Dominique Marie

Indirubin, the active constituent of a Chinese antileukaemia medicine,inhibits cyclin-dependent kinases

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. Here we identify indirubin and its analogues as potent inhibitors of cyclin-dependent kinases (CDKs). The crystal structure of CDK2 in complex with indirubin derivatives shows that indirubin interacts with the kinase’s ATP-binding site through van der Waals interactions and three hydrogen bonds. Indirubin-3′-monoxime inhibits the proliferation of a large range of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. These results have implications for therapeutic optimization of indigoids.

A cytometric exercise in plant DNA histograms, with 2C values for 70 species

Summary— An introduction is given to the literature concerning methods and objectives for cytometric DNA analysis of plant nuclei. This area has gained relevance with applications in plant breeding and seed production industries, where laboratories unfamiliar with cytometry are adopting the method. An extensive graphical guide to interpreting DNA histograms and their problems is given. Conversely, cytometry laboratories unfamiliar with plant sciences will find herein a guide, and references, to adapt their methods to plant material. A table of 2C values reassessed by flow cytometry for 70 plant species, plus the genome composition (GC%) in many instances, is also included.

Growth of Prochlorococcus, a Photosynthetic Prokaryote, in the Equatorial Pacific Ocean

The cell cycle of Prochlorococcus, a prokaryote that accounts for a sizable fraction of the photosynthetic biomass in the eastern equatorial Pacific, progressed in phase with the daily light cycle. DNA replication occurred in the afternoon and cell division occurred at night. Growth rates were maximal (about one doubling per day) at 30 meters and decreased toward the surface and the bottom of the ocean. Estimated Prochlorococcus production varied between 174 and 498 milligrams of carbon per square meter per day and accounted for 5 to 19 percent of total gross primary production at the equator. Because Prochlorococcus multiplies close to its maximum possible rate, it is probably not severely nutrient-limited in this region of the oceans.

Clade-Specific 16S Ribosomal DNA Oligonucleotides Reveal the Predominance of a Single Marine Synechococcus Clade throughout a Stratified Water Column in the Red Sea

ABSTRACT Phylogenetic relationships among members of the marine Synechococcus genus were determined following sequencing of the 16S ribosomal DNA (rDNA) from 31 novel cultured isolates from the Red Sea and several other oceanic environments. This revealed a large genetic diversity within the marine Synechococcus cluster consistent with earlier work but also identified three novel clades not previously recognized. Phylogenetic analyses showed one clade, containing halotolerant isolates lacking phycoerythrin (PE) and including strains capable, or not, of utilizing nitrate as the sole N source, which clustered within the MC-A (Synechococcus subcluster 5.1) lineage. Two copies of the 16S rRNA gene are present in marine Synechococcus genomes, and cloning and sequencing of these copies from Synechococcus sp. strain WH 7803 and genomic information from Synechococcus sp. strain WH 8102 reveal these to be identical. Based on the 16S rDNA sequence information, clade-specific oligonucleotides for the marine Synechococcus genus were designed and their specificity was optimized. Using dot blot hybridization technology, these probes were used to determine the in situ community structure of marine Synechococcus populations in the Red Sea at the time of a Synechococcus maximum during April 1999. A predominance of genotypes representative of a single clade was found, and these genotypes were common among strains isolated into culture. Conversely, strains lacking PE, which were also relatively easily isolated into culture, represented only a minor component of the Synechococcus population. Genotypes corresponding to well-studied laboratory strains also appeared to be poorly represented in this stratified water column in the Red Sea.

A Single Species, Micromonas pusilla (Prasinophyceae), Dominates the Eukaryotic Picoplankton in the Western English Channel

ABSTRACT The class Prasinophyceae (Chlorophyta) contains several photosynthetic picoeukaryotic species described from cultured isolates. The ecology of these organisms and their contributions to the picoeukaryotic community in aquatic ecosystems have received little consideration. We have designed and tested eight new 18S ribosomal DNA oligonucleotide probes specific for different Prasinophyceae clades, genera, and species. Using fluorescent in situ hybridization associated with tyramide signal amplification, these probes, along with more general probes, have been applied to samples from a marine coastal site off Roscoff (France) collected every 2 weeks between July 2000 and September 2001. The abundance of eukaryotic picoplankton remained high (>103 cells ml−1) during the sampling period, with maxima in summer (up to 2 × 104 cells ml−1), and a single green algal species, Micromonas pusilla (Prasinophyceae), dominated the community all year round. Members of the order Prasinococcales and the species Bathycoccus prasinos (Mamiellales) displayed sporadic occurrences, while the abundances of all other Prasinophyceae groups targeted remained negligible.

Flow cytometric detection of viruses

Representatives from several different virus families (Baculoviridae, Herpesviridae, Myoviridae, Phycodnaviridae, Picornaviridae, Podoviridae, Retroviridae, and Siphoviridae) were stained using a variety of highly fluorescent nucleic acid specific dyes (SYBR Green I, SYBR Green II, OliGreen, PicoGreen) and examined using a standard flow cytometer equipped with a standard 15 mW argon-ion laser. The highest green fluorescence intensities were obtained using SYBR Green I. DNA viruses with genome sizes between 48.5 and 300 kb could easily be detected. The fluorescence signals of the small genome-sized RNA viruses (7.4-14.5 kb) were found at the limit of detection. No significant linear relationship could be found between genome size and the green fluorescence intensity of the SYBR Green I stained virus preparations. To our knowledge, this is the first report of detecting and discriminating between a wide range of different viruses directly using flow cytometry. This rapid and precise assay represents a new and promising tool in the field of virology.

Enumeration of Phytoplankton, Bacteria, and Viruses in Marine Samples

For many years, a small but dedicated group of scientists have been using flow cytometry for the evaluation of marine microorganisms. One of these scientists now provides us with a detailed series of protocols in this area, spelling out the variations in method and instrument operation that are crucial to the successful extraction of quality flow data from marine organisms. In addition, the use of a number of less frequently employed fluorescent probes gives some insight into alternative staining procedures. As our collection of microbiologically oriented techniques increases, this knowledge database becomes invaluable.

Vertical structure of picophytoplankton at different trophic sites of the tropical northeastern Atlantic Ocean

The vertical structure and chlorophyllous pigments of picophytoplanktonic populations of the northeastern Atlantic Ocean were studied by flow cytometry and spectrofluorometry. Three sites (EU, MESO and OLIGO) on a hydrological gradient from near coastal, eutrophic waters to offshore, oligotrophic waters were occupied in October 1991 (except EU), June 1992 and December 1992. The population structure of the EU site (20°32′N 18°34′W) was greatly influenced by a permanent, wind-induced upwelling. The latter was well developed in June, and an important nutrient enrichment of surface waters ensued (> 10 mM NO2+NO3). The latter favored the blooming of diatoms, but picophytoplanktonic populations remained low. In December the upwelling was less developed, and there was a dramatic increase of the cell abundances of both prokaryotic (Prochlorococcus and Synechococcus) and picoeukaryotic populations at the EU site. Cells of all groups were concentrated and homogeneously distributed in the upper, 30–35 m thick, mixed layer. Similar population structures were observed in June and December at the MESO site (18°29′N 21°05′W). In these cases, both Synechococcus cyanobacteria (with concentrations of up to 5 × 105 cells ml−1 during winter) and picoeukaryotes (typically 1–2 × 104 cells ml−1) made significant contributions to the integrated picophytoplanktonic biomass in terms of carbon (166–333 μg C cm−2 and 92–155 μg C cm−2, respectively). Prochlorococcus made a smaller contribution (6–48 μg C cm−2), as also indicated by a low ratio of divinyl-chlorophyll a to total chlorophyll a (≤22%). The population structure observed in October at the MESO site was much more variable, even at the time-scale of hours. At the OLIGO site (21°02′N 31°08′W), the relative contribution of Prochlorococcus to picophytoplankton carbon and total chlorophyll standing stocks increased dramatically (> 50%), mainly as a result of a sharp decrease of both Synechococcus and picoeukaryotes cell concentrations down to a few thousands cells per ml. There was little seasonal change in the vertical structure of any of the three populations at this site. From analyses of cell cycle distributions during a 31 h time-series, growth rates were estimated for Prochlorococcus as 0.41 day−1 at mid-depth (80 m) and 0.39 day−1 in the deep chlorophyll maximum.

Control of Toxic Marine Dinoflagellate Blooms by Serial Parasitic Killers

The marine dinoflagellates commonly responsible for toxic red tides are parasitized by other dinoflagellate species. Using culture-independent environmental ribosomal RNA sequences and fluorescence markers, we identified host-specific infections among several species. Each parasitoid produces 60 to 400 offspring, leading to extraordinarily rapid control of the hosts population. During 3 consecutive years of observation in a natural estuary, all dinoflagellates observed were chronically infected, and a given host species was infected by a single genetically distinct parasite year after year. Our observations in natural ecosystems suggest that although bloom-forming dinoflagellates may escape control by grazing organisms, they eventually succumb to parasite attack.

Diel variability of photosynthetic picoplankton in the equatorial Pacific

The diel variability in cell abundance, light scatter, and pigment fluorescence of three autotrophic picoplankton groups (Prochlorococcus, Synechococcus, picoeukaryotes) measured by flow cytometry was investigated in surface waters of the equatorial Pacific Ocean (5°S, 150°W) during 5 days with about 1 hour temporal resolution. The diel variability of vertical profiles was examined at the same station on days 2 and 4. Prochlorococcus division rate was also estimated from cell cycle measurements. A more limited data set was obtained at a station located in very oligotrophic waters (16°S, 150°W). All three picoplankton populations exhibited very marked diel variability. Cell division was highly synchronized but not phased identically for all three populations: Synechococcus divided first, followed 2 hours later by Prochlorococcus and 7 hours later by picoeukaryotes. Cells grew in size only once the sun had risen, but growth did continue in the dark for a short period. Growth processes occurred in parallel at the top and the bottom of the mixed layer, inducing uniform profiles for cell abundance and scatter. For chlorophyll fluorescence, in contrast, prokaryotes displayed opposite patterns during the light period between surface (decrease due to very strong quenching) and depth (increase). This created steep vertical gradients during the day that vanished at night because of convective mixing. In the top 25 m, strong light intensities (including UV radiation) had very pronounced detrimental effects on prokaryotes, especially on Prochlorococcus, inducing fluorescence quenching, slowed down growth, and retardation of DNA synthesis.