Cryogenic sensor enabling broad-band and traceable power measurements.

Recently, great progress has been made in the field of ultrasensitive microwave detectors, reaching even the threshold for utilization in circuit quantum electrodynamics. However, cryogenic sensors lack the compatibility with broad-band metrologically traceable power absorption measurements at ultralow powers, which restricts their range of applications. Here, we demonstrate such measurements using an ultralow-noise nanobolometer, which we extend by an additional direct-current (dc) heater input. The tracing of the absorbed power relies on comparing the response of the bolometer between radio frequency and dc-heating powers traced to the Josephson voltage and quantum Hall resistance. To illustrate this technique, we demonstrate two different methods of dc-substitution to calibrate the power that is delivered to the base temperature stage of a dilution refrigerator using our in situ power sensor. As an example, we demonstrate the ability to accurately measure the attenuation of a coaxial input line between the frequencies of 50 MHz and 7 GHz with an uncertainty down to 0.1 dB at a typical input power of -114 dBm.

Bolometer operating at the threshold for circuit quantum electrodynamics.

Radiation sensors based on the heating effect of absorbed radiation are typically simple to operate and flexible in terms of input frequency, so they are widely used in gas detection1, security2, terahertz imaging3, astrophysical observations4 and medical applications5. Several important applications are currently emerging from quantum technology and especially from electrical circuits that behave quantum mechanically, that is, circuit quantum electrodynamics6. This field has given rise to single-photon microwave detectors7-9 and a quantum computer that is superior to classical supercomputers for certain tasks10. Thermal sensors hold potential for enhancing such devices because they do not add quantum noise and they are smaller, simpler and consume about six orders of magnitude less power than the frequently used travelling-wave parametric amplifiers11. However, despite great progress in the speed12 and noise levels13 of thermal sensors, no bolometer has previously met the threshold for circuit quantum electrodynamics, which lies at a time constant of a few hundred nanoseconds and a simultaneous energy resolution of the order of 10h gigahertz (where h is the Planck constant). Here we experimentally demonstrate a bolometer that operates at this threshold, with a noise-equivalent power of 30 zeptowatts per square-root hertz, comparable to the lowest value reported so far13, at a thermal time constant two orders of magnitude shorter, at 500 nanoseconds. Both of these values are measured directly on the same device, giving an accurate estimation of 30h gigahertz for the calorimetric energy resolution. These improvements stem from the use of a graphene monolayer with extremely low specific heat14 as the active material. The minimum observed time constant of 200 nanoseconds is well below the dephasing times of roughly 100 microseconds reported for superconducting qubits15 and matches the timescales of currently used readout schemes16,17, thus enabling circuit quantum electrodynamics applications for bolometers.

Air drying optimization of Saccharomyces cerevisiae through its water-glycerol dehydration properties.

AIMS: This study describes the different stages of optimization in an original drying process for yeasts, which allows the retrieval of dried samples of Saccharomyces cerevisiae CBS 1171 with maximum viability. METHODS AND RESULTS: The process involves the addition of wheat flour to yeast pellets, followed by mixing and then air-drying in a fluidized bed dryer. The sensitivity to the osmotic stress was first studied in a water-glycerol solution and the observed results were then applied to the drying process. This study have shown that the yeast was quite resistant to osmotic stress and pointed out the existence of zones of sensitivity where viability dramatically decrease as function of final osmotic pressure and temperature of the treatment. Thus, for dehydration until low osmotic pressure (133 MPa, i.e. a(w) = 0.38) results have shown that viability was better when temperature of the treatment was less than 8 degrees C or higher than 25 degrees C. Moreover, kinetic of dehydration was found to greatly influence cells recovery. CONCLUSIONS: These observations allowed the choice of parameters of dehydration of yeasts with an original drying process which involve the mix of the yeasts with wheat flour and then drying in a fluidized bed. SIGNIFICANCE AND IMPACT OF THE STUDY: This process dried rapidly the yeasts to less than 220 MPa (aw < or = 0.2) with whole cell recovery and good fermentative capabilities.

Photochemical rearrangement of oxaziridines and nitrones in the hexahydroindole series: a convenient synthetic route to 1-azabicyclo[5.2.0]nonan-2-ones as novel RGD mimetics.

[reaction: see text] Photolysis of oxaziridines a or nitrones b provides a convenient synthetic route to fused bicyclic lactams c adequately substituted on both cycles A and B as scaffolds for mimicking conformationally constrained beta-turn peptides as in the tripeptide RGD signaling motif of fibronectin.

Toxic effects of wastewaters collected at upstream and downstream sites of a purification station in cultures of rainbow trout hepatocytes.

The toxic effects of wastewater samples, collected in December 1998, from upstream (U) and downstream (D) sites of the purification station of the town of Nice (South-East France on the coast of the Mediterranean Sea) were assessed undiluted and at various dilutions (75%, 50%, and 25% of collected water sample), on trout hepatocyte cultures treated for 48 or 72 h. Chemical contamination (PCBs, PAHs, Cd, Cu, Pb, and Zn) was also evaluated by chemical analysis. The water samples from the upstream site were more cytotoxic than those from the downstream site. The induction of CYP1A enzyme and metallothioneins (MTs) were selected as specific indicators of exposure to organic contaminants and metals, respectively. CYP1A-related EROD activity as well as protein expression were found to be greatly induced after 72 h exposure of the hepatocytes to the undiluted water samples (U(100%) and D(100%)), but CYP1A1 mRNA was significantly overexpressed only by samples from the upstream site. Maximal MT levels were reached after 48 h of treatment with the least concentrated water samples (U(25%) and D(25%)). Glutathione S-transferase (GST) activities were similarly increased under the same conditions. On the other hand, there was no significant glutathione peroxidase (GPx) activity response. Induction of apoptosis was analyzed by using as markers both the fragmentation of the nuclear DNA into oligonucleosomal-length fragments recognized as a "DNA ladder" and the activation of DEVD (Asp-Glu-Val-Asp)-dependent protease considered as the central mediator of programmed cell death. Significant DNA cleavage was only detectable after 72-h exposure to the most concentrated water samples from upstream sites (U(75%) and U(100%)). DEVD-dependent protease activities were significantly increased, mainly in cells exposed to U(75%) and D(25%) for 72 h. In addition, pollution-related DNA damage assessed by using the Comet assay was approximatively 1.5 times greater than that of the control level with the undiluted water samples U(100%) and D(100%), after 72-h and 48-h exposure, respectively. The present study shows that such a multibiomarker-based approach could provide complementary information, for aquatic pollution monitoring, about the early biochemical effects in cells exposed to complex chemical pollution and could be considered as early warning systems to aquatic pollution.

Differential induction of glutathione S-transferases in the clam Ruditapes decussatus exposed to organic compounds.

Studies of glutathione S-transferase (GST) induction were performed in the Mediterranean clam Ruditapes decussatus after controlled exposure to organics in holding tanks. Clams were treated with phenobarbital (PB), benzo[a]pyrene (BaP), and 2,2-bis-(p-chlorophenyl)-1,1-dichlorethylene (p,p'-DDE). Three different substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid (ETHA), and paranitrobenzene chloride (PNBC), were used to determine GST activities in order to distinguish the isoenzymes induced by contamination. The isoforms conjugating ETHA were significantly induced by treatment with PB and BaP, whereas exposure to p,p'-DDE induced isoforms conjugating CDNB and ETHA. An antibody against affinity-purified GSTs from R. decussatus was prepared by injection into rabbit. The serum containing the antibody gave a positive reaction with both the purified GSTs from R. decussatus and the low molecular weight GSTs from rat. Subcellular fractions from both control and treated animals were analyzed by Western blot. Cytosolic extracts from clams contaminated with PB and p,p'-DDE showed a 24-kDa band in addition to the 26-kDa band recognized by the antibody. Results of these studies suggest that, in R. decussatus, organics may induce GSTs belonging to the pi class.

Structure-activity relationship for bromoindole carbaldehydes: effects on the sea urchin embryo cell cycle.

Natural derivatives of indole-3-carbaldehyde were isolated from the tropical marine ascidian Stomoza murravi. A series of 13 derivatives, three natural and 10 synthetic (brominated and N-methylated), were examined for their effects on cell division of sea urchin eggs. These derivatives were shown to inhibit the first mitotic cycle in a concentration-dependent manner. By comparing the IC50 values with the structure of the various molecules, we were able to determine that bromination increased the cytotoxicity of the compound with a maximum occurring when bromine was added to carbon number 2, while addition of N-methylation was shown to markedly reduce the cytotoxicity of these same compounds brominated at carbon 2 only. Biological activity of this family of compounds has been characterized, via detailed study of addition of the most active derivative, 2,5,6-tribromoindole-3-carbaldehyde, on macromolecule synthesis and cytoskeleton reorganization during the first mitotic cycle of fertilized sea urchin eggs. Fluorescence localization of chromatin and microtubules revealed that 2,5,6-tribromoindole-3-carbaldehyde allowed pronuclei migration and fusion but prevented the condensation of chromatin, nuclear envelope breakdown, and bipolar mitotic spindle assembly, inducing an arrest of sea urchin embryogenesis at the beginning of mitosis. It is postulated here that this phenotype is likely to be due to a strong inhibition of DNA replication and protein synthesis.

Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygene species.

Cadmium poses a serious environmental threat in aquatic ecosystems but the mechanisms of its toxicity remain unclear. The purpose of this work was first to determine whether cadmium induced apoptosis in trout hepatocytes, second to determine whether or not reactive oxygen species (ROS) were involved in cadmium-induced apoptosis and genotoxicity. Hepatocytes exposed to increasing cadmium concentrations (in the range of 1-10 microM) showed a molecular hallmark of apoptosis which is the fragmentation of the nuclear DNA into oligonucleosomal-length fragments, resulting from an activation of endogenous endonucleases and recognized as a 'DNA ladder' on conventional agarose gel electrophoresis. Exposure of hepatocytes to cadmium led clearly to the DEVD-dependent protease activation, acting upstream from the endonucleases and considered as central mediators of apoptosis. DNA strand breaks in cadmium-treated trout hepatocytes was assessed using the comet assay, a rapid and sensitive single-cell gel electrophoresis technique used to detect DNA primary damage in individual cells. Simultaneous treatment of trout hepatocytes with cadmium and the nitroxide radical TEMPO used as a ROS scavenger, reduced significantly DNA fragmentation, DEVD-related protease activity and DNA strand breaks formation. These results lead to a working hypothesis that cadmium-induced apoptosis and DNA strand breaks in trout hepatocytes are partially triggered by the generation of ROS. Additional studies are required for proposing a mechanistic model of cadmium-induced apoptosis and genotoxicity in trout liver cells, in underlying the balance between DNA damage and cellular defence systems in fish.

Leukotriene B4 photoaffinity probes: design, synthesis and evaluation of new arylazide-1,3-disubstituted cyclohexanes.

The synthesis and the binding affinities of new leukotriene B4 receptor photoaffinity probes, where a 1,3-disubstituted cyclohexane ring replaces the conjugated delta6,7 and delta8,9 double bonds of the natural eicosanoid, are described. One enantiomeric compound, 4b alpha, is specifically cross-linked upon photolysis to the recombinant leukotriene B4 receptor from human origin (h-BLTR) solubilized in a micellar medium. This probe appears as a good candidate for identifying the ligand binding site of this receptor.

Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.

Sulfation is a major modification of many molecules in eukaryotes that is dependent on the enzymatic synthesis of an activated sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). While sulfate activation has long been assumed to occur in the cytosol, we show in this study that human PAPS synthetase 1 (PAPSS1), a bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase enzyme sufficient for PAPS synthesis, accumulates in the nucleus of mammalian cells. Nuclear targeting of the enzyme is mediated by its APS kinase domain and requires a catalytically dispensable 21 amino acid sequence at the amino terminus. Human PAPSS1 and Drosophila melanogaster PAPSS localize to the nucleus in yeast and relieve the methionine auxotrophy of ATP sulfurylase- or APS kinase-deficient strains, suggesting that PAPSS1 is fully functional in vivo when targeted to the nucleus. A second PAPS synthetase gene, designated PAPSS2, has recently been described, mutations of which are responsible for abnormal skeletal development in human spondyloepimetaphyseal dysplasia and murine brachymorphism. We found that PAPSS2, which localizes to the cytoplasm when ectopically expressed in mammalian cells, is relocated to the nucleus when coexpressed with PAPSS1. Taken together, these results indicate that a sulfation pathway might exist in the nucleus of eukaryotic cells. -Besset, S., Vincourt, J.-B., Amalric, F., Girard, J.-P. Nuclear localization of PAPS synthetase 1: a sulfate activation pathway in the nucleus of eukaryotic cells.

The nitroxide stable radical tempo prevents metal-induced inhibition of CYP1A1 expression and induction.

Heavy metals are known to provoke oxidative stress in fish liver cells. Because H2O2, OH*- and intracellular superoxide are involved in this oxidation, we investigated the effect of nitroxide radical, 2,2,6,6-tetramethylpiperidinyl-N-oxyl (abbreviated as TEMPO), a cell-permeable agent possessing antioxidant properties, on CYP1A expression in trout (Oncorhynchus mykiss) hepatocytes. 3-methylcholanthrene (3-MC) induced the CYP1A-related EROD activity. This induction was inhibited by concomitant exposure to Cd (II), Cu (II), Pb (II) or Zn (II). CYP1A mRNA levels were also reduced. Simultaneous treatment with 3-MC, a heavy metal and TEMPO suppressed both the inhibition of EROD activity and the decrease of CYP1A mRNA expression. These results suggest a working hypothesis that heavy metals produce multiple oxidative effects, including generation of hydroxyl radicals, which could down-regulate CYP1A1 expression. This metal-induced inhibition was prevented by TEMPO, which might protect trout hepatocytes by scavenging free radicals and thus preventing their inhibitory effects on CYP1A induction and expression.

Heterogeneity of endothelial cells: the specialized phenotype of human high endothelial venules characterized by suppression subtractive hybridization.

High endothelial venules (HEVs) are specialized postcapillary venules, found in lymphoid organs and chronically inflamed tissues, that support high levels of lymphocyte extravasation from the blood. Molecular characterization of HEV endothelial cells (HEVECs) has been hampered by difficulties in their purification and in vitro maintenance. To overcome these limitations, we developed a strategy combining the use of freshly purified HEVECs ( approximately 98% positive for the HEV-specific marker MECA-79) and the recently described polymerase chain reaction (PCR)-based cDNA subtraction cloning procedure called suppression subtractive hybridization (SSH). Subtracted probes prepared by SSH from small amounts of total RNA were used to screen a HEVEC cDNA library. This resulted in cloning of 22 cDNAs preferentially expressed in HEVECs, which encode the promiscuous chemokine receptor DARC, mitochondrial components, and matricellular proteins. The latter included hevin, thrombospondin-1, and mac25/IGFBP-rP1, which is a secreted growth factor-binding protein previously found to accumulate specifically in tumor blood vessels. Biochemical and histochemical analysis confirmed the identification of mac25 and DARC as novel markers of the HEVECs. Ultrastructural immunolocalization revealed a noticeable association of mac25 and MECA-79 antigens with microvillous processes near the endothelial cell junctions, suggesting a role for mac25 in the control of lymphocyte emigration. This study shows that PCR-based SSH is useful for cloning of differentially expressed genes in very small samples.

Molecular cloning and functional analysis of SUT-1, a sulfate transporter from human high endothelial venules.

High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na(+)-coupled anion transporters and exhibits 40-50% amino acid identity with the rat renal Na(+)/sulfate cotransporter, NaSi-1, as well as with the human and rat Na(+)/dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na(+)-dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription-PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na(+)-coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV.

Tissue-specific induction and inactivation of cytochrome P450 catalysing lauric acid hydroxylation in the sea bass, Dicentrarchus labrax.

Microsomal cytochrome P450-dependent lauric acid hydroxylase activities were characterized in liver, kidney, and intestinal mucosa of the sea bass (Dicentrarchus labrax). Microsomes from these organs generated (omega-1)-hydroxylauric acid and a mixture of positional isomers including (omega)-, (omega-2)-, (omega-3)- and (omega-4)-hydroxylauric acids, which were identified by RP-HPLC and GC-MS analysis. Peroxisome proliferators, such as clofibrate and especially di(2-ethylhexyl) phthalate, increased kidney microsomal lauric acid hydroxylase activities. The synthesis of 11-hydroxylauric acid was enhanced 5.3-fold in kidney microsomes. Liver microsomal lauric acid hydroxylase activities were weakly affected and no significant induction was found in small intestine microsomes from clofibrate or di(2-ethylhexyl) phthalate-treated fish. The differences in lauric acid metabolisation and the tissue-specific induction by peroxisome proliferators suggest the involvement of several P450s in this reaction. Incubations of liver and kidney microsomes with lauric acid analogues (11- or 10-dodecynoic acids) resulted in a time- and concentration-dependent loss of lauric acid hydroxylase activities. The induction of these activities in fish by phthalates, which are widely-distributed environmental pollutants, may be taken into consideration for the development of new biomarkers.

Cloning of CYP4F7, a kidney-specific P450 in the sea bass Dicentrarchus labrax.

A cDNA sequence coding for a cytochrome P450 of the CYP4F subfamily was isolated from total RNA of sea bass kidney by rapid amplification of cDNA ends. The full length sequence coded for a protein of 526 amino acids. The amino acid sequence shared 39% to 56% residue identities with the mammalian CYP4F sequences, and thus was named CYP4F7 (accession number AF123541). RNA blot analysis using CYP4F7 cDNA as a probe indicated that the corresponding mRNA was only detected in kidney. Expression in the kidney was constitutive, and no induction of this mRNA was detected in this or other tissues, with any of the inducers tested, including peroxisome proliferators.

Mechanisms of cytotoxicity by cosmetic ingredients in sea urchin eggs.

The acute cytotoxicities of four cosmetic ingredients: a preservative, imidazolidinylurea (IU) and three mild surfactants, cocamido propyl hydroxy sultaine (CAS), magnesium laureth sulfate (Mg LES), and decyl glucoside (APG) were studied using sea urchin eggs. The cellular targets of these compounds were identified by studying the effects on calcium homeostasis, intracellular pH, sodium and potassium contents, protein and DNA synthesis, and protein phosphorylation. These compounds inhibited the first cleavage of sea urchin eggs in a dose-dependent fashion with half maximal doses (IC50) from 30 microg/ml for Mg LES, 60 microg/ml for IU, 83 microg/ml for CAS, to above 400 microg/ml for APG. The time at which a compound showed the greatest toxicity to the cell cycle was definable for APG (between 20 and 50 min postfertilization) and IU (from fertilization to 50 min later); the other compounds being toxic throughout division. Compounds exhibited toxicity to a wide range of cellular targets. IU, the least toxic, mainly operates through inhibition of protein and DNA syntheses. CAS and Mg LES produced nonspecific cytotoxicity related to alterations of membrane and endomembrane permeabilities resulting in ionic disequilibrium (Na+, K+, Ca2+) and inhibition of intracellular storage of Ca2+. The APG effect mainly involved intracellular pH and DNA synthesis, a hypothesis suggested by the narrow postfertilization period of maximal toxicity.

Caulerpenyne blocks MBP kinase activation controlling mitosis in sea urchin eggs.

In a previous study, we demonstrated that caulerpenyne (Cyn), a natural sesquiterpene having an antiproliferative potency, blocked the mitotic cycle of sea urchin embryos at metaphase and inhibited the phosphorylation of several proteins, but did not affect histone H1 kinase activation (Pesando et al, 1998, Eur. J. Cell Biol. 77, 19-26). Here, we show that concentrations of Cyn that blocked the first division of the sea urchin Paracentrotus lividus embryos in a metaphase-like stage (45 microM) also inhibited the stimulation of mitogen-activated protein kinase (MAPK) activity in vivo as measured in treated egg extracts using myelin basic protein (MBP) as a substrate (MBPK). However, Cyn had no effect on MBP phosphorylation when added in vitro to an untreated egg extract taken at the time of metaphase, suggesting that Cyn acts on an upstream activation process. PD 98059 (40 microM), a previously characterized specific synthetic inhibitor of MAPK/extracellular signal-regulated kinase-1 (MEK1), also blocked sea urchin eggs at metaphase in a way very similar to Cyn. Both molecules induced similar inhibitory effects on MBP kinase activation in vivo, but had no direct effect on MBP kinase activity in vitro, whereas they did not affect H1 kinase activation neither in vivo nor in vitro. As a comparison, butyrolactone 1 (100 microM), a known inhibitor of H1 kinase activity, did inhibit H1 kinase of sea urchin eggs in vivo and in vitro, and blocked the sea urchin embryo mitotic cycle much before metaphase. Immunoblots of mitotic extracts, treated with anti-active MAP-kinase antibody, showed that both Cyn and PD 98059 reduced the phosphorylation of p42 MAP kinase (Erk2) in vivo. Our overall results suggest that Cyn blocks the sea urchin embryo mitotic cycle at metaphase by inhibiting an upstream phosphorylation event in the MBPK activation pathway. They also show that H1 kinase and MBPK activation can be dissociated from each other in this model system.

Synthesis and structure-activity relationships of new 1, 3-disubstituted cyclohexanes as structurally rigid leukotriene B(4) receptor antagonists.

A series of 1-hydroxy-3-¿3-hydroxy-7-phenyl-1-hepten-1-yl cyclohexane acetic acid derivatives was designed based on postulated active conformation of leukotriene B(4) (LTB(4)) and evaluated as human cell surface LTB(4) receptor (BLTR) antagonists. Binding was determined through ¿(3)HLTB(4) displacement from human neutrophils and receptor antagonistic assays by in vitro measurements of inhibition of leukocyte chemotaxis induced by LTB(4). On the basis of these assays, a structure-affinity relationship was investigated. Optimization of the acid chain length and omega-substitution of a phenyl group on the lipophilic tail were shown to be critical for binding activity. These modifications led to the discovery of compounds with submicromolar potency and selective BLTR antagonism. The most potent compound 3balpha (IC(50) = 250 nM) was found to significantly inhibit oedema formation in a topical model of phorbolester-induced inflammation. Substantial improvement of in vitro potency was achieved by modification of the carboxylic acid function leading to the identification of the N,N-dimethylamide series. Compound 5balpha, free of agonist activity, displayed higher potency in receptor binding with an IC(50) of 40 nM. These results support the hypothesis that the spatial relationship between the carboxylic acid and allylic hydroxyl functions is crucial for high binding affinity with BLTR.

Caulerpenyne interferes with microtubule-dependent events during the first mitotic cycle of sea urchin eggs.

Caulerpenyne (Cyn), the major secondary metabolite synthesized by the green alga Caulerpa taxifolia proliferating in the Mediterranean Sea, is a cytotoxic sesquiterpene. As this compound has an antiproliferative potency by inhibiting division of many types of cells, we examined the precise effects of Cyn during the early development of the sea urchin Paracentrotus lividus. Whereas Cyn (60 microM) had no effect on fertilization, it blocked the first cell division in the same manner whether added before or after fertilization, provided the drug was added before or during metaphase. Immunofluorescence localization revealed that Cyn had no effect on the microtubular sperm aster formation, pronuclei migration and fusion, chromosome condensation, nuclear envelope breakdown, and bipolar mitotic spindle assembly. However, mitosis was blocked in a metaphase-like stage at which most chromosomes were aligned at the equatorial plate, while a few of them had not even migrated towards the metaphase plate. When added after the metaphase-anaphase transition, the first division occurred normally but the second division was inhibited with the same phenotype as described above. We previously showed that Cyn did not affect protein synthesis or H1 kinase activation or deactivation (Pesando et al., 1996, Aquat. Toxicol. 35, 139), but that it partially inhibited DNA synthesis. Our results establish that Cyn does not affect the microfilament-dependent processes of fertilization and cytokinesis and allows the beginning of mitosis, but prevents normal DNA replication and results in metaphase-like arrest of sea urchin embryos.