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The distance between nodes of Ranvier, referred to as internode length, positively correlates with axon diameter, and is optimized during development to ensure maximal neuronal conduction velocity. Following myelin loss, internode length is reestablished through remyelination. However, remyelination results in short internode lengths and reduced conduction rates. We analyzed the potential role of neurofilament phosphorylation in regulating internode length during remyelination and myelination. Following ethidium bromide induced demyelination, levels of neurofilament medium (NF-M) and heavy (NF-H) phosphorylation were unaffected. Preventing NF-M lysine-serine-proline (KSP) repeat phosphorylation increased internode length by 30% after remyelination. To further analyze the role of NF-M phosphorylation in regulating internode length, gene replacement was used to produce mice in which all KSP serine residues were replaced with glutamate to mimic constitutive phosphorylation. Mimicking constitutive KSP phosphorylation reduced internode length by 16% during myelination and motor nerve conduction velocity by ~27% without altering sensory nerve structure or function. Our results suggest that NF-M KSP phosphorylation is part of a cooperative mechanism between axons and Schwann cells that together determine internode length, and suggest motor and sensory axons utilize different mechanisms to establish internode length.

Molecular and cellular mechanisms underlying the peripheral conditioning lesion remain unsolved. We show here that injection of a chemical demyelinating agent, ethidium bromide, into the sciatic nerve induces a similar set of regeneration-associated genes and promotes a 2.7-fold greater extent of sensory axon regeneration in the spinal cord than sciatic nerve crush. We found that more severe peripheral demyelination correlates with more severe functional and electrophysiological deficits, but more robust central regeneration. Ethidium bromide injection does not activate macrophages at the demyelinated sciatic nerve site, as observed after nerve crush, but briefly activates macrophages in the dorsal root ganglion. This study provides a new method for investigating the underlying mechanisms of the conditioning response and suggests that loss of the peripheral myelin may be a major signal to change the intrinsic growth state of adult sensory neurons and promote regeneration.

Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells. © 2012 Society of Photo-Optical Instrumentation Engineers (SPIE).

Herein, we study the nanomechanical characteristics of single DNA molecules in the presence of DNA binders, including intercalating agents (ethidium bromide and doxorubicin), a minor groove binder (netropsin) and a typical alkylating damaging agent (cisplatin). We have used magnetic tweezers manipulation techniques, which allow us to measure the contour and persistence lengths together with the bending and torsional properties of DNA. For each drug, the specific variations of the nanomechanical properties induced in the DNA have been compared. We observed that the presence of drugs causes a specific variation in the DNA extension, a shift in the natural twist and a modification of bending dependence on the imposed twist. By introducing a naive model, we have justified an anomalous correlation of torsion data observed in the presence of intercalators. Finally, a data analysis criterion for discriminating between different molecular interactions among DNA and drugs has been suggested.

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors.

OBJECTIVES: Evaluation of novel synthetic analogues of piperine as inhibitors of multidrug efflux pump NorA of Staphylococcus aureus.METHODS: A library of piperine-derived compounds was evaluated for their potential to inhibit ethidium bromide efflux in NorA-overexpressing S. aureus SA 1199B. The active compounds were then individually combined with ciprofloxacin to study the potentiation of ciprofloxacin's activity.RESULTS: Based on the efflux inhibition assay, a library of 200 compounds was screened. Three piperine analogues, namely SK-20, SK-56 and SK-29, were found to be the most potent inhibitors of the NorA efflux pump. These inhibitors acted in a synergistic manner with ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. These analogues were 2- to 4-fold more potent than piperine at a significantly lower minimal effective concentration. Furthermore, these inhibitors also significantly suppressed the in vitro emergence of ciprofloxacin-resistant S. aureus.CONCLUSIONS: A newly identified class of compounds derived from a natural amide, piperine, is more potent than the parent molecule in potentiating the activity of ciprofloxacin through the inhibition of the NorA efflux pump. These molecules may prove useful in augmenting the antibacterial activities of fluoroquinolones in a clinical setting.

Based on our recent findings that piperine is a potent Staphylococcus aureus NorA efflux pump inhibitor (EPI), 38 piperine analogs were synthesized and bioevaluated for their EPI activity. Twenty-five of them were found active with potentiating activity equivalent or more than known EPIs like reserpine, carsonic acid and verapamil. The inhibitory mechanism of the compounds was confirmed by efflux inhibition assay using ethidium bromide as NorA substrate. The present communication describes the synthesis, bioevaluation and structure related activity of these efflux pump inhibitors.

OBJECTIVES: Evaluation of novel synthetic analogues of piperine as inhibitors of multidrug efflux pump NorA of Staphylococcus aureus.METHODS: A library of piperine-derived compounds was evaluated for their potential to inhibit ethidium bromide efflux in NorA-overexpressing S. aureus SA 1199B. The active compounds were then individually combined with ciprofloxacin to study the potentiation of ciprofloxacin's activity.RESULTS: Based on the efflux inhibition assay, a library of 200 compounds was screened. Three piperine analogues, namely SK-20, SK-56 and SK-29, were found to be the most potent inhibitors of the NorA efflux pump. These inhibitors acted in a synergistic manner with ciprofloxacin, by substantially increasing its activity against both NorA-overexpressing and wild-type S. aureus isolates. These analogues were 2- to 4-fold more potent than piperine at a significantly lower minimal effective concentration. Furthermore, these inhibitors also significantly suppressed the in vitro emergence of ciprofloxacin-resistant S. aureus.CONCLUSIONS: A newly identified class of compounds derived from a natural amide, piperine, is more potent than the parent molecule in potentiating the activity of ciprofloxacin through the inhibition of the NorA efflux pump. These molecules may prove useful in augmenting the antibacterial activities of fluoroquinolones in a clinical setting.

We have compared the formation of pores in rat submandibular acinar cells in response to 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (Bz-ATP) and maitotoxin. Bz-ATP (100 microM) permeabilized the cells to ethidium bromide. The uptake of ethidium increased to 29+/-1% of maximal uptake in 10 min. DL-Propranolol (300 microM) inhibited the Bz-ATP-induced uptake of ethidium bromide by 40% without affecting the P2X(7)-gated cation channel. The inhibitory effect of DL-propranolol on the formation of pores by Bz-ATP was reproduced by D-propranolol, an optical isomer with very poor beta-blocking activity. Tenidap, an antiinflammatory drug, enhanced the permeabilization in response to Bz-ATP. Propanolol inhibited the response to tenidap plus Bz-ATP. The effect of propranolol was reproduced by labetolol, a beta-adrenergic antagonist with membrane-stabilizing properties, but not by atenolol, which blocks beta-adrenergic receptors but has no effect on the stability of the membrane. In the presence of extracellular calcium, maitotoxin also increased the uptake of ethidium bromide. Tenidap had no effect on this response, which was delayed by propranolol. In conclusion, we have shown that propranolol, in a range of 10-300 microM, inhibits the pore-forming activity of the P2X(7) receptor without affecting the opening of the cation channel coupled to this receptor. This inhibition is not related to its beta-adrenergic blocking activity but rather to its membrane-stabilizing properties. Propranolol also delays the uptake of ethidium bromide in response to maitotoxin. This is in agreement with the current view that P2X(7) agonists and maitotoxin share a common pore., Comparative Study, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

We have compared the formation of pores in rat submandibular acinar cells in response to 2',3'-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (Bz-ATP) and maitotoxin. Bz-ATP (100 microM) permeabilized the cells to ethidium bromide. The uptake of ethidium increased to 29+/-1% of maximal uptake in 10 min. DL-Propranolol (300 microM) inhibited the Bz-ATP-induced uptake of ethidium bromide by 40% without affecting the P2X(7)-gated cation channel. The inhibitory effect of DL-propranolol on the formation of pores by Bz-ATP was reproduced by D-propranolol, an optical isomer with very poor beta-blocking activity. Tenidap, an antiinflammatory drug, enhanced the permeabilization in response to Bz-ATP. Propanolol inhibited the response to tenidap plus Bz-ATP. The effect of propranolol was reproduced by labetolol, a beta-adrenergic antagonist with membrane-stabilizing properties, but not by atenolol, which blocks beta-adrenergic receptors but has no effect on the stability of the membrane. In the presence of extracellular calcium, maitotoxin also increased the uptake of ethidium bromide. Tenidap had no effect on this response, which was delayed by propranolol. In conclusion, we have shown that propranolol, in a range of 10-300 microM, inhibits the pore-forming activity of the P2X(7) receptor without affecting the opening of the cation channel coupled to this receptor. This inhibition is not related to its beta-adrenergic blocking activity but rather to its membrane-stabilizing properties. Propranolol also delays the uptake of ethidium bromide in response to maitotoxin. This is in agreement with the current view that P2X(7) agonists and maitotoxin share a common pore., Comparative Study, Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

HD caused apoptosis and to a lesser degree, necrosis in a wide variety of cell types. As the concentration of HD was increased the proportion of necrotic cells became more predominant. HD produced a modest elevation of intracellular calcium levels in J774 and CHO-Kl cells, as well as in human skin keratinocytes, although this rise did not seem causally related to HD induced cell death. However, very recent evidence indicates that HD induced DNA fragmentation may not he directly related to cytotoxicity. HD also activated caspase-3 in CHO-K1 cells, but neither specific nor general caspase inhibitors nor proteosome inhibitors reduced HD cytotoxicity. These results suggest that the toxicity of HD is fundamentally different than apoptotic stimulae such as dexamethasone or X-irradiation, in which the apoptosis induced by both of these treatments can he reduced by the above mentioned protease inhibitors. Subtypes of the P2X(sub 7) receptors were identified in neuronal synaptosomes, J774 cells and CHO-Kl cells. In CHO-K1 cells P2X(sub 7) receptor subtypes were identified and the specific P2X(sub 7) inhibitor, oxidized ATP, was found effective in reducing HD induced DNA fragmentation and the appearance of soluble DNA. However, this treatment appeared only to shunt the mechanism of cell death from being predominantly apoptotic, to more necrotic in nature; however, the overall cytotoxicity remained unchanged., Original contains color plates: All DTIC reproductions will be in black and white.

The permeabilizing effect of P2X(7) agonists was tested in rat submandibular acinar cells using the uptake of ethidium bromide as an index. The uptake of ethidium bromide by acini incubated at 37 degrees C in the presence of 1 mM ATP increased with time and reached after 5 min about 10% of maximal uptake measured in the presence of digitonin. The response to ATP was dose-dependent (half-maximal concentration around 40 microM) and it was decreased when the temperature was lowered to 25 degrees C. Benzoyl-ATP reproduced the response to ATP (half-maximal concentration around 10 microM). UTP or 2-methylthioATP had no effect. The permeabilization in response to ATP was blocked by oxidized ATP and by magnesium and inhibited by Coomassie blue. ATP increased the activity of a calcium-insensitive phospholipase A(2) (iPLA(2)). Bromoenol lactone (BEL) inhibited the iPLA(2) stimulated by ATP but potentiated the uptake of ethidium bromide in response to the purinergic agonist. From these results it is concluded that the activation of P2X(7) receptors permeabilizes rat submandibular acinar cells. The pore-forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA(2) by the receptor., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

The permeabilizing effect of P2X(7) agonists was tested in rat submandibular acinar cells using the uptake of ethidium bromide as an index. The uptake of ethidium bromide by acini incubated at 37 degrees C in the presence of 1 mM ATP increased with time and reached after 5 min about 10% of maximal uptake measured in the presence of digitonin. The response to ATP was dose-dependent (half-maximal concentration around 40 microM) and it was decreased when the temperature was lowered to 25 degrees C. Benzoyl-ATP reproduced the response to ATP (half-maximal concentration around 10 microM). UTP or 2-methylthioATP had no effect. The permeabilization in response to ATP was blocked by oxidized ATP and by magnesium and inhibited by Coomassie blue. ATP increased the activity of a calcium-insensitive phospholipase A(2) (iPLA(2)). Bromoenol lactone (BEL) inhibited the iPLA(2) stimulated by ATP but potentiated the uptake of ethidium bromide in response to the purinergic agonist. From these results it is concluded that the activation of P2X(7) receptors permeabilizes rat submandibular acinar cells. The pore-forming activity of the receptor might be negatively regulated by the concomitant activation of the iPLA(2) by the receptor., Journal Article, Research Support, Non-U.S. Gov't, FLWIN, info:eu-repo/semantics/published

Exposure to HD of a selected number of phylogenetically different cell types (human skin keratinocytes, J774 cells, CRO-K1 cells, thymocytes and chick neurons) resulted in concentration-dependent cytotoxicity. HD induced apoptosis was shown to be common to all cell types tested as illustrated by annexin staining, production of DNA fragments, soluble DNA assay, morphology (acridine/ethidium bromide staining) and positive TUNEL reaction. Apoptosis was prevalent at lower concentrations, while necrosis was relatively more evident at higher HD concentrations. Calcium levels were elevated in HD exposed human keratinocytes, J774 cells and CHO-K1 cells at concentrations of HD that ultimately caused cell death. HD induced cytotoxicity (apoptosis) was unaffected by manipulation of calcium levels prior to exposure, suggesting no cause-effect relationship in keratinocytes. The role of calcium in other cell types is currently being examined. Studies using molecular and immunohistochemical techniques to identify ATP receptor types in HD sensitive cells revealed novel findings, including the presence of P2X1 and P2X7 receptors on vas deferens, and on immature thymocytes. ATP induced calcium influx in synaptosomes was demonstrated following activation of a novel CNS P2X7 receptor. HD did not potentiate this influx. The effects of caspase inhibitors and the role of cytoprotective genes in HD toxicity are currently being investigated.

In the presence of double helical polynucleotides (sodium poly(dA-dT).poly(dA-dT) or calf thymus DNA), the efficiency of oxidative or reductive electron transfer between photoexcited ruthenium(II) chelates Ru(tap)2(hat)2+ or Ru(phen)2+(3) (where tap = 1,4,5,8-tetraazaphenanthrene, hat = 1,4,5,8,9,12-hexaazatriphenylene, and phen = 1,10-phenanthroline) and appropriate cationic quenchers (ethidium, Ru(NH3)3+(6), methyl viologen, or M(phen)3+(3), where M = Co, Rh, Cr) increases 1-2 orders of magnitude compared to the efficiency of the same quenching in microhomogeneous aqueous medium (kq = 0.3-1.8 x 10(9) M-1 s-1). The enhancement is more pronounced when the binding constant of the quencher (10(3) less than Kb less than 10(6) M-1) is large. Similar trends are found when the biopolymers are replaced by sodium poly(styrenesulfonate) (PSS). The accelerated electron transfer process is proposed to be due mainly to the effect of accumulation of the reagents in the electrostatic field of the polymer; if corrections for this effect are introduced (e.g. ratioing [quencher]/[polynucleotide]), the reaction rate becomes essentially independent of the polymer concentration. Based upon a model for electron transfer reaction of the complexes within a small cylindrical interface around the DNA helix, calculations of the bimolecular electron transfer rate constants are computed to be 10(3) times smaller when the reactants are bound to the double-stranded polynucleotides and decreased mobility of the cationic species is apparent. The effect is less pronounced if a simpler polyelectrolyte (PSS) is employed. Emission lifetimes of the Ru(II) polypyridyls bound to the DNA (0.32-2 microseconds, double exponential decays) are discussed as well., Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S., FLWNA, info:eu-repo/semantics/published

In the presence of double helical polynucleotides (sodium poly(dA-dT).poly(dA-dT) or calf thymus DNA), the efficiency of oxidative or reductive electron transfer between photoexcited ruthenium(II) chelates Ru(tap)2(hat)2+ or Ru(phen)2+(3) (where tap = 1,4,5,8-tetraazaphenanthrene, hat = 1,4,5,8,9,12-hexaazatriphenylene, and phen = 1,10-phenanthroline) and appropriate cationic quenchers (ethidium, Ru(NH3)3+(6), methyl viologen, or M(phen)3+(3), where M = Co, Rh, Cr) increases 1-2 orders of magnitude compared to the efficiency of the same quenching in microhomogeneous aqueous medium (kq = 0.3-1.8 x 10(9) M-1 s-1). The enhancement is more pronounced when the binding constant of the quencher (10(3) less than Kb less than 10(6) M-1) is large. Similar trends are found when the biopolymers are replaced by sodium poly(styrenesulfonate) (PSS). The accelerated electron transfer process is proposed to be due mainly to the effect of accumulation of the reagents in the electrostatic field of the polymer; if corrections for this effect are introduced (e.g. ratioing [quencher]/[polynucleotide]), the reaction rate becomes essentially independent of the polymer concentration. Based upon a model for electron transfer reaction of the complexes within a small cylindrical interface around the DNA helix, calculations of the bimolecular electron transfer rate constants are computed to be 10(3) times smaller when the reactants are bound to the double-stranded polynucleotides and decreased mobility of the cationic species is apparent. The effect is less pronounced if a simpler polyelectrolyte (PSS) is employed. Emission lifetimes of the Ru(II) polypyridyls bound to the DNA (0.32-2 microseconds, double exponential decays) are discussed as well., Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S., FLWNA, info:eu-repo/semantics/published

This report is divided into three sections dealing with the development and use of fluorescent probes of the nicotinic acetylcholine receptor (AcChR). Section I summarized the purification and characterization of four mono-fluorescein cobra a-toxin derivatives. Section II deals with the use of the about derivatives to define the approximate orientation of cobra a-toxin on the acetylcholine receptor. Section III summarizes our measurements of the intersite distances between the binding sites for acetylcholine and phencyclidine on the nicotinic acetylcholine receptor.

The axoplasmic migration of ribosomes has been detected in the visual system of the chick. Monocular injection of radioactive uridine or an amino acid mixture was followed by sedimentation analysis in sucrose or cesium sulfate density gradients, of ribosomes prepared from the retinae of injected eyes and the left and right optic lobes. By this means both RNA and protein components of ribosomes were found to migrate from the retina to the innervated contralateral optic lobe. Following denervation of the distal nerve segment by eye removal, the stability of the transported RNA was reduced, suggesting its presynaptic location. The transport of RNA was not significantly imparied by intraocular injection of inhibitors of informational RNA or mitochondrial RNA synthesis prior to injection of radioactive uridine but was depressed by a low dose of actinomycin D.

The axoplasmic migration of ribosomes has been detected in the visual system of the chick. Monocular injection of radioactive uridine or an amino acid mixture was followed by sedimentation analysis in sucrose or cesium sulfate density gradients, of ribosomes prepared from the retinae of injected eyes and the left and right optic lobes. By this means both RNA and protein components of ribosomes were found to migrate from the retina to the innervated contralateral optic lobe. Following denervation of the distal nerve segment by eye removal, the stability of the transported RNA was reduced, suggesting its presynaptic location. The transport of RNA was not significantly imparied by intraocular injection of inhibitors of informational RNA or mitochondrial RNA synthesis prior to injection of radioactive uridine but was depressed by a low dose of actinomycin D.

This work is an ultrastructural and cytochemical study of a structure observed in the nucleolus of Allium cepa meristematic cells after nucleolar disgregation, by a continuous treatment of 12 h with ethidium bromide. The ultrastructural and cytochemical data allow us to consider this structure as the intranucleolar chromatin collapsed by the effect of the drug.