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Background: This study introduces a newly created strain (Rhodococcus equiEtBr25) by exposing R. equi ATCC 33701 to ethidium bromide (EtBr), a substrate for MDR transporters. Such an approach allowed us to investigate the resulting phenotype and genetic mechanisms underlying the efflux-mediated resistance in R. equi., Methods: R. equi ATCC 33701 was stimulated with increasing concentrations of EtBr. The antimicrobial susceptibility of the parental strain and R. equiEtBr25 was investigated in the presence/absence of efflux pump inhibitors (EPIs). EtBr efflux was evaluated by EtBr-agar method and flow cytometry. The presence of efflux pump genes was determined by conventional PCR before to quantify the expression of 30 genes coding for membrane transporters by qPCR. The presence of erm(46) and mutations in 23S rRNA, and gyrA/gyrB was assessed by PCR and DNA sequencing to exclude the occurrence of resistance mechanisms other than efflux., Results: R. equi EtBr25 showed an increased EtBr efflux. Against this strain, the activity of EtBr, azithromycin and ciprofloxacin was more affected than that of rifampicin and azithromycin/rifampicin combinations. Resistances were reversed by combining the antimicrobials with EPIs. Gene expression analysis detected a marked up-regulation of REQ_RS13460 encoding for a Major Facilitator Superfamily (MFS) transporter. G→A transition occurred in the transcriptional repressor tetR/acrR adjacent to REQ_RS13460., Conclusions: Exposure of R. equi to EtBr unmasked an efflux-mediated defence against azithromycin and ciprofloxacin, which seemingly correlates with the overexpression of a specific MFS transporter. This genotype may mirror an insidious low-level resistance of clinically important isolates that could be countered by EPI-based therapies., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: The oxazolidinone class of antibiotics such as linezolid have a narrow spectrum of activity that targets Gram-positive bacteria. We hypothesized that the poor activity of linezolid in Gram-negative bacteria is in part caused by relatively low intracellular concentration due to efflux., Methods: Using whole cell accumulation assays we estimated the intracellular concentration of linezolid in Escherichia coli and other Enterobacteriaceae. We included test strains with enhanced RND-type multidrug efflux pump activity and with genetic inactivation of the pump or functional inhibition by carbonyl cyanide m-chlorophenylhydrazone as inhibitor of the proton motive force or 1-(1-naphthylmethyl)-piperazine (NMP), an efflux pump inhibitor., Results: Consistent with susceptibility studies, enhanced pump activity caused decreased accumulation, and pump inactivation and inhibition caused increased accumulation, of linezolid. The accumulation levels in test strains of E. coli, Citrobacter freundii and Enterobacter aerogenes with functional pumps were lower than in control strains of Staphylococcus aureus and Enterococcus faecium, but were higher after pump inactivation and correlated with ethidium bromide and pyronin Y accumulation., Conclusions: The intracellular concentration of linezolid is comparatively low owing to efficient efflux of the drug and could be increased substantially by inhibition of RND-type efflux pumps.

Demyelination of CNS axons occurs in a number of pathological conditions, including multiple sclerosis and contusion-type spinal cord injury. The demyelination can be repaired by remyelination in both humans and rodents, and even within the CNS remyelination can be achieved by endogenous and/or exogenous Schwann cells, the myelinating cells of the PNS. Remyelinated axons can often conduct impulses securely, but the organization of ion channels at long-term remyelinated nodes is not known. In the present study, the expression of voltage-gated sodium (Na(v)) and potassium (K(v)) channels along central axons remyelinated by endogenous Schwann cells has been studied in lesions induced more than 1 year previously by the intraspinal injection of ethidium bromide (EB). The expression of the channels at long-term nodes formed by Schwann cell remyelination has been compared with that present in nascent nodes formed in the adult at 18 and 23 days post-EB injection. Immunohistochemical studies revealed that long-term nodes formed by Schwann cell remyelination exhibit a clustering of Na(v)1.6 sodium channels within the nodal membrane, with the Shaker-type potassium channel K(v)1.2 segregated within the juxtaparanodal region, similar to the arrangement at normal mature CNS nodes. Na(v)1.2 was not detected at nodes formed by Schwann cells at any stage of their development. Moreover, Na(v)1.6, but not Na(v)1.2, was clustered at nascent nodes formed by remyelinating Schwann cells 18 and 23 days following EB injection. These observations show that endogenous Schwann cells can establish and maintain nodes of Ranvier on central axons for over one year, and that the nodes exhibit an apparently normal distribution of sodium and potassium channels, with Na(v)1.6 the predominant subtype of sodium channel present at such nodes at all stages of their development.

The most conspicuous internal structures of the endospore of Rhinosporidium seeberi are the 10-16 spherical, 1.0-1.5-microm bodies that have been termed electron-dense bodies (EDB) or lipid bodies (LB); some authors have regarded them as nutritive stores of lipid or protein while others have regarded them as DNA-containing, ultimate generative units of R. seeberi. The literature is reviewed as supporting either view. We report, for the first time, (i) reactions of the endospores with the salt MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) and (ii) ultrastructural appearances; and suggest that both views on the nature of spherical bodies are valid (i.e. the endospore contains both EDB, and lipid or protein bodies). Well-marked reduction of the MTT with the production of deep-purple staining was seen in a proportion of the spherical bodies, probably the EDB, suggesting that they are actively metabolizing, viable elements with dehydrogenase activity, and that these bodies are the thick-walled electron-dense bodies described as EDB and visualized in the transmission electron microphotograph illustrated in this paper. The spherical bodies showed fluorescent labeling with acridine orange and with ethidium bromide supporting the idea that they contained nucleic acids. TMRE (tetramethyl rhodamine ethyl ester), a mitochondrion-specific dye, also labeled the intra-endosporial spherical bodies. Other bodies (LB) of a similar size that were MTT-non-reducing, electron lucent, and have no organized structure, are probably the lipid or protein containing, inert, nutritive storage bodies suggested by previous authors.

Objectives: This prospective study focused on the evaluation of anti-parasitic therapy in congenital Chagas' disease, diagnosed and monitored by PCR and conventional diagnosis., Materials and Methods: We studied 152 children born to seroreactive mothers, living in a non-endemic area. Fifty infants aged 0-6 months (GA) were diagnosed by microhaematocrit and PCR and 102 children aged 7 months to 17 years (GB) were diagnosed by serology and PCR. Forty treated patients were monitored for 2 or 3 years by PCR and conventional methods. A competitive-quantitative PCR was used to determine pre-therapy parasitic loads and follow their post-treatment evolution., Results: In GA, the sensitivities of the PCR and microhaematocrit were 100% and 82.4% and their specificities 97% and 100%, respectively. In GB, the sensitivity of the PCR was 73.8% with a specificity of 100%. Pre-therapy parasitic loads ranged from 12.5 to 125,000 and 12.5 to 125 parasite genomic equivalents/mL of blood in GA and GB, respectively. PCR turned negative in all treated pre-therapy PCR positive patients before or at the end of treatment, which was followed by their seronegativation in 10/10 GA, in 3/5 children initiating therapy at 7 months to 2 years of age but in 0/16 initiating therapy at an older age. Two out of the latter patients were occasionally PCR positive during post-treatment, suggesting no parasitological response. Out of nine pre-therapy PCR negative patients, four turned seronegative after treatment, suggesting that in undetermined patients, undetectable parasitic burdens may lead to better post-treatment prognosis., Conclusions: PCR was useful for sensitive diagnosis and therapy monitoring, allowing early detection of refractory cases.

Exposure to lead causes decreased fertility in women. In the present study, we examined the in vitro effects of lead on cytochrome p450 aromatase (p450 arom) and on estrogen receptor beta (ERbeta), two key proteins for the human ovary. Aromatase is required for the bioconversion of androgen to estradiol; ERbeta mediates estrogen effects in granulosa cells. Granulosa cells were collected from women undergoing in vitro fertilization and then cultured with 10 microM lead acetate. Using atomic absorption spectrometry, we showed that lead accumulated in cells. Aromatase activity as measured by a tritiated water production assay was significantly reduced. Using semiquantitative reverse transcription-polymerase chain reaction and Western blotting procedures, we showed that p450 arom and ERbeta mRNA and protein content were both significantly reduced. Adding 10 microg/ml of cycloheximide, a protein inhibitor, did not eliminate the effects of lead. The present results support the hypothesis that the action of lead on fertility in women may result, in part, from the down-regulation of p450 arom and ERbeta gene transcription in ovarian granulosa.

It has been hypothesized that the progressive failure of remyelination in chronic multiple sclerosis is, in part, the consequence of repeated episodes of demyelination at the same site, eventually depleting oligodendrocyte progenitor cells (OPCs) and exhausting the remyelinating capacity. We investigated the effect of previous focal, ethidium bromide-induced demyelination of brain stem white matter (with intervening recovery) on the efficiency of the remyelination process during second and third subsequent episodes of demyelination, and the OPC response during a second episode of demyelination. Previous focal demyelinating lesions followed by recovery did not result in any retardation of the remyelination process, nor did they alter the proportion of Schwann cell versus oligodendrocyte remyelination. The OPC response during remyelination was quantified by in situ hybridization using a probe to platelet-derived growth factor-alpha receptor (PDGF alpha R), an OPC-expressed mRNA. Following recovery from focal, toxin-induced CNS demyelination, the OPC density returned to levels equivalent to those in normal white matter. Further more, there was no depletion of OPCs following repeated episodes of focal, toxin-induced CNS demyelination at the same site. These results indicate that repeated CNS demyelination, which has the opportunity to repair in the intervening period, is not characterized by impaired remyelination or depletion of OPCs.

To study the structure of partially replicated plasmids, we cloned the Escherichia coli polar replication terminator TerE in its active orientation at different locations in the ColE1 vector pBR18. The resulting plasmids, pBR18-TerE@StyI and pBR18-TerE@EcoRI, were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis and electron microscopy. Replication forks stop at the Ter-TUS complex, leading to the accumulation of specific replication intermediates with a mass 1.26 times the mass of non-replicating plasmids for pBR18-TerE@StyI and 1.57 times for pBR18-TerE@EcoRI. The number of knotted bubbles detected after digestion with ScaI and the number and electrophoretic mobility of undigested partially replicated topoisomers reflect the changes in plasmid topology that occur in DNA molecules replicated to different extents. Exposure to increasing concentrations of chloroquine or ethidium bromide revealed that partially replicated topoisomers (CCCRIs) do not sustain positive supercoiling as efficiently as their non-replicating counterparts. It was suggested that this occurs because in partially replicated plasmids a positive DeltaLk is absorbed by regression of the replication fork. Indeed, we showed by electron microscopy that, at least in the presence of chloroquine, some of the CCCRIs of pBR18-Ter@StyI formed Holliday-like junction structures characteristic of reversed forks. However, not all the positive supercoiling was absorbed by fork reversal in the presence of high concentrations of ethidium bromide.

The link between cell adhesion to extracellular matrix and integrin-mediated survival signals has been established in several physiological systems, and roles for the cytokines tumor necrosis factor alpha (TNF alpha) and transforming growth factor alpha (TGF alpha) have been suggested. TGF alpha stimulates fibronectin production in hen granulosa cells and is an important survival factor during follicular maturation. In contrast, the role of TNF alpha and its possible interaction with TGF alpha in the regulation of granulosa cell fate (death versus survival) during ovarian follicular development have not been fully elucidated. The object of the current study was to determine if TNF alpha and TGF alpha interact in the regulation of hen granulosa cell fibronectin and integrin content in the context of cell death and survival during follicular development. TGF alpha (0.1 or 10 ng/ml), but not TNF alpha (0.1 or 10 ng/ml), increased both cellular and secreted fibronectin content in granulosa cell cultures of F5,6 but not F1 follicles. The expression of integrin beta(3) subunit was also stimulated by TGF alpha in a follicular stage-dependent manner, and culture of F5,6 granulosa cells with TNF alpha in the presence of maximal stimulatory concentrations of TGF alpha potentiated this response. TGF alpha increased both F5,6 and F1 granulosa cell [(3)H]thymidine incorporation but not 3-(4,5-dimethylthiazol-2-yl)3,5-diphenyl tetrazolium bromide (MTT) metabolism. Although TNF alpha had no effect on [(3)H]thymidine incorporation irrespective of the presence of the growth factor, MTT metabolism was higher in F5,6 granulosa cells cultured for 24 h with both TNF alpha and TGF alpha than with either cytokine alone. Incubation of F5,6 granulosa cells for 48 and 72 h resulted in a TGF alpha-inhibited loss of cellular adhesion and detachment of granulosa cells from the growth surface. Although TNF alpha alone had no effect on cell morphology, it facilitated the reorganization of the granulosa cells into multicellular follicle-like structures in the presence of the growth factor. DNA degradation significantly increased between 0 and 72 h of culture in the absence of the cytokine but was suppressed by the addition of TGF alpha but not of TNF alpha. However, fluorometric analysis indicated that the primary type of cell death exhibited by F5,6 granulosa cells during extended culture and attenuated by the presence of TNF alpha and TGF alpha was necrosis and not apoptosis. The current study demonstrates that TNF alpha and TGF alpha interact in the regulation of granulosa cell integrin content and cell survival in vitro in a follicular stage-dependent manner. These findings suggest that follicular development is accompanied by a change in the intraovarian role of TNF alpha; it is atretogenic prior to follicular selection but prevents follicular demise during preovulatory growth.

We have been investigated the possibility of B-Z transition in ZnTMPyP-DNA interaction based on the observation of spectroscopic data. In this study, we found drastic change in the AFM image of supercoiled plasmid DNA when it was interacted with TMPyPs indicating that the considerable amount of unwinding of double helix or B-Z transition is induced by the drug-DNA interaction. Such phenomena were not observed for other cationic drugs examined.

As demonstrated by several studies, the pan-inhibitory peptide somatostatin (SS) is implicated in a large variety of physiological processes in the gastrointestinal tractus. SS inhibits hormonal and gastric acid secretions, and decreases gastric and intestinal motility, mesenteric blood flow and intestinal absorption. In vitro and in vivo studies showed also that the antiproliferative potency of SS analogs may be a target to improve the prognosis of colorectal cancer. Here we report the expression profile of the five SS receptor subtypes (hsst1-5) mRNAs in a large set of tumoral and normal colon. Using reverse transcription-PCR, we showed that hsst5, hsst1 and hsst2 mRNA subtypes were the most frequently expressed hsst mRNA subtypes in normal and pathological colon. Interestingly, we found that the frequency of hsst5 mRNA expression in the left colon was significantly higher in tumors than in normal samples: 81. 2% (13/16) and 36.4% (4/11) respectively (0.025>P>0.01, chi2 test with Yates' correction). We did not find any influence of Dukes' stage on hsst mRNAs expression. Of interest, no loss of hsst2 and hsst5 mRNA expression in advanced stages was noted. Some differences in the frequency of expression of hsst mRNAs according to the origin of the tissue (left or right colon) were evident. The expression of hsst5 and hsst2 mRNA in advanced colorectal carcinoma associated with the development of new SS analogs boost the relevance of colorectal cancer treatment by somatostatin analogs.

Rapid bacterial detection and viability measurements have been greatly enhanced by recent advances in the use of fluorescent stains in cytometry. It has previously been shown that four physiological states can be distinguished: reproductively viable, metabolically active, intact and permeabilized. Previous sorting experiments have shown that not all intact cells readily grow, but some intact cells can grow even when they fail to show metabolic activity, as determined by esterase turnover. To circumvent the limitations imposed by active dye extrusion or cell dormancy on viability measurements used to date (e.g., enzyme activity or cell polarization), a fast triple fluorochrome staining procedure has been developed that takes account of these problems. This allows further cellular characterization of intact cells by: active exclusion of ethidium bromide (EB) (metabolically active cells), uptake of EB but exclusion of bis-oxonol (BOX) (de-energized but with a polarized cell membrane) and uptake of both dyes (depolarized). Permeabilized cells were identified by propidium iodide (PI) uptake. The method was validated using an electronically programmable single cell sorter (EPICS Elite) and aged Salmonella typhimurium cells. Reproductive viability was determined by sorting single cells to their staining pattern directly onto agar plates. Most polarized cells could be recovered as well as a significant fraction of the depolarized cells, demonstrating that depolarization is a sensitive measure of cell damage but a poor indicator of cell death.

We have induced a local conformational transition by positive torsional stress in small synthetic circular DNA molecules containing cruciforms with immobile or tetramobile branched junctions. The immobile species correspond to the extruded and intruded extrema of the tetramobile junction. Under normal conditions the sequences of all the branched species prevent them from being re-absorbed into the circle. We have induced positive stress by addition of ethidium to the circle, in a low ionic strength medium. Alterations in gel electrophoretic mobility under increasing concentrations of ethidium suggest that the cruciforms undergo a transition under torsional stress. The product of this transition contains mispaired nucleotides, but interwound backbones. By comparing the electrophoretic mobilities of circles containing these structures with that of a completely complementary circle of the same length, we conclude that the twist in the mispairing region is similiar to that of completely paired species.

This report describes a method for the construction of a mimic molecule for monitoring PCR. A blue cheese homogenate spiked with Yersinia enterocolitica was used to demonstrate how false-negative PCR results may be avoided using the mimic. Further, the possibility of estimating bacterial numbers using the mimic was illustrated by varying the target concentration.

A fiber optic biosensor was used for the fluorimetric detection of T/AT triple-helical DNA formation. The surfaces of two sets of fused silica optical fibers were functionalized with hexaethylene oxide linkers from which decaadenylic acid oligonucleotides were grown in the 3'to 5'and 5'to 3'direction, respectively, using a DNA synthesizer. Fluorescence studies of hybridization showed unequivocal hybridization between oligomers immobilized on the fibers and complementary oligonucleotides from the solution phase, as detected by fluorescence from intercalated ethidium bromide. The complementary oligonucleotide, dT10, which was expected to Watson-Crick hybridize upon cooling the system below the duplex melting temperature ( T m), provided a fluorescence intensity with a negative temperature coefficient. Upon further cooling, to the point where the pyrimidine motif T*AT triple-helix formation occurred, a fluorescence intensity change with a positive temperature coefficient was observed. The reverse-Hoogsteen T.AT triplex, which is known to form with branched nucleic acids, provided a corresponding decrease in fluorescence intensity with decreasing temperature. Full analytical signal evolution was attainable in minutes.

A 6-yr-old boy presented with muscle weakness, lactic acidemia, and insulin-dependent diabetes mellitus (IDDM). Using PCR and restriction enzyme analysis, he was found to have the classical A3248G mitochondrial DNA (mtDNA) mutation frequently associated with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). The mutation was confirmed by sequencing muscle mtDNA. The mutation in mtDNA from muscle, lymphoblasts, and blood was clearly demonstrable by standard methods using ethidium bromide staining. His mother also had IDDM, but no A3243G mutation could be detected in her blood or transformed lymphoblasts using the same PCR technique. When PCR was carried out in the presence of [32P]deoxycytidine triphosphate, subsequent autoradiography detected the presence of the mutation at low levels in mtDNA from the mother's lymphoblasts and blood. Study of the mother's muscle showed a mitochondrial myopathy, despite the fact that she was asymptomatic. We emphasize that the increased sensitivity of radiolabeled PCR may be necessary to detect small percentages of heteroplasmic A3243G mtDNA mutation in blood from diabetic subjects. Otherwise the incidence of mtDNA mutations in both IDDM and non-insulin dependent diabetes may be underestimated.

Demyelinating lesions induced by intraspinal injection of gliotoxin have been studied for many years in order to gain insights into reasons for failure of remyelination and to improve understanding of the axonal conduction disorders in multiple sclerosis. Although the electrophysiological correlates of experimental demyelination and remyelination are well established, the behavioural effects have not been investigated. In this study we aimed to determine whether behavioural deficits could be detected during spinal cord demyelination, and furthermore, whether remyelination was associated with return of lost function. We used injections of the gliotoxin ethidium bromide into the dorsal funiculus of the cervical spinal cord of the rat to induce zones of demyelination and compared the effects on locomotion with those resulting from saline injections. The resulting locomotor deficits were quantified by analysis of foot placement during traverse of a horizontal 18 mm diameter wooden beam. Following ethidium bromide injection there was a decrease in security of foot placement, that recovered by approximately 5 weeks post-injection. In a second experiment, remyelination was prevented by exposure of the spinal cord to 40 Gy of X-irradiation. Behavioural deficits were induced as before, but the animals failed to recover throughout the duration of the experiment. Saline-injected animals in both experiments exhibited minimal deficits and quickly recovered. We conclude that demyelination produces detectable behavioural deficits which disappear following spontaneous remyelination.

Because the down-regulation by progesterone of cystic fibrosis transmembrane conductance regulator (CFTR) expression could be a useful specific marker to define the state of implant receptivity in endometrium, a competitive reverse transcription-polymerase chain reaction (RT-PCR) was developed for quantifying the CFTR mRNA concentration in human endometrial samples. A competitor RNA was constructed with the same sequence as the CFTR sequence except for a 20-nucleotide insertion in the middle. The amplified products were separated by polyacrylamide gel electrophoresis. The ratio of CFTR band areas to competitor band areas provided the basis of quantification. Using this competitive RT-PCR, we measured CFTR mRNA in human endometrial samples taken at different periods of the menstrual cycle, in endometriosis, and in hyperplasia. Results show that the method is suitable for measuring the concentration of CFTR mRNA.

The expression of the mRNA for the inhibin/activin subunits (alpha and beta A) in the granulosa layer of the five largest preovulatory follicles of the hen was investigated. Total RNA from the granulosa layer of the F5 (the fifth largest) to F1 (the largest) follicles was extracted and analyzed by Northern blot analysis using homologous chicken inhibin alpha and beta A subunit cDNA probes. RNA loading was quantified by a cDNA probe of bovine 18S rRNA. Results showed that for the chicken inhibin alpha subunit mRNA signals (n = 3), the mean relative intensity for the F1, F2, F3, and F4 follicles was 0.50 +/- 0.10 ( +/- SEM,), 0.52 +/- 0.08, 0.59 +/- 0.06, and 0.81 +/- 0.04, respectively, compared to a mean relative intensity of 1.00 (p < 0.05) for the F5 follicle. For the beta A subunit mRNA signals (n = 3), the mean relative intensity for the F5, F4, F3, and F2 follicles was 0.25 +/- 0.06, 0.28 +/- 0.15, 0.40 +/- 0.17, and 0.48 +/- 0.10 (p < 0.05) for the F1 follicle. The inhibin alpha subunit was also estimated to be more abundantly expressed among follicles in the granulosa layer than was the beta A subunit. Our data indicate that the expression of inhibin alpha and beta A subunits is differentially regulated in the hen granulosa layer during follicular development. Expression of the alpha subunit is reduced with follicular development whereas inhibin beta A subunit expression is dramatically enhanced. In addition, the granulosa layer of the large preovulatory follicles may produce more inhibin alpha subunit than beta A subunit, and the F1 follicle may be the primary source of the beta A subunit for dimeric inhibin and/or activin in the hen.

It has been shown recently that some analogues of clinically ineffective trans-diamminedichloroplatinum (II) (transplatin) exhibit antitumor activity. This finding has inverted the empirical structure-antitumor activity relationships delineated for platinum(II) complexes, according to which only the cis geometry of leaving ligands in the bifunctional platinum complexes is therapeutically active. As a result, interactions of trans platinum compounds with DNA, which is the main pharmacological target of platinum anticancer drugs, are of great interest. The present paper describes the DNA binding of antitumor trans-[PtCl(2)(E-imino ether)(2)] complex (trans-EE) in a cell-free medium, which has been investigated using three experimental approaches. They involve thiourea as a probe of monofunctional DNA adducts of platinum (II) complexes with two leaving ligands in the trans configuration, ethidium bromide as a probe for distinguishing between monofunctional and bifunctional DNA adducts of platinum complexes and HPLC analysis of the platinated DNA enzymatically digested to nucleosides. The results show that bifunctional trans-EE preferentially forms monofunctional adducts at guanine residues in double-helical DNA even when DNA is incubated with the platinum complex for a relatively long time (48 h at 37 degrees C in 10 mM NaCIO(4). It implies that antitumor trans-EE modifies DNA in a different way than clinically ineffective transplatin, which forms prevalent amount of bifunctional DNA adducts after 48 h. This result has been interpreted to mean that the major adduct of trans-EE, occurring in DNA even after long reaction times, is a monofunctional adduct in which the reactivity of the second leaving group is markedly reduced. It has been suggested that the different properties of the adducts formed on DNA by transplatin and trans-EE are relevant to their distinct clinical efficacy.

Telomeric DNA in Saccharomyces is organized into a non-nucleosomal chromatin structure called the telosome that can be released from chromosome ends in soluble form by nuclease digestion (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210). The protein-DNA interactions of soluble telosomes were investigated by monitoring isolated telomeric DNA fragments for the retention of bound protein using both gel mobility shift and nitrocellulose filter-binding assays. Telosomal proteins remained associated with telomeric DNA at concentrations of ethidium bromide that dissociated nucleosomes. The protein-DNA interactions in the yeast telosome were also disrupted by much lower salt concentrations than those known to disrupt either the interactions of ciliate terminus-binding proteins with telomeric DNA or the interactions of histones with DNA in nucleosomes. Taken together, these data corroborate previously published nuclease mapping data indicating that telosomes are distinct in structure from conventional nucleosomes. These data also indicate that yeast do not possess telomere binding proteins similar to those detected in ciliates that remain tightly bound to telomeric DNA even in high salt. In addition, the characteristic gel mobility shift of soluble telosomes could be mimicked by complexes formed in vitro with yeast telomeric DNA and recombinant Rap1p suggesting that Rap1p, a known component of soluble yeast telosomes (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210; Conrad, M. N., Wright, J. H., Wolf, A. J. and Zakian, V. A. (1990) Cell 63, 739-750), is likely to be the major structural protein bound directly to yeast telomeric DNA.

Two series of tethered nitrogen mustards based on the minor groove-binding and A/T sequence-specific natural product distamycin have been synthesized and evaluated. The conjugates, which have a modified dimethylamino C-terminus, are comprised of one, two or three pyrrole carboxamide units linked to either benzoic acid mustard (BAM) or chlorambucil (CHL). The DNA binding properties, in vitro cytotoxicities and DNA cross-linking abilities were determined for each of the conjugates. The conjugates were found to bind preferentially to poly(dA.dT) compared to poly(dG.dC) DNA by ethidium displacement and circular dichroism. The di- and tripyrrole conjugates had higher binding affinities than the monopyrrole conjugates. All the conjugates were more cytotoxic than the nitrogen mustards themselves. Cytotoxicity increased with the increase from one to three pyrrole units and the CHL conjugates were more cytotoxic than the corresponding BAM analogues. The CHL conjugates were able to cross-link plasmid DNA at a 10-fold lower dose than CHL itself. The BAM conjugates showed < 10% cross-linking at doses which gave 100% cross-linking with the CHL conjugates. In cells, the CHL conjugates showed significant cross-linking at the IC50 values, while the BAM conjugates showed no evidence of cross-link formation even at 10 times the IC50 value. These results are discussed in reference to a series of previously reported GC-recognizing imidazole analogues possessing the same nitrogen mustard groups.

The DNA-binding properties of six candidate nitroarene-functionalized oligopeptides 8-13 (i.e. 5-nitrofuran, 2-nitroimidazole and 4-nitrobenzene derivatives) with potential hypoxia-selective activity, developed as analogues of the archetypal minor groove-binding ligands, netropsin 1 and distamycin 2, have been examined using an extended molecular mechanics and dynamics (MM/MD) modelling approach. The energies calculated for interaction with the d(CGCGAATTCGCG) duplex, or d(CGCAAATTTGCG)2 in the case of the elongated nitrobenzene 13, correlate with fluorimetric data obtained by indirect competitive displacement of ethidium bromide from double-stranded calf thymus DNA. The results suggest that the mode of interaction for these agents resembles the behavior of structurally unrelated bis(amidine) derivatives that also bind to AT-rich stretches of duplex B-DNA via the minor groove. Analysis of the binding behaviour indicates that the interactions are determined by enthalpic rather than entropic energy terms, suggesting that specific or differential hydration effects for the DNA and ligand may be unimportant. A rational structure-activity relationship is established for the interaction of this family of oligopeptides with DNA that also encompass the bis(amidine) class of ligands.

We present an original application of competitive polymerase chain reaction (PCR) for measuring oncogene amplification in DNA from human tumors by simultaneous PCR amplification of genomic DNA with fixed amounts of an internal standard (competitor DNA). Competitors share the same sequence as the target genes but contain an additional 15- to 20-base-pair insert, which allows resolution of the amplified products after polyacrylamide gel electrophoresis and ethidium bromide staining. The gene copy number is derived from the ratio between the intensities of the bands corresponding to the amplified products. Using this procedure, we measured c-erbB-2 amplification in breast and bladder carcinomas in both fresh tumor tissues and paraffin-embedded tissue samples and assessed the precision, sensitivity, and accuracy of the assay. Competitive PCR is a simple, reliable, and accurate method for the evaluation of c-erbB-2 amplification and is potentially suitable for use in the clinical laboratory.

The primary or secondary structure of single-stranded nucleic acids has been investigated with fluorescent oligonucleotides, i.e., oligonucleotides covalently linked to a fluorescent dye. Five different chromophores were used: 2-methoxy-6-chloro-9-amino-acridine, coumarin 500, fluorescein, rhodamine and ethidium. The chemical synthesis of derivatized oligonucleotides is described. Hybridization of two fluorescent oligonucleotides to adjacent nucleic acid sequences led to fluorescence excitation energy transfer between the donor and the acceptor dyes. This phenomenon was used to probe primary and secondary structures of DNA fragments and the orientation of oligodeoxynucleotides synthesized with the alpha-anomers of nucleoside units. Fluorescence energy transfer can be used to reveal the formation of hairpin structures and the translocation of genes between two chromosomes.

A major event in enhancing sperm chromatin stability is the replacement of the histones by protamines during spermiogenesis. In this study, we present results indicating that chromomycin A3 (CMA3) can be used to show protamine deficiency in sperm chromatin. Fixed chromatin of mature mouse spermatozoa showed high fluorescence after treatment with ethidium bromide (EB), but was completely unstained after treatment with CMA3. The same chromatin was found to be highly resistant to in situ nick-translation. In contrast, a substantial fraction of human spermatozoa were positive for CMA3. The accessibility of CMA3 to the DNA of human sperm was eliminated if the slides were previously treated with protamine in situ. This treatment did not affect the accessibility of EB to the chromatin. Individual human sperm samples revealed a substantial frequency of spermatozoa with endogenous nicks, which was found to be the same as the frequency of spermatozoa responding positively to CMA3 staining. Treatment of preparations with protamines prevented the identification of the endogenous nicks. These data as a whole suggest that CMA3 could represent a useful tool for the detection of protamine deficiency in sperm chromatin. Furthermore, confirmation of experiments relating sensitivity to nick translation and positivity to CMA3 may allow an indirect in situ visualization of nicked and partially denatured DNA, which could correlate with certain forms of male factor infertility.

We describe the adaptation and evaluation of an error-compensating method for kinetic determinations of deoxyribonucleic acids (DNAs). The DNA is first reacted with ethidium bromide to produce a fluorescent intercalation complex. Subsequent treatment of the complex with DNase catalyzes hydrolysis of the DNA, causing a time-dependent decrease in fluorescence, which is monitored. A model for two-component parallel first-order processes is fit to the decay curve to predict the total change in fluorescence expected if the process were monitored to equilibrium. The predicted change in fluorescence response varies linearly with DNA concentration with an intercept corresponding to 0.13 mg/L DNA. Results by the predictive method are 47-, 58-, and 250-fold less dependent on DNase activity, temperature, and ethidium bromide concentration, respectively, than are results for an initial-rate method utilizing the same data. Moreover, the predictive method yields a significantly wider linear range than the initial-rate method, and is much less affected by blank fluorescence and RNA interference than is an equilibrium method based on the reaction of DNA with ethidium bromide alone.

A combination of calorimetric and spectroscopic techniques was used to evaluate the thermodynamic behavior of a set of DNA hairpins with the sequence d(GCGCTnGCGC), where n = 3, 5 and 7, and the interaction of each hairpin with ethidium. All three hairpins melt in two-state monomolecular transitions, with tm's ranging from 79.1 degrees C (T3) to 57.5 degrees C (T7), and transition enthalpies of approximately 38.5 kcal mol-1. Standard thermodynamic profiles at 20 degrees C reveal that the lower stability of the T5 and T7 hairpins corresponds to a delta G degree term of +0.5 kcal mol-1 per thymine residue, due to the entropic ordering of the thymine loops and uptake of counterions. Deconvolution of the ethidium-hairpin calorimetric titration curves indicate two sets of binding sites that correspond to one ligand in the stem with binding affinity, Kb, of approximately 1.8 x 10(6) M-1, and two ligands in the loops with Kb of approximately 4.3 x 10(4) M-1. However, the binding enthalpy, delta Hb, ranges from -8.6 (T3) to -11.6 kcal mol-1 (T7) for the stem site, and -6.6 (T3) to -12.7 kcal mol-1 (T7) for the loop site. Relative to the T3 hairpin, we obtained an overall thermodynamic contribution (per dT residue) of delta delta Hb = delta(T delta Sb) = -0.7(5) kcal mol-1 for the stem sites and delta delta Hb = delta(T delta Sb) = -1.5 kcal mol-1 for the loop sites. Therefore, the induced structural perturbations of ethidium binding results in a differential compensation of favorable stacking interactions with the unfavorable ordering of the ligands.

A method for RNA amplification in an immobilized medium is described. The medium contains a complete set of nucleotide substrates and purified Q beta replicase, an enzyme capable of exponentially amplifying RNAs under isothermal conditions. RNA amplification in the immobilized medium results in the formation of separate 'colonies', each comprising the progeny of a single RNA molecule (a clone). The colonies were visualized by staining with ethidium bromide, by utilizing radioactive substrates, and by hybridization with sequence-specific labeled probes. The number and identity of the RNA colonies corresponded to that of the RNAs seeded. When a mixture of different RNA species was seeded, these species were found in different colonies. Possible implementations of this technique include a search for recombinant RNAs, very sensitive nucleic acid diagnostics, and gene cloning in vitro.

The presence of prolactin (PRL) mRNA in the mammary gland, placenta, and pituitary gland of lactating and pregnant rats was investigated by polymerase chain reaction (PCR). Polyadenylated RNA was prepared from total RNA samples by oligo(dT)-cellulose chromatography, and complementary cDNAs were synthesized. A standardized amount of cDNA from each sample was used as the template in a Taq PCR under high-stringency conditions. PCR amplified a signal with the predicted size of approximately 375 bp in mammary and pituitary glands of lactating and pregnant rats, and in placentae of pregnant rats. This band specifically hybridized with a probe overlapping the entire sequence of the mature rat (r) PRL mRNA in Southern blot analysis. When the rPRL-specific primers were used, PCR revealed no signal in the liver or in lactating mammary gland explants cultured in vitro for 48 h, while the same cDNA preparations gave strong signals for beta-actin. The viability of the mammary gland explants was also suggested by their ability to secrete immunoreactive casein in vitro. PRL mRNA was localized in the epithelium of alveoli and ducts of the lactating mammary gland by in situ hybridization. These data provide evidence that the PRL gene is expressed in the mammary gland of pregnant and lactating rats, and suggest that the mammary gland might contribute to PRL in milk by de novo synthesis. Thus, while the placenta is an exogenous source of PRL-like activities for the fetus in utero, the mammary gland might take over this function after birth.

An efficient method is described for the purification of Ti plasmid DNA from Agrobacterium. The procedure is based on the relative binding capacity of ethidium bromide to supercoiled plasmid DNA and linear DNA and on the high solubility of ethidium bromide in phenol. Following treatment with ethidium bromide, more than 87% of linear chromosomal DNA and most of the RNA was present in the phenol phase, while 91% of Ti plasmid DNA was recovered from the aqueous phase. The Ti plasmid DNA was sufficiently pure for restriction endonuclease analysis and cloning. The procedure is simple, fast and provides eight times higher yield than the standard isopycnic ultracentrifugation method.

Following targeted disruption of the unique CYC1 gene, the petite-negative yeast, Kluyveromyces lactis, was found to grow fermentatively in the absence of cytochrome c-mediated respiration. This observation encouraged us to seek mitochondrial mutants by treatment of K. lactis with ethidium bromide at the highest concentration permitting survival. By this technique, we isolated four mtDNA mutants, three lacking mtDNA and one with a deleted mitochondrial genome. In the three isolates lacking mtDNA, a nuclear mutation is present that permits petite formation. The three mutations occur at two different loci, designated MGI1 and MGI2 (for Mitochondrial Genome Integrity). The mgi mutations convert K. lactis into a petite-positive yeast. Like bakers' yeast, the mgi mutants spontaneously produce petites with deletions in mtDNA and lose this genome at high frequency on treatment with ethidium bromide. We suggest that the MGI gene products are required for maintaining the integrity of the mitochondrial genome and that, petite-positive yeasts may be naturally altered in one or other of these genes.

Murine epidermal Langerhans cells (LC) have been demonstrated to stimulate a vigorous T cell response to Leishmania major, a cause of human cutaneous leishmaniasis. It was therefore of interest to analyze whether LC can take up viable parasites. Epidermal cells were obtained from mouse ear skin for incubation with L. major and subsequent detection of intracellular parasites by cytochemistry. Freshly isolated LC, but not cultured LC, phagocytosed L. major and the uptake was inhibited by antibodies to the complement receptor type 3. Electron microscopic studies revealed the presence of viable amastigotes within LC. Moreover, with double-labeling techniques, L. major-containing LC could also be detected in infected skin. The results demonstrate that LC can internalize L. major. Since the number of organisms per infected LC remained consistently low, the prime task of LC may not be the promotion of parasite spreading but the presentation of L. major antigen to T cells and, thus, the regulation of the cellular immunity during cutaneous leishmaniasis.

The fission yeast Schizosaccharomyces pombe has never been found to give rise to viable cells totally lacking mitochondrial DNA (rho(o)). This paper describes the isolation of rho(o) strains of S. pombe by very long term incubation of cells in liquid medium containing glucose, potassium acetate and ethidium bromide. Once isolated, the rho(o) strains did not require potassium acetate or any other novel growth factors. These nonrespiring strains contained no mitochondrial DNA (mtDNA) detectable either by gel-blot hybridization using as probe a clone containing the entire S. pombe mtDNA, or by 1',6-diamidino-2-phenylindole staining of whole cells. Induction of rho(o) derivatives of standard laboratory strains was not reproducible from culture to culture. The cause of this irreproducibility appears to be that growth of the rho(o) strains of S. pombe depended on nuclear mutations that occurred in some, but not all, of the initial cultures. Two independent rho(o) isolates contained mutations in unlinked genes, termed ptp1-1 and ptp2-1. These mutations allowed reproducible ethidium bromide induction of viable rho(o) strains. No other phenotypes were associated with ptp mutations in rho+ strains.

Novel DNA binding ligands (1 and its stereoisomer 2) which contain three potentially intercalating units in a linear molecular skeleton were prepared. From a study of the displacement of ethidium bromide from several natural and synthetic polynucleotides, both compounds 1 and 2 were found to show an AT base-pair preference in the interaction with DNAs with 2 a slightly higher affinity for DNA. This result is in sharp contrast to that for acridine and anthraquinone, because these two compounds exhibit a GC-preference.

Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [32P]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0-125 microM cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75 microM. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences.

A Sfi I restriction map of the fission yeast Schizosaccharomyces pombe genome was aligned with the Not I restriction map. There are 16 Sfi I sites in the S. pombe genome. Three Sfi I sites are on chromosome III which is devoid of Not I sites. The sizes of the entire genome and individual chromosomes, calculated from the Sfi I fragment sizes, are consistent with that calculated from the Not I fragment sizes. The Sfi I map provides greater physical characterization of the S. pombe genome and further validates the use of S. pombe chromosomal DNA as size standard. These maps have allowed detection of polymorphism on all three chromosomes.

The SpeI/DpnI map of the 5.9 Mb Pseudomonas aeruginosa PAO (DSM 1707) genome was refined by two-dimensional (2D) pulsed-field gel electrophoresis techniques (PFGE) which allow the complete and consistent physical mapping of any bacterial genome of interest. Single restriction digests were repetitively separated by PFGE employing different pulse times and ramps in order to detect all bands with optimum resolution. Fragment order was evaluated from the pattern of 2D PFGE gels: 1. Partial-complete digestion. A partial restriction digest was separated in the first dimension, redigested to completion, and subsequently perpendicularly resolved in the second dimension. 2D-gel comparisons of the ethidium bromide stain of all fragments and of the autoradiogram of end-labeled partial digestion fragments was nearly sufficient for the construction of the macrorestriction map. 2. Reciprocal gels. A complete restriction digest with enzyme A was run in the first dimension, redigested with enzyme B, and separated in the second orthogonal direction. The order of restriction digests was reverse on the second gel. In case of two rare-cutters, fragments were visualized by ethidium bromide staining or hybridization with genomic DNA. If a frequent and a rare cutter were employed, linking fragments were identified by end-labeling of the first digest. 3. A few small fragments were isolated by preparative PFGE and used as a probe for Southern analysis.--38 SpeI and 15 DpnI fragments were positioned on the map. The zero point was relocated to the 'origin of replication'. The anonymous mapping techniques described herein are unbiased by repetitive DNA, unclonable genomic regions, unfavourable location of restriction sites, or cloning artifacts as frequently encountered in other top-down or bottom-up approaches.