Le Pecq Research Articles

Evidence for Proteomic and Metabolic Adaptations Associated with Alterations of Seed Yield and Quality in Sulfur-limited Brassica napus L

In Brassica napus, seed yield and quality are related to sulfate availability, but the seed metabolic changes in response to sulfate limitation remain largely unknown. To address this question, proteomics and biochemical studies were carried out on mature seeds obtained from plants grown under low sulfate applied at the bolting (LS32), early flowering (LS53), or start of pod filling (LS70) stage. The protein quality of all low-sulfate seeds was reduced and associated with a reduction of S-rich seed storage protein accumulation (as Cruciferin Cru4) and an increase of S-poor seed storage protein (as Cruciferin BnC1). This compensation allowed the protein content to be maintained in LS70 and LS53 seeds but was not sufficient to maintain the protein content in LS32 seeds. The lipid content and quality of LS53 and LS32 seeds were also affected, and these effects were primarily associated with a reduction of C18-derivative accumulation. Proteomics changes related to lipid storage, carbohydrate metabolism, and energy (reduction of caleosins, phosphoglycerate kinase, malate synthase, ATP-synthase β-subunit, and thiazole biosynthetic enzyme THI1 and accumulation of β-glucosidase and citrate synthase) provide insights into processes that may contribute to decreased oil content and altered lipid composition (in favor of long-chain fatty acids in LS53 and LS32 seeds). These data indicate that metabolic changes associated with S limitation responses affect seed storage protein composition and lipid quality. Proteins involved in plant stress response, such as dehydroascorbate reductase and Cu/Zn-superoxide dismutase, were also accumulated in LS53 and LS32 seeds, and this might be a consequence of reduced glutathione content under low S availability. LS32 treatment also resulted in (i) reduced germination vigor, as evidenced by lower germination indexes, (ii) reduced seed germination capacity, related to a lower seed viability, and (iii) a strong decrease of glyoxysomal malate synthase, which is essential for the use of fatty acids during seedling establishment.

Calcium and Calmodulin-dependent Serine/Threonine Protein Kinase Type II (CaMKII)-mediated Intramolecular Opening of Integrin Cytoplasmic Domain-associated Protein-1 (ICAP-1α) Negatively Regulates β1 Integrins

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the β integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of β1A integrins, limits both talin and kindlin interaction with β1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the β1 integrin tail. ICAP-1α direct interaction with the β1 integrin tail and the modulation of β1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of β1 integrin activation and cell adhesion.

Essential Role of Nuclear Factor of Activated T Cells (NFAT)-mediated Wnt Signaling in Osteoblast Differentiation Induced by Strontium Ranelate

The antiosteoporotic treatment strontium ranelate (SrRan) was shown to increase bone mass and strength by dissociating bone resorption and bone formation. To identify the molecular mechanisms of action of SrRan on osteoblasts, we investigated its effects on calcineurin-NFAT (nuclear factor of activated T cells) signaling, an important calcium sensitive pathway controlling bone formation. Using murine MC3T3-E1 and primary murine osteoblasts, we demonstrate that SrRan induces NFATc1 nuclear translocation, as shown by immunocytochemical and Western blot analyses. Molecular analysis showed that SrRan increased NFATc1 transactivation in osteoblasts, an effect that was fully abrogated by the calcineurin inhibitors cyclosporin A or FK506, confirming that SrRan activates NFATc1 signaling in osteoblasts. This has functional implications because calcineurin inhibitors blunted the enhanced osteoblast replication and expression of the osteoblast phenotypic markers Runx2, alkaline phosphatase, and type I collagen induced by SrRan. We further found that SrRan increased the expression of Wnt3a and Wnt5a as well as beta-catenin transcriptional activity in osteoblasts, and these effects were abolished by calcineurin inhibitors. The Wnt inhibitors sFRP1 and DKK1 abolished SrRan-induced osteoblast gene expression. Furthermore, blunting the Wnt5a receptor Ryk or RhoA that acts downstream of Ryk abrogated cell proliferation and osteoblast gene expression induced by SrRan. These results indicate that activation of NFATc1 and downstream canonical and non-canonical Wnt signaling pathways mediate SrRan-induced osteoblastic cell replication and differentiation, which provides novel insights into the mechanisms of action of this antiosteoporotic agent in osteoblastogenesis.

Proteomics Analysis of Bladder Cancer Exosomes

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.

Evaluation of randomly amplified polymorphic DNA (RAPD) for discrimination of Coxiella burnetii ruminant strains isolated in France

Coxiella burnetii is the causative agent of Q fever, a worldwide zoonosis. Ruminant species represent the main reservoir of the bacterium, as high levels of the bacteria are shed in birth products and other excreta. Human infection generally occurs after inhalation of contaminated aerosols [1]. The availability of methods to study the distribution and the spread of a given C. burnetii strain, from different geographical areas or hosts, would promote a better understanding of the epidemiology of this pathogen. Several genetic typing methods for C. burnetii have been evaluated and appeared to be useful tools for epidemiological and phylogenetic purposes. However, RAPD [2,3] has never been evaluated for C. burnetii typing. In this work, a RAPD protocol was used to evaluate the genetic diversity among C. burnetii ruminant strains in comparison to published MLVA data [4]. Ten French C. burnetii isolates obtained from goats, sheep, cows and the reference strain Nine Mile, were used for RAPD typing (Table 1). Whole DNA was extracted using the Qiagen Dneasy kit (Courtaboeuf, France), following the manufacturer’s protocol. To assess optimal RAPD conditions several parameters were tested, for instance using a range of MgCl2 concentrations (from 1.5 to 5.5 mM), or different brands of Taq polymerase. RAPD PCRs were performed using a set of four 10-mer primers (P4M, 5¢-AAGACGCCGT-3¢; PR5, 5¢-AGTCGTCCCC-3¢; PR10A, 5¢AGGGCCGTCT3¢; and PR12A, 5¢-CAGCTCACGA-3¢). Unless specified, the optimised PCR conditions were, for a final volume of 25 lL in dH2O: 1X Taq polymerase buffer (without MgCl2); 3 mM MgCl2; 0.2 mM each dNTPs; 1U of Taq polymerase; 0.5 lM 10-mer primer; and 10 ng of DNA (all the reagents were purchased from Invitrogen, Cergy Pontoise, France). Negative controls consisting of dH2O only were included in each run. Amplifications were performed in an Eppendorf Mastercycler (Le Pecq, France), programmed for an initial denaturation step of 5 min at 94 C, 45 cycles of 96 C for 30 s, 37 C for 30 s, 72 C for 90 s and a final extension step at 72 C for 5 min. The PCR products were separated on 0.8% agarose gel containing ethidium bromide, visualised and photographed under UV light. Gel analysis was performed using the Quantity One 1-D Analysis software from Bio-Rad (Marnes la Coquette, France). An RAPD type was defined after the combination of the patterns obtained with the four primers used in this study (Table 1). The reproducibility of the method was evaluated by repeating the same assay several times, by different manipulators, as well as by using independent DNA preparations. Briefly, RAPD profiles were stable when varying MgCl2 concentration. However, a higher yield of amplification, estimated by the intensity of the bands observed on the gels, was observed using 2.5–3.5 mM MgCl2. This yield decreased when using higher concentrations of MgCl2 (data not shown). A MgCl2 concentration of 3 mM was therefore chosen for subsequent analyses. Different RAPD patterns were observed when different brands of Taq polymerases were used (data not shown). The Hot start platinum Taq polymerase (Invitrogen) produced the best patterns in our hands and was chosen for all the experiments. After optimisation of our RAPD protocol, the use of each primer generated distinct polymorphisms allowing differentiation of the studied strains. Strains from neighbouring flocks, that were indistinguishable using MLVA analysis, could be discriminated using RAPD typing (Table 1). For instance, CbB2 and CbB5 are two strains obtained in 2001 from neighbouring flocks, but were isolated from cows displaying different clinical signs (Table 1). CbB2 was isolated from a case of metritis and CbB5 from an aborted cow. Corresponding author and reprint requests: Dr V. Duquesne, AFSSA, Sophia Antipolis, Unite de Pathologie des Petits Ruminants, 105 Route des Chappes, BP111, 06902 SophiaAntipolis, France E-mail. v.duquesne@afssa.fr

E-PTFE (Gore-Tex®) implant with or without low-dosage mitomycin-C as an adjuvant in penetrating glaucoma surgery: 2 year randomized clinical trial

E-PTFE (Gore-Tex^®) implant with or without low-dosage mitomycin-C as an adjuvant in penetrating glaucoma surgery: 2 year randomized clinical trial

The Elastin Receptor Complex Transduces Signals through the Catalytic Activity of Its Neu-1 Subunit

The binding of elastin peptides on the elastin receptor complex leads to the formation of intracellular signals but how this is achieved remains totally unknown. Using pharmacological inhibitors of the enzymatic activities of its subunits, we show here that the elastin peptide-driven ERK1/2 activation and subsequent pro-MMP-1 production, observed in skin fibroblasts when they are cultured in the presence of these peptides, rely on a membrane-bound sialidase activity. As lactose blocked this effect, the elastin receptor sialidase subunit, Neu-1, seemed to be involved. The use of a catalytically inactive form of Neu-1 and the small interfering RNA-mediated decrease of Neu-1 expression strongly support this view. Finally, we report that N-acetyl neuraminic acid can reproduce the effects of elastin peptides on both ERK1/2 activation and pro-MMP-1 production. Altogether, our results indicate that the enzymatic activity of the Neu-1 subunit of the elastin receptor complex is responsible for its signal transduction, presumably through sialic acid generation from undetermined substrates.

Novel Role of Cold/Menthol-sensitive Transient Receptor Potential Melastatine Family Member 8 (TRPM8) in the Activation of Store-operated Channels in LNCaP Human Prostate Cancer Epithelial Cells

Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (I(cold/menthol)) that shows inward rectification and high Ca(2+) selectivity, which are dramatically different properties from "classical" TRPM8-mediated I(cold/menthol). Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both I(cold/menthol) and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca(2+) release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca(2+) release channel, potentially involved in a number of Ca(2+)- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis.

Maturation and Release of Interleukin-1β by Lipopolysaccharide-primed Mouse Schwann Cells Require the Stimulation of P2X7 Receptors

The P2X7 receptor, mainly expressed by immune cells, is a ionotropic receptor activated by high concentration of extracellular ATP. It is involved in several processes relevant to immunomodulation and inflammation. Among these processes, the production of extracellular interleukin-1beta (IL-1beta), a pro-inflammatory cytokine, plays a major role in the activation of the cytokine network. We have investigated the role of P2X7 receptor and of an associated calcium-activated potassium conductance (BK channels) in IL-1beta maturation and releasing processes by Schwann cells. Lipopolysaccharide-primed Schwann cells synthesized large amounts of pro-IL-1beta but did not release detectable amounts of pro or mature IL-1beta. ATP on its own had no effect on the synthesis of pro-IL-1beta, but a co-treatment with lipopolysaccharide and ATP led to the maturation and the release of IL-1beta by Schwann cells. Both mechanisms were blocked by oxidized ATP. IL-1beta-converting enzyme (ICE), the caspase responsible for the maturation of pro-IL-1beta in IL-1beta, was activated by P2X7 receptor stimulation. The specific inhibition of ICE by the caspase inhibitor Ac-Tyr-Val-Ala-Asp-aldehyde blocked the maturation of IL-1beta. In searching for a link between the P2X7 receptor and the activation of ICE, we found that enhancing potassium efflux from Schwann cells upregulated the production of IL-1beta, while strongly reducing potassium efflux led to opposite effects. Blocking BK channels actually modulated IL-1beta release. Taken together, these results show that P2X7 receptor stimulation and associated BK channels, through the activation of ICE, leads to the maturation and the release of IL-1beta by immune-challenged Schwann cells.

Peroxisome Proliferator-activated Receptor γ (PPARγ) as a Molecular Target for the Soy Phytoestrogen Genistein

The principal soy phytoestrogen genistein has an array of biological actions. It binds to estrogen receptor (ER) alpha and beta and has ER-mediated estrogenic effects. In addition, it has antiestrogenic effects as well as non-ER-mediated effects such as inhibition of tyrosine kinase. Because of its complex biological actions, the molecular mechanisms of action of genistein are poorly understood. Here we show that genistein dose-dependently increases estrogenic transcriptional activity in mesenchymal progenitor cells, but its biological effects on osteogenesis and adipogenesis are different. At low concentrations (< or =1 microm), genistein acts as estrogen, stimulating osteogenesis and inhibiting adipogenesis. At high concentrations (>1 microm), however, genistein acts as a ligand of PPARgamma, leading to up-regulation of adipogenesis and down-regulation of osteogenesis. Transfection experiments show that activation of PPARgamma by genistein at the micromolar concentrations down-regulates its estrogenic transcriptional activity, while activation of ERalpha or ERbeta by genistein down-regulates PPARgamma transcriptional activity. Genistein concurrently activates two different transcriptional factors, ERs and PPARgamma, which have opposite effects on osteogenesis or adipogenesis. As a result, the balance between activated ERs and PPARgamma determines the biological effects of genistein on osteogenesis and adipogenesis. Our findings may explain distinct effects of genistein in different tissues.

Removal of Natural Organic Matter by Coagulation-Flocculation: A Pyrolysis-GC-MS Study

The removal of the natural organic matter (NOM) in the Seine river water sampled at Le Pecq (France) was carried out by using three coagulants: ferric chloride, aluminum sulfate, and aluminum polychlorosulfate (PACS). The efficiency of the coagulation-flocculation process depended on the pH. The best results were obtained at pH = 5.5 with ferric chloride and pH = 7.0 with the Al based coagulants aluminum sulfate and PACS.The composition of NOM in the raw and clarified water as well as in the flocs was analyzed by pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). Using this technique on the flocs formed with ferric chloride, a selectivity of this coagulant for polyhydroxyaromatics (PHA) was shown. A preferential binding was also observed between PACS and polysaccharides (PS). No such trend could be observed with aluminum sulfate. These results were confirmed and refined by Py-GC-MS analyses on the different mass fractions of the clarified water obtained by gel permeation chromatography.

Synchronized Spontaneous Ca2+ Transients in Acute Anterior Pituitary Slices

We investigated the organization of spontaneous rises in cytosolic free Ca2+ concentration ([Ca2+]i) due to electrical activity in acute pituitary slices. Real time confocal imaging revealed that 73% of the cells generated fast peaking spontaneous [Ca2+]i transients. Strikingly, groups of apposing cells enhanced their [Ca2+]i in synchrony with a speed of coactivation >1,000 microm/s. Single-cell injection of Neurobiotin or Lucifer yellow labeled clusters of cells, which corresponded to coactive cells. Halothane, a gap junction blocker, markedly reduced the spread of tracers. Coupling between excitable cells was mainly homologous in nature, with a prevalence of growth hormone-containing cells. We conclude that spontaneously active endocrine cells are either single units or arranged in synchronized gap junction-coupled assemblies scattered throughout the anterior pituitary gland. Synchrony between spontaneously excitable cells may help shape the patterns of basal secretion.

Peroxidase-catalyzed covalent binding of the antitumor drug N2-methyl-9-hydroxyellipticinium to DNA in vitro.

In the presence of DNA, the antitumor drug N2-methyl-9-hydroxyellipticinium (elliptinium; NMHE) [Le Pecq, J. B., Gosse, C., Dat-Xuong, N., & Paoletti, C. (1975) C. R. Seances Acad. Sci., Ser. D 281, 1365-1367] is oxidized by the horseradish peroxidase-hydrogen peroxide (HRP-H2O2) system to the quinone imine derivative N2-methyl-9-oxoellipticinium (NMOE) [Auclair, C., & Paoletti, C. (1981) J. Med. Chem. 24, 289-295], which interacts with DNA according to the intercalation mode. When excess H2O2 was used, the major part of the quinone imine was further oxidized to the o-quinone N2-methyl-9,10-dioxoellipticinium [Bernadou, J., Meunier, G., Paoletti, C., & Meunier, B. (1983) J. Med. Chem. 26, 574-579]. In the presence of stoichiometric amounts of H2O2 (H2O2/NMHE = 1), NMOE reacts with DNA, yielding a fluorescent compound irreversibly linked to the nucleic acid, which is related to the covalent binding of the ellipticinium chromophore. Under optimal reaction conditions, NMHE binding occurs according to a first-order process (k = 4.3 X 10(-3) min-1) with a linear increase with respect to drug to nucleotide ratio up to a maximum binding of 1 NMHE per 20 base pairs (r = 0.05). The fluorescence spectra (ex, 330 nm; em, 548 nm) of NMHE bound to DNA, the occurrence of energy transfer from the DNA to the drug, and the DNA length increase of the DNA-NMHE adduct suggest that the binding occurs at the intercalating site with limited denaturation of the DNA helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Roundtable – Trends in Ozonation

This discussion took place June 25, 1985, during AWWA's annual conference in Washington, D. C. Philip C. Singer, professor in the department of environmental sciences and engineering. University of North Carolina, Chapel Hill, moderated the panel and also assisted with editing the transcript. Other participants were: François Fiessinger, director of the central laboratory of the Lyonnaise des Eaux, Le Pecq, France; Wilfred LePage, superintendent of water treatment for the city of Monroe, Mich.; Paul W. Prendiville, senior vice‐president of Camp Dresser &amp; McKee, Boston, Mass.; Rip G. Rice, president of Rip G. Rice Inc., Ashton, Md.; Heinrich Sontheimer, professor at the University of Karlsruhe, West Germany; and Alexander J. Varas, associate with Hazen and Sawyer, New York City.

Inhibition of tRNA aminoacylation by ethidium dimer and several other bifunctional intercalators with or without antitumor activity

The effect of various bifunctional intercalators with or without antitumor activity on the tRNA aminoacylation step was examined. The ethidium dimer appears as the strongest inhibitor of aminoacylation of E. coli methionine, phenylalanine and leucine tRNA and of yeast phenylalanine tRNA. Ethidium dimer binds to tRNA through two classes of sites, I and II ( K I > 10 9 M −1, K II − 10 6M −1) (C.R. Reinhardt, Biophys. J. 25, 44242 (1979)]. Surprisingly, binding of the drug to the high affinity class of sites causes only partial inhibition of tRNA aminoacylation, while full inhibition requires saturation of the second class of sites. This inhibition is reversed by displacement of the drug by an excess of DNA. In the studied systems, the 7H pyridocarbazole dimers 1 and 2 which have antitumor activity [B.P. Roques, D. Pelaprat, J. Le Guen, G. Porcher, C. Gosse and J. B. Le Pecq, Biochem. Pharmac. 28, 1811 (1979); D. Pelaprat, A. Delbarre, J. Le Guen, J. B. Le Pecq and B. P. Roques, J. med. Chem. 23, 1336 (1980)] inhibit aminoacylation at much higher concentrations than that of ethidium dimer. This behavior is discussed in relation to the particular structures of these drugs.

Torsion Dynamics and Depolarization of Fluorescence of Linear Macromolecules I. Theory and Application to DNAt

A model consisting of a series of N+ 1 identical rigid rods connected at their ends by torsion springs and undergoing rotational brownian motion about their fixed symmetry axes is proposed as a model for the torsion dynamics of DNA. The fluorescence anisotropy bfbnund flunrophores is expressed in terms of angular correlation functions, the subsequent calculation of which is virtually identical to that recently presented for the dynamic structure factor of tIse RouseùZimm model. Tin complete time course of the fluorescence anisotropy is divided into four zones, (i) initial exponential decay zone, (ii) iotermediate zone, (iii) longest internal mude zone, and (iv) uniform mode zone. Simple approximate expressions for the lluoresccnee anisotropy are derived for the initial exponential decay zone and for the intermediate zone. The mag- nitude of end-effects, due to enhanced amplitudes of motion near the chain ends, in the intermediate zone is investigated botls numerically and analytically, and the domain of validity of the approximate expression pertinent to that zone is de- termined to be 100-fold smaller than previously supposed. The fluorescence anisotropy decay data of Wahl, Paoletti and Le Pecq for ethidium bound to DNA is interpreted in terms of this nsodel. Although relaxations in zones (iii) and (iv) can be ruled out, it is possible to obtain equally good fits to the decay dala either in zone (i) witls parameters pertinent to a rod-length of 86 base-pairs or in zone (ii) with parameters pertinent to a rod-length of I base pair. However, other data, in- cluding the steady-state polarization anisotropy in concentrated sucrose solution, definitely favor the zone (i) fit with a rod-length of 86 base-pairs, thus providing evidence for isolated torsion joints, or inhomogeneities in the torsional rigidity, nf that particular calf-thymus DNA. A comparison of twisting energies compused from the present optimum torsion con- stants with the measured free-energies of supercoil formation is also given.

DNA bifunctional intercalators. I. Synthesis and conformational properties of an ethidium homodimer and of an acridine ethidium heterodimer.

An ethidium homodimer and an acridine ethidium heterodimer have been synthesized. The ethidium and the acridine chromophore were introduced in such bifunctional intercalators in order to allow the fluorometric study of the interaction of such molecules with DNA, which is reported in the companion paper (Gaugain, B., Barbet, J., Capelle, N., Roques, B.P., & Le Pecq, J.B.(1978) Biochemistry 17 (following paper in this issue)). In the preparation of the acridine-ethidium dimer, we report the use of acetyl groups as new protecting agents in the phenanthridine series. Conformational studies of these molecules by visible absorption and NMR spectroscopy indicate that these dimers exist in equilibrium between folded and unfolded conformations and that this equilibrium is pH and temperature dependent. Models for the geometry of the folded forms are proposed.

A rapid fluorometric method for the determination of DNA in human semen.

Laboratory of Reproductive Pharmacology, Department of Pharmacology, New York Medical College, Valhalla, New York 10595, U.S.A. (Received 3rd June 1974) The dye, ethidium bromide, markedly increases its fluorescence quantum efficiency when bound to double-stranded undenatured nucleic acid (Le Pecq, Yat & Paoletti, 1964; Le Pecq & Paoletti, 1966). When added to sperm suspensions, ethidium bromide binds almost exclusively to the sperm head and induces an intense fluorescence (Edelman & Millette, 1971). In this report, a method for DNA assay in human semen and washed sperm suspensions is described. The method takes advantage of these earlier findings and permits quantitative estimates of DNA and, indirectly, of sperm counts to be made in less than 15 min. Semen specimens were obtained from medical students. Sperm counts were made in quadruplicate using a haemocytometer, as described by Freund & Carol (1964). The procedure for preparing washed sperm suspensions and the composition of the

Unwinding the DNA helix by intercalation

Data relating to the effect of intercalating drugs on the winding of the DNA helix is re-considered. Analyses by Paoletti & Le Pecq (1971) of fluorescence depolarization, X-ray diffraction and molecular model building are reappraised. It is concluded that the helix is unwound by ~12 ° as proposed by Fuller & Waring (1964). This refutes the recent suggestion by Paoletti & Le Pecq (1971) that the intercalation winds the helix by ~13 °.

Fluorimetric determination of DNA interstrand crosslinks

The increase of the fluorescence intensity of ethidium bromide on binding to DNA provides a new way to DNA interstrand crosslinks estimation. When there are no strand breaks, the method is as accurate as the classical isopycnic ultracentrifugation, but faster and much less expensive. One determination requires 30 μg of DNA using the standard fluorescence cells provided by Zeiss. In addition, the measurement of the mean number of crosslinks per DNA molecule does not require precise knowledge of the DNA concentration nor extensive purification of the DNA preparation (J.-B. Le Pecq and C. Paoletti, Anal. Biochem., 17 (1966) 100). The method is particularly useful in association with CsCl density gradient analysis when singlestrand breaks are present in the crosslinked DNA preparation as a correction can then be applied to obtain a more accurate value for Z, the mean number of crosslinks per DNA molecule.