic vesicles show identical temperature variations of their order parameter (S = 0.58 at 35 degrees C and S = 0.72 AT 22 DEGREES C). Myelin appears more rigid (S = 0.66 at 35 degrees C and S = 0.76 at 22 degrees C). A discontinuity of the order parameter variation as a function of temperature, is observed between 14.5 degrees C and l9.5 degrees C with the three types of membranes. (2) The hydrophobic core of these membranes is very fluid. No transition temperature is observed. The measured values of the spin label rotation correlation times and rotational activation energies are 2.1 and 2.8 ns at 35 degrees C and 3.1 and 3.6 kcal/mol respectively for synaptosomal plasma membranes and myelin. (3) Ca2+ enhances the membrane rigidity (12+/-0.7% increase of the order parameter at 35 degrees C in the presence of 10(-3) M Ca2+) and increases the transition temperature. At a lower extend, similar effects are observed with Mg2+.     

Characterization of phospholipase A2 and acyltransferase activities in purified zymogen granule membranes

Phospholipase A2 and acyltransferase activities were identified in membranes associated with purified pancreatic zymogen granules. In homogenate and granule membranes, phospholipase activity was linearly related to protein concentration and was Ca2(+)-dependent with an alkaline pH optimum. The Ca2+ sensitivity was observed over the range of concentrations through which intracellular ionic Ca2+ is elevated by physiological stimuli in intact cells. Intact zymogen granules and granule membranes also demonstrated reacylating activity in the presence and absence of an exogenous acceptor. Reacylating activity was related to the concentration of lyosphospholipid added and was optimally activated at alkaline pH. A more rapid rate of reacylation was observed when [14C]arachidonoyl CoA was employed as the donor molecule rather than [3H]arachidonate (plus coenzyme A); this suggests the absence of acyl-CoA synthetase in the purified granule membranes. We conclude that granule membrane phospholipase A2 and acyltransferases may be involved in arachidonic acid turnover in exocrine pancreas and perhaps in membrane fusion events associated with exocytosis.     

Photoaffinity labeling of the tetrodotoxin binding component ofElectrophorus electricus electroplax

Radioactive azide derivatives of tetrodotoxin (TTX) were synthesized using 2-nitro-4-azidephenyl-[3H]beta alanine for the purpose of photolabeling of the Na channel. Three azide derivatives, N1, N2 and N3, were separated by ion exchange chromatography on Bio-Rex 70 resin and reversed phase high performance liquid chromatography. N3 was more stable and obtained at a higher yield than the other two derivatives. Bioactivity of N3 was one-twentieth of that of TTX. N3 showed reversible binding to membranes of Electrophorus electricus electroplax in the dark with Kd = 30 nM and B max = 5.2 pmol/mg protein. By photoirradiation, irreversible binding of N3 to the membranes was observed. A N3 binding component was solubilized by lubrol PX and partially purified from the electroplax membranes by Sephadex G25 and Sepharose 6B column chromatography. The component, purified 500 fold from the starting membranes, showed molecular weight of 10,000.     

Long-term storage of industrial microbial strains

Morphofunctional characteristics of industrial microorganisms belonging to different genera, species, and strains were investigated after 15 to 20 years of storage in liquid nitrogen. The taxonomic position of microorganisms, the cell physiological state prior to storage, and the cryoconservation regime were found to affect microbial cryoresistance. Protective media, density of cell suspensions, freezing rate, and heating temperature are the parameters important for development of efficient technologies for cryoconservation of industrial microorganisms at ?196°C.     

Characterization of Imido [8-3H] Guanosine 5′-Triphosphate Binding Sites to Rat Brain Membranes

Besides their well-defined intracellular roles in transmembrane signals transduction, guanine derivatives play important roles by acting from the outside of neural cell membranes. These roles are mediated by two different pool sites in cell membranes: G proteins, which bind to specific (GDP and GTP) intracellular guanine derivatives, and sites that bind to extracellular guanine derivatives. In this study we investigated some methodological characteristics of both guanine derivatives binding sites (intracellular and extracellular) in rat brain neural membranes. By investigating the binding of a poorly hydrolyzed GTP analogue and the adenylate cyclase activity in neural membranes, we observed some distinctiveness of guanine derivatives binding sites: stability to washing procedures (extracellular) and modulation of adenylate cyclase activity (intracellular). These results allow dealing with each site separately, which could be useful for discriminating the roles of extracellular and intracellular guanine derivatives in the central nervous system.     

The influence of ferrylhemoglobin and methemoglobin on the human erythrocyte membrane

The aim of the study was to examine and compare the effects of methemoglobin (metHb) and ferrylhemoglobin (ferrylHb) on the erythrocyte membrane. Kinetic studies of the decay of ferrylhemoglobin (*HbFe(IV)=O denotes ferryl derivative of hemoglobin present 5 min after initiation of the reaction of metHb with H(2)O(2); ferrylHb) showed that autoredecay of this derivative is slower than its decay in the presence of whole erythrocytes and erythrocyte membranes. It provides evidence for interactions between ferrylHb and the erythrocyte membrane. Both hemoglobin derivatives induced small changes in the structure and function of the erythrocyte membrane which were more pronounced for ferrylHb. The amount of ferrylHb bound to erythrocyte membranes increased with incubation time and, after 2 h, was twice that of membrane-bound metHb. The incubation of erythrocytes with metHb or ferrylHb did not influence osmotic fragility and did not initiate peroxidation of membrane lipids in whole erythrocytes as well as in isolated erythrocyte membranes. Membrane acetylcholinesterase activity increased by about 10% after treatment of whole erythrocytes with both metHb and ferrylHb. ESR spectra of membrane-bound maleimide spin label demonstrated minor changes in the conformation of label-binding proteins in ferrylHb-treated erythrocyte membranes. The fluidity of the membrane surface layer decreased slightly after incubation of erythrocytes and isolated erythrocyte membranes with ferrylHb and metHb. In whole erythrocytes, these changes were not stable and disappeared during longer incubation.     

Antimicrobial properties of analgesic kyotorphin peptides unraveled through atomic force microscopy

Antimicrobial peptides (AMPs) are promising candidates as alternatives to conventional antibiotics for the treatment of resistant pathogens. In the last decades, new AMPs have been found from the cleavage of intact proteins with no antibacterial activity themselves. Bovine hemoglobin hydrolysis, for instance, results in AMPs and the minimal antimicrobial peptide sequence was defined as Tyr-Arg plus a positively charged amino acid residue. The Tyr-Arg dipeptide alone, known as kyotorphin (KTP), is an endogenous analgesic neuropeptide but has no antimicrobial activity itself. In previous studies new KTP derivatives combining C-terminal amidation and Ibuprofen (Ib) - KTP-NH(2), IbKTP, IbKTP-NH(2) - were designed in order to improve KTP brain targeting. Those modifications succeeded in enhancing peptide-cell membrane affinity towards fluid anionic lipids and higher analgesic activity after systemic injection resulted therefrom. Here, we investigated if this affinity for anionic lipid membranes also translates into antimicrobial activity because bacteria have anionic membranes. Atomic force microscopy revealed that KTP derivatives perturbed Staphylococcus aureus membrane structure by inducing membrane blebbing, disruption and lysis. In addition, these peptides bind to red blood cells but are non-hemolytic. From the KTP derivatives tested, amidated KTP proves to be the most active antibacterial agent. The combination of analgesia and antibacterial activities with absence of toxicity is highly appealing from the clinical point of view and broadens the therapeutic potential and application of kyotorphin peptides.     

Introduction of alkynyl chains on C-8 of adenosine led to very selective antagonists of the A(3) adenosine receptor.

Some 8-alkynyladenosines were synthesized and evaluated for their adenosine receptor activity, utilizing radioligand binding studies (A(1), A(2A), A(3)) or adenylyl cyclase activity assays (A(2B)). Furthermore, the maximal induction of guanosine 5'-(gamma-thio)triphosphate ([35S]GTPgammaS) binding to G proteins and the inhibition of NECA-stimulated binding, in membranes of CHO cells which express the human A(3) receptor, were used to determine the intrinsic activity of these nucleosides at the A(3) adenosine receptor. The results showed that these new adenosine derivatives are very selective ligands for the A(3) receptor subtype and behave as adenosine antagonists, since they do not stimulate basal [35S]GTPgammaS binding, but inhibit NECA-stimulated binding. This is the first report that adenosine derivatives, with unmodified ribose moiety, are adenosine receptor antagonists.     

Induction of Cholinergic Expression in Developing Spinal Cord Cultures

The induction of choline acetyltransferase (ChAT) by cAMP derivatives was studied in dissociated spinal cord cultures. Dibutyryl cAMP (dbcAMP) and 8-bromo cAMP (1 mM) produced a 2-3-fold stimulation of ChAT activity in developing cultures whereas 8-bromo cGMP had no effect. A phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine, also increased (2-fold) ChAT activity in immature cultures. Significant elevations in ChAT were detected after 2 h incubation with dbcAMP. Maximum enzyme induction was observed 24 h after dbcAMP supplementation to the culture medium. Developmental studies revealed that ChAT could be induced on days 2-16 in culture. The largest induction of ChAT activity was observed on day 7 in culture. After day 19, when control enzyme activity attained levels of mature cultures, cAMP-mediated ChAT induction was no longer observed. Cycloheximide and actinomycin D blocked ChAT induction whereas basal enzyme activity remained unaffected. Culture protein content was not changed after 1-day exposure to dbcAMP. 125I-Tetanus toxin fixation after dbcAMP treatment revealed a 20% decrease from control in neuronal surface during days 7-9 in culture. These data indicated that cAMP derivatives produced a rapid increase in cholinergic expression during a specific period of development in spinal cord cultures. There appears to be specificity to this effect, as total neuronal surface does not respond in the same manner as ChAT activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+ -ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+ -ATPase and the Mg2+ -ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membrane-bound Mg2+ -ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20 degrees C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+ -ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at -20 degrees C, which was characteristic of hepatoma plasma membrane Mg2+ -ATPase. With solubilized Mg2+ -ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+ -ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at -20 degrees C.     

Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.