Stablilzation of two-standard ribohomopolymer helices and destabilzation of a three-stranded helix by ethidium bromide.
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ABSTRACT:
SUBMITTER: Waring MJ
PROVIDER: S-EPMC1168409 | biostudies-other | 1974 Nov
REPOSITORIES: biostudies-other
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Project description:Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing.
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Project description:Alanine-based peptides of defined sequence and length show measurable helix contents, allowing them to be used as a model system both for analyzing the mechanism of helix formation and for investigating the contributions of side-chain interactions to protein stability. Extensive characterization of many peptide sequences with varying amino acid contents indicates that the favorable helicity of alanine-based peptides can be attributed to the large helix-stabilizing propensity of alanine. Based on their analysis of alanine-rich sequences N-terminally linked to a synthetic helix-inducing template, Kemp and coworkers [Kemp, D. S., Boyd, J. G. & Muendel, C. C. (1991) Nature (London) 352, 451-454; Kemp, D. S., Oslick, S. L. & Allen, T. J. (1996) J. Am. Chem. Soc. 118, 4249-4255] argue that alanine is helix-indifferent, however, and that the favorable helix contents of alanine-based peptides must have some other explanation. Here, we show that the helix contents of template-nucleated sequences are influenced strongly by properties of the template-helix junction. A model in which the helix propensities of residues at the template-peptide junction are treated separately brings the results from alanine-based peptides and template-nucleated helices into agreement. The resulting model provides a physically plausible resolution of the discrepancies between the two systems and allows the helix contents of both template-nucleated and standard peptide helices to be predicted by using a single set of helix propensities. Helix formation in both standard peptides and template-peptide conjugates can be attributed to the large intrinsic helix-forming tendency of alanine.
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Project description:Ethidium Bromide (EB) is a commonly used dye in a deoxyribonucleic acid (DNA) study. Upon an intercalation, this dye significantly increases its brightness and fluorescence lifetime. In this report we have studied the time-resolved fluorescence properties of EB existing simultaneously in free and DNA-bound forms in the solution. Fluorescence intensity decays were fitted globally to a double exponential model with lifetimes corresponding to free (1.6ns) and bound (22ns) forms, and molar fractions were determined for all used solutions. Anisotropy decays displayed characteristic time dependence with an initial rapid decline followed by recovery and slow decay. The short-lived fraction associated with free EB molecules decreases faster than long-lived fraction associated with EB bound to DNA. Consequently, contribution from fast rotation leads to initial rapid decay in anisotropy. On the other hand bound fraction, due to slow rotation helps recover anisotropy in time. This effect of associated anisotropy decays in systems such as EB free/EB-DNA is clearly visible in a wide range of concentrations, and should be taken into account in polarization assays and biomolecule dynamics studies.
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Project description: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.
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Project description:Ethidium bromide (EtBr) is widely used in most laboratories to detect nucleic acids in gel electrophoresis applications. It is a well-known carcinogenic and mutagenic agent, which can affect biotic components of the place in which it is disposed. Usually the gel-waste is either buried in the ground or incinerated, whereas the liquid waste is disposed of down the sink following the recommended methods of treatment. The recommended methods do not involve biological potential, but rather make use of chemicals, which may further deteriorate soil and water quality. The present study identifies and characterizes the EtBr-degrading bacterial isolates BR3 and BR4. A bibliographic review of the risk status of using these isolates for the treatment of lab waste in laboratory settings is also presented. BR3 was identified as Proteus terrae N5/687 (LN680103) and BR4 as Morganella morganii subsp. morganii ATCC 25830 (AJ301681) with 99.9% and 99.48% similarity, respectively, using an EzBioCloud microbial identifier. The literature revealed the bacterium Proteus terrae as a non-pathogenic and natural microflora of humans, but Morganella morganii as an opportunistic pathogen. These organisms belong to risk group II. Screening the sensitivity of these isolates to antibiotics revealed a sufficient number of antibiotics, which can be used to control them, if required. BR3 and BR4 exhibited resistance to individual antibiotics, ampicillin and vancomycin, whereas only BR3 was resistant to tetracycline. The current investigation, along with earlier reported work on these isolates, identifies BR3 as a useful isolate in the industrial application for the degradation of EtBr. Identical and related microorganisms, which are available in the culture collection repositories, can also be explored for such potential to formulate a microbial consortium for the bioremediation of ethidium bromide prior to its disposal.
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