Feasibility of assessing the community composition of prasinophytes at the Helgoland Roads sampling site with a DNA microarray

The microalgal class Prasinophyceae (Chlorophyta) contains several picoeukaryotic species, which are known to be common in temperate and cold waters and have been observed to constitute major fractions of marine picoplankton. However, reliable detection and classification of prasinophytes are mainly hampered by their small size and few morphological markers. Consequently, very little is known about the abundance and ecology of the members of this class. In order to facilitate the assessment of the abundance of the Prasinophyceae, we have designed and evaluated an 18S rRNA gene-targeted oligonucleotide microarray consisting of 21 probes targeting different taxonomic levels of prasinophytes. The microarray contains both previously published probes from other hybridization methods and new probes, which were designed for novel prasinophyte groups. The evaluation of the probe set was done under stringent conditions with 18S PCR fragments from 20 unialgal reference cultures used as positive targets. This microarray has been applied to assess the community composition of prasinophytes at Helgoland, an island in the North Sea where time series data are collected and analyzed daily but only for the nano- and microplankton-size fractions. There is no identification of prasinophytes other than to record them numerically in the flagellate fraction. The samples were collected every 2 weeks between February 2004 and December 2006. The study here demonstrates the potential of DNA microarrays to be applied as a tool for quick general monitoring of this important picoplanktonic algal group.     

Choice of Pore Size Can Introduce Artefacts when Filtering Picoeukaryotes for Molecular Biodiversity Studies

Published results of studies based on samples size fractionated by sequential filtration (e.g. 0.2–3 μm) indicate that many ciliate, dinoflagellate and rhizarian phylotypes are found among marine picoeukaryotes. This is somewhat surprising as these protists are typically known as being large organisms (often >10 μm) and no picoplanktonic species have so far been identified. Here, the abundances of ciliate and dinoflagellate phylotypes in published molecular studies of picoeukaryotes are shown to correlate negatively with the pore size chosen for the end filter in the sequential filtrations (i.e. the filter used to collect the microbial biomass). This suggests that extracellular DNA adhering to small particles may be the source of ciliate and dinoflagellate phylotypes in picoplanktonic size fractions. This hypothesis was confirmed using real-time qPCR, which revealed significantly less dinoflagellate 18S rDNA in a 0.8–3-μm size fraction compared to 0.2–3 μm. On average, the abundance of putative extracellular phylotypes decreased by 84–89 % when a 0.8-?μm end filter was used rather than a 0.2-μm end filter. A 0.8-μm filter is, however, not sufficient to retain all picoeukaryotic cells. Thus, selection of filter pore size involves a trade-off between avoiding artefacts generated by extracellular DNA and sampling the entire picoeukaryotic community. In contrast to ciliate and dinoflagellate phylotypes, rhizarian phylotypes in the picoplankton size range do not display a pattern consistent with an extracellular origin. This is likely due to the documented existence of picoplanktonic swarmer cells within this group.     

Picoeukaryotic Plankton Diversity at the Helgoland Time Series Site as Assessed by Three Molecular Methods

We analyzed picoeukaryote assemblages in the German Bight at the Helgoland time series site by sequencing cloned eukaryotic 18S rRNA genes in six genetic libraries plus one library from the Orkney Islands from a cruise of opportunity. The libraries were constructed from environmental samples collected at different periods of the year. The same samples were also analyzed using a fingerprinting technique, single-strand conformational polymorphism (SSCP), and DNA microarrays with class-level oligonucleotide probes. One hundred unique clones were analyzed from each library, thus insuring over 85% coverage of the library. The V4 region of the 18S rRNA gene was sequenced from each of these clones, thus providing the most discrimination among the clones. The nonphotosynthetic picoeukaryotic component dominated over the photosynthetic one and was represented by the ciliates at 45% and group II alveolates at 42%. Prasinophytes dominated the photosynthetic group at 40%, but other picoplankton groups, such as bolidomonads and chrysophytes, were also present. Totally novel groups were found in the cryptomonads and in the dinoflagellates. A new algal group sister to the cryptophyte nuclear gene and the glaucocystophytes was also found. These three groups have been found in other picoeukaryotic planktonic clone libraries. SSCP analyses at closer time intervals suggest that clone libraries should be made at weekly intervals if succession in the picoeukaryotic plankton community is to be monitored accurately. A comparison of annual samples suggests thatthere appears to be an annual cycle with regard to species composition. Microarray analysis supported the clone library data and offered a faster means of community analysis, which can be performed with similar accuracy and with higher throughput for a more in-depth analysis.     

The transcriptional control of TGF-beta in human osteoblast-like cells is distinct from that of IL-1 beta.

Transforming growth factor beta (TGF-beta) and interleukin 1 (IL-1) are among the most potent osteotropic cytokines. The expression of mRNA for both TGF-beta and IL-1 beta was studied in human osteoblast-like cells in vitro. These cells constitutively expressed TGF-beta but not IL-1 beta mRNA. Treatment of the cells with the systemic hormones 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (10(-8) M) and parathyroid hormone (10(-7) M) induced an increase in TGF-beta mRNA but failed to stimulate the production of IL-1-beta mRNA. Retinoic acid (10(-8) M) had no effect on either mRNA species. The cytokines IL-1 alpha (200 pg/ml), tumour necrosis factor alpha (TNF-alpha) (17 ng/ml) and bacterial lipopolysaccharide (LPS) (500 ng/ml) stimulated the production of IL-1 beta mRNA after 6-8 hours. This was followed by an increase in protein production after 24 hours. In contrast, the production of TGF-beta mRNA remained constant after treatment with these agents. Treatment of the cells with hydrocortisone (10(-8) M) resulted in the suppression of both TGF-beta and IL-1 beta mRNA. However, when the stimulating agent 1,25-(OH)2D3 was added in conjunction with hydrocortisone the mRNA expression of TGF-beta mRNA returned to 70% of the stimulated level. In contrast, the addition of the stimulatory agent IL-1 alpha to hydrocortisone-treated cells resulted in no increase in IL-1 beta mRNA. In-situ hybridization demonstrated both TGF-beta and IL-1 beta mRNA at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoplasmic membrane polarization in Gram-positive and Gram-negative bacteria grown in the absence and presence of tetracycline

The ability of numerous diverse compounds and ions to cross the bacterial cytoplasmic membrane by diffusion and active transport is highly dependent on cytoplasmic membrane fluidity, which can be measured using fluorescent probes to estimate membrane polarization values. However, membrane polarization data are lacking for most bacterial species. The cytoplasmic membrane polarization values for Arthrobacter sp. ATCC 21908, Bacillus cereus NRC 3045, Pseudomonas fluorescens R2F, Pseudomonas putida NRC 2986 and Escherichia coli C600 bacterial cells were spectrofluorometrically measured over a temperature range from 10 to 50 degrees C, and in the absence and presence of 1 microg/ml tetracycline, using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to obtain new information on their membrane fluidity. At an assay temperature of 10 degrees C, E. coli cells grown in the absence of tetracycline exhibited the highest cytoplasmic membrane polarization value (least fluid membrane) of 0.446, followed by values of 0.392, 0.371, 0.344 and 0.293, respectively, for B. cereus, Arthrobacter sp., P. fluorescens and P. putida. At an assay temperature of 30 degrees C, the polarization values ranged from 0.357 to 0.288 for cells grown in the absence of tetracycline, regardless of the species. B. cereus grown in the presence of 1 microg/ml tetracycline had lower polarization values than when grown in the absence of this antibiotic at all assay temperatures. Regardless of the absence or presence of 1 microg/ml tetracycline in the growth medium, all bacterial species generally exhibited a more fluid membrane as the assay temperature increased from 10 to 50 degrees C. To our knowledge, these are some of the first cytoplasmic membrane polarization values reported for these Gram-negative and Gram-positive bacteria over a broad temperature range and also for cells grown in the presence of tetracycline.     

Design and evaluation of 16S rRNA-targeted peptide nucleic acid probes for whole-cell detection of members of the genus Listeria

Six fluorescein-labeled peptide nucleic acid oligomers targeting Listeria-specific sequences on the 16S ribosomal subunit were evaluated for their abilities to hybridize to whole cells by fluorescence in situ hybridization (FISH). Four of these probes yielded weak or no fluorescent signals after hybridization and were not investigated further. The remaining two FISH-compatible probes, LisUn-3 and LisUn-11, were evaluated for their reactivities against 22 Listeria strains and 17 other bacterial strains belonging to 10 closely related genera. Hybridization with BacUni-1, a domain-specific eubacterial probe, was used as a positive control for target accessibility in both Listeria spp. and nontarget cells. RNase T1 treatment of select cell types was used to confirm that positive fluorescence responses were rRNA dependent and to examine the extent of nonspecific staining of nontarget cells. Both LisUn-3 and LisUn-11 yielded rapid, bright, and genus-specific hybridizations at probe concentrations of approximately 100 pmol ml(-1). LisUn-11 was the brightest probe and stained all six Listeria species. LisUn-3 hybridized with all Listeria spp. except for L. grayi, for which it had two mismatched bases. A simple ethanolic fixation yielded superior results with Listeria spp. compared to fixation in 10% buffered formalin and was applicable to all cell types studied. This study highlights the advantages of peptide nucleic acid probes for FISH-based detection of gram-positive bacteria and provides new tools for the rapid detection of Listeria spp. These probes may be useful for the routine monitoring of food production environments in support of efforts to control L. monocytogenes.     

Genetic diversity of picoeukaryotes in eight lakes differing in trophic status

The genetic diversity of picoeukaryotes (0.2-5.0 μm) was investigated in 8 lakes differing in trophic status in Nanjing, China. Denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing of 18S rRNA genes were applied to analyze the picoeukaryotic communities. DGGE analysis showed that among the 8 lakes, the diversity of picoeukaryotes was greatest in the mesotrophic Lake Nan (24 bands) and least in the oligotrophic Lake Qian (12 bands). Cluster analysis of DGGE profiles revealed that the 8 lakes were grouped into 2 distinct clusters. Cluster 1 contained lakes Mochou, Zixia, Huashen, Nan, Pipa, and Qian, while cluster 2 contained lakes Xuanwu and Baijia. Clone libraries were constructed from the mesotrophic Lake Xuanwu and the oligotrophic Lake Zixia, and the 2 libraries were compared using the program LIBSHUFF. This analysis indicated that the picoeukaryotic community composition differed significantly between the 2 lakes (p = 0.001). A total of 25 operational taxonomic units were detected; 18 (62 clones) were related to known eukaryotic groups, while 7 (30 clones) were not affiliated with any known eukaryotic group. Alveolates and stramenopiles were the dominant groups in Lake Xuanwu, while alveolates and chlorophyta predominated in Lake Zixia. Multivariate statistical analysis indicated that the differences in the picoeukaryotic community composition of the 8 lakes might be related to trophic status and top-down regulation by metazooplankton.     

Effect of petrochemical sludge concentrations on microbial communities during soil bioremediation

Qualitative and quantitative changes of microbial communities in soil microcosms during bioremediation were determined throughout one year. The soil was contaminated with 0%, 2.5%, 5%, 10% (wt/wt) of petrochemical sludge containing polynuclear aromatic hydrocarbons. We analyzed the hydrocarbon concentration in the microcosms, the number of cultivable bacteria using CFU and most probable number assays, the community structure using denaturing gradient gel electrophoresis, and the metabolic activity of soil using dehydrogenase activity and substrate-induced respiration assays. After one year of treatment, the chemical analysis suggested that the hydrocarbon elimination process was over. The biological analysis, however, showed that the contaminated microcosms suffered under long-term disturbance. The number of heterotrophic bacteria that increased after sludge addition (up to 10(8)-10(9) cells ml(-1)) has not returned to the level of the control soil (2-6 x 10(7) cells ml(-1)). The community structure in the contaminated soils differed considerably from that in the control. The substrate-induced respiration of the contaminated soils was significantly lower (approximately 10-fold) and the dehydrogenase activity was significantly higher (20-40-fold) compared to the control. Changes in the community structure of soils depended on the amount of added sludge. The species, which were predominant in the sludge community, could not be detected in the contaminated soils.     

Dormancy as a survival strategy of the fish pathogen Streptococcus parauberis in the marine environment

The fate of Streptococcus parauberis in seawater and sediment microcosms at different temperatures (6 and 22 degrees C) was investigated by comparing the survival dynamics of 2 strains of this bacterial species, isolated respectively from diseased turbot and cattle. The turbot and the bovine isolate showed similar survival kinetics, remaining culturable for approximately 1 mo in water and 6 mo in sediment. A slight influence of temperature on the stability of the cells was observed, in that the number of culturable cells was about 1 log10 unit higher at 6 than at 22 degrees C. During the starvation period, the metabolic activity of the cells, after suffering a strong reduction during the first 12 d, stabilized at levels ranging from 20 to 40% of the initial values. However, in all the microcosms, the acridine orange (AO) and 4',6-diamidino-2-phenilindole (DAPI) counts remained at about 10(5) cells ml(-1) throughout the experimental period, even when cells became undetectable by standard plate count methods. The addition of fresh medium to microcosms containing nonculturable cells induced the return to culturability of S. parauberis strains. On the basis of these results, it seems that S. parauberis has the ability to enter into a viable but nonculturable (VBNC) state. Dormant cells of the turbot isolate maintained their infectivity and pathogenic potential for fish.     

Eukaryotic picoplankton communities of the Mediterranean Sea in summer assessed by molecular approaches (DGGE, TTGE, QPCR)

The composition and abundance of eukaryotic picoplankton (defined here as cells smaller than 3 mum) was investigated in the Morocco upwelling and throughout the Mediterranean Sea in late summer using flow cytometry and molecular methods (gradient gel electrophoresis and quantitative PCR). The picoplankton displayed characteristics typical of oligotrophic oceanic areas with concentrations down to 1000 cells mL(-1) in the Eastern Basin. The most abundant eukaryotic sequences recovered by gradient gel electrophoresis were related to uncultivated marine groups: alveolates I (16%) and II (26%) and a newly discovered group (env Nansha, 17%) for which sequences have been recently obtained from the South China Sea and that could be related to Acantharians. Prasinophyceae (photosynthetic green algae) accounted for 10% of the sequences, whereas Cercozoa, Stramenopiles, Polycystinea, dinoflagellates and ciliates provided minor contributions. The use of quantitative PCR coupled with taxon-specific primers allowed us to estimate the relative abundance of several taxa belonging to the Prasinophyceae. Of the three genera assessed, Bathycoccus appeared as the most abundant, forming localized maxima at depth.     

Cardiac function and critical swimming speed of the winter flounder (Pleuronectes americanus) at two temperatures

Using Transonic flow probes and a uniquely designed swimming flume, we directly measured cardiac parameters (Q, cardiac output; SV, stroke volume; and fH, heart rate) in winter flounder (Pleuronectes americanus) before and during critical swim speed (Ucrit) tests at 4 and 10 degrees C. Resting Q, SV and fH averaged 9.8 ml min(-1) kg(-1), 0.5 ml kg(-1) (1.0 ml g ventricle(-1)) and 21 beats min(-1) at 4 degrees C and 15.5 ml min(-1) kg(-1), 0.5 ml kg(-1) (0.95 ml g ventricle(-1)) and 34 beats min(-1) at 10 degrees C (Q10 values of 2.13, 0.91 and 2.35, for Q, SV and fH, respectively). Cardiac output, SV and fH increased by approx. 170%, 70% and 60% at both temperatures during the Ucrit test. However, cardiac parameters generally reached near maximal levels almost immediately upon swimming and remained at these levels until Ucrit (0.65 +/- 0.06 bl s(-1) at 4 degrees C and 0.73 +/ -0.07 bl s(-1) at 10 degrees C). This rapid rise in cardiac function to near maximal levels did not appear to be the result of stress alone, as Q only fell slightly when flounder were swum for 75 min at < 0.4 bl s(-1), speeds at which they appeared to swim comfortably. Our results suggest that both Q and Ucrit have been significantly overestimated in flatfishes, and that "lift-off"/slow swimming is energetically expensive. Furthermore, they show that maximum and resting stroke volume (per g of ventricle) are extremely high in the flounder as compared with other teleosts.     

Gene expression during 3T3-L1 adipocyte differentiation. Characterization of initial responses to the inducing agents and changes during commitment to differentiation.

The mouse 3T3-L1 fibroblastic cell line rapidly differentiates to an adipocyte phenotype when post-confluent cells are treated for 48 h in fetal calf serum-containing medium supplemented with 1 microM dexamethasone (D), 0.5 mM methylisobutylxanthine (M) and 10 micrograms/ml insulin (I). D and I act synergistically to commit the cells to differentiate 24-48 h after initiating treatment, and this is blocked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. In order to identify cellular proteins involved in the differentiation process we analyzed differentiating 3T3-L1 cells using two-dimensional electrophoresis on large format gels. We observed changes in over 300 proteins during differentiation (over 100 within 5 h of initiating differentiation) and many of these are also changed at the level of mRNA (by analysis of in vitro translation products). About 75% of the initial changes were maximally induced by treatment with a combination of M and I, while no more than 10 proteins and their corresponding mRNAs were maximally induced by D within 3.5 h. Another 10 proteins were synergistically regulated by the combination of all three agents (DMI) within 3.5 h. Additional species were induced at later times. Five of these were synergistically induced by treatments that lead to differentiation, were first expressed at elevated levels during commitment and remained elevated in fully differentiated adipocytes. One or more of these proteins could well have a functional role in the commitment to and/or expression of the adipocyte differentiation program.     

Screening the Sargasso Sea metagenome for data to investigate genome evolution in Ostreococcus (Prasinophyceae, Chlorophyta)

The Sargasso Sea water shotgun sequencing unveiled an unprecedented glimpse of marine prokaryotic diversity and gene content. The sequence data was gathered from 0.8 microm filtered surface water extracts, and revealed picoeukaryotic (cell size<2 microm) sequences alongside the prokaryotic data. We used the available genome sequence of the picoeukaryote Ostreococcus tauri (Prasinophyceae, Chlorophyta) as a benchmark for the eukaryotic sequence content of the Sargasso Sea metagenome. Sequence data from at least two new Ostreococcus strains were identified and analyzed, and showed a bias towards higher coverage of the AT-rich organellar genomes. The Ostreococcus nuclear sequence data retrieved from the Sargasso metagenome is divided onto 731 scaffolds of average size 3917 bp, and covers 23% of the complete nuclear genome and 14% of the total number of protein coding genes in O. tauri. We used this environmental Ostreococcus sequence data to estimate the level of constraint on intronic and intergenic sequences in this compact genome.     

Prasinoxanthin-constaining Prasinophyceae Discovered in Jiaozhou Bay, China

The class Prasinophyceae （Chlorophyta） contains some photosynthetic eukaryotic ultraplankton species characterized by containing prasinoxanthin. The existence and abundance of these organisms can be estimated by the diagnostic pigment. We detected the unique pigments of prasinoxanthin-containing Prasinophyceae in Jiaozhou Bay, China using high performance liquid chromatography （HPLC）. This was the first finding of this kind in Chinese seas. Using the ratio of prasinoxanthin to chlorophyll a, the abundance of prasinoxanthin-containing Prasinophyceae has been calculated. The average contribution of prasinoxanthin-containing Prasinophyceae to the chlorophyll a pool was 8.5% and 17.0% in May and August 2004 in Jiaozhou Bay, and the maximums were 25.9% and 36.3%. Size fractionated pigment analysis suggested that more than 80% of prasinoxanthin were in the fraction of 2-20 μm. According to the results of pigment and morphological analysis, the possible genera of prasinoxanthin-containing Prasinophyceae and the reasons for causing this high abundant phytoplankton in Jiaozhou Bay were discussed. This kind of phytoplankton can not be discovered in traditional biological investigation, but its contribution to the coastal ecosystem is significant enough to be studied further.     

The phytoplankton of the internal Gulf of Olbia (North-East Sardinia) between July 1992 and July 1993

Abstract

The density variations and phytoplankton structure of the internal Gulf of Olbia (north-east Sardinia) from July 1992 to July 1993 are described.

The most important class was the Bacillariophyceae, because it accounted for the maximum values and because it dominated over about one-third of the study cycle. The picoplankton was present practically all the year round with the greatest development from September to March. Prymnesiophyceae, Dinophyceae, Cyanophyceae and Euglenophyceae were found more numerously in summer and autumn 1992 than in the rest of the year, while Bacillariophyceae, Chlorophyceae and Chrysophyceae were mostly found in spring and summer 1993.

Among the 16 species that expressed the highest mean yearly density values (greater than 1×10⁴ cell I^-1), 10 were Bacillariophyceae, 2 Dinophyceae, 3 Chrysophyceae and 1 Chlorophycea. The Bacillariophyceae have also been the dominant class in floristic terms (49 taxa on 95 total), followed by the Dinophyceae (26), the Chlorophyceae (5), the Chrysophyceae (5), the Prymnesiophyceae (4), the Prasinophyceae (2), the Euglenophyceae (2) and the Cyanophyceae (2).     

Distribution and growth of aerobic anoxygenic phototrophs in the Mediterranean Sea

The distribution of aerobic anoxygenic phototrophs (AAPs) was surveyed in various regions of the Mediterranean Sea in spring and summer. These phototrophic bacteria were present within the euphotic layer at all sampled stations. The AAP abundances increased with increasing trophic status ranging from 2.5 × 10(3) cells per ml in oligotrophic Eastern Mediterranean up to 90 × 10(3) cells per ml in the Bay of Villefranche. Aerobic anoxygenic phototrophs made up on average 1-4% of total prokaryotes in low nutrient areas, whereas in coastal and more productive stations these organisms represented 3-11% of total prokaryotes. Diel bacteriochlorophyll a decay measurements showed that AAP community in the Western Mediterranean grew rapidly, at rates from 1.13 to 1.42 day(-1). The lower AAP abundances registered in the most oligotrophic waters suggest that they are relatively poor competitors under nutrient limiting conditions. Instead, AAPs appear to be metabolically active organisms, which thrive better in more eutrophic environments providing the necessary substrates to maintain high growth rates.     

Involvement of nucleotide excision repair (NER) system in repair of mono ADP-ribosylated dG adducts produced by pierisin-1, a cytotoxic protein from cabbage butterfly

Pierisin-1, a cytotoxic protein from the cabbage butterfly (Pieris rapae), induces apoptosis in mammalian cell lines. Binding of its C-terminal region to glycosphingolipid Gb3 and Gb4 receptors on cell membrane is necessary for incorporation into cells, while the N-terminal polypeptide catalyzes transfer of the ADP-ribose moiety of NAD at N2 of dG in DNA. Resulting DNA adducts cause mutation if they are present at low levels. If the DNA damage is more severe, the cells undergo apoptosis. In the present study, we examined the repair system for ADP-ribosylated dG adducts using nucleotide excision repair (NER) mutants of Chinese hamster ovary (CHO) cell lines. Pierisin-1 showed cytotoxic effects in all cases: IC50 values of them were; 650 ng/ml for AA8 (wild), 230 ng/ml for UV5, 190 ng/ml for UV20, 260 ng/ml for UV41, and 240 ng/ml for UV135. Thus, wild-type AA8 proved most resistant to pierisin-1-induced cytotoxicity. When these CHO cell lines were treated with pierisin-1, the adduct levels of ADP-ribosylated dG increased to 2.5-4.8/10(5) nucleotides time-dependently in all cell lines at 12 h. After removal of pierisin-1, the adduct levels remained constant or increased to 4-14/10(5) nucleotides in all NER mutant cells (UV5, UV20, UV41, UV135), while those rapidly decreased to 0.27/10(5) nucleotides in the repair proficient AA8 cells for 24 h. From these results, it is suggested that the NER system is involved in the repair of ADP-ribosylated dG adducts in DNA.     

Inhibin and beta type transforming growth factor (TGF beta) have opposite modulating effects on the follicle stimulating hormone (FSH)-induced aromatase activity of cultured rat granulosa cells

We have recently observed that attomolar concentration of exogenously added TGF beta, a molecule structurally related to inhibin, can stimulate the basal secretion of FSH in a pituitary cell culture. Inhibin purified from porcine follicular fluid antagonizes this activity of TGF beta. To understand further the homeostatic regulatory properties of inhibin and TGF beta we have investigated whether the aromatase activity of ovarian granulosa cells is also subject to intra-ovarian modulation by these peptides. Granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were cultured for 2 days with androstenedione (10(-7) M) as a substrate, oFSH (2 ng), and different amounts of TGF beta or inhibin. Basal estrogen secretion was negligible and remained unaffected by treatment with purified TGF beta or inhibin (10 ng/ml), whereas treatment with oFSH (2 ng/ml) produced a 100-fold increase in estrogen accumulation. The concurrent application of increasing concentrations (10 pg-10 ng/ml) of TGF beta produced dose-dependent increments in the FSH-stimulated accumulation of estrogen with a ED50 of 0.3 +/- 0.02 ng/ml. On the other hand, concurrent incubation of FSH with inhibin ranging from 10 pg to 10 ng/ml decreases the FSH-mediated estrogen secretion. TGF beta antagonizes the inhibition of inhibin on aromatase activity. These findings suggest that inhibin and TGF beta, two closely related molecules, play novel and opposite roles in modulating the follicular functions.     

Two bacteria phylotypes are predominant in the Suiyo seamount hydrothermal plume

Microbial diversity and populations in a hydrothermal plume that was present inside the caldera of the Suiyo Seamount, a submarine volcano on the Izu-Bonin Arc, were investigated by performing a phylogenetic analysis of the 16S rRNA gene and by using fluorescence in situ hybridization (FISH). Corresponding to transmissivity, an indicator of turbidity, the vertical total cell count as determined by 4',6'-diamidino-2-phenylindole (DAPI) staining varied from 5.6 x 10(4) to 1.1 x 10(5) cells ml(-1), and the apparent plume layer was assessed to be at a depth of 1,050 to 1,200 m inside the caldera and to contain 1.0 x 10(5) to 1.1 x 10(5) cells ml(-1). From microbial samples collected in the plume by an in situ filtration system, the following two major phylogenetic groups, which were closely related to sulfur-oxidizing microbes, were obtained: the SUP05 group belonging to the gamma subclass of the Proteobacteria (13 of 20 clones) and the SUP01 group belonging to the epsilon subclass of the Proteobacteria (5 of 20 clones). Specific oligonucleotide probes for these groups (SUP05-187 and SUP01-63) were designed and were used with various water samples obtained from the Suiyo Seamount. In the apparent plume layer, up to 66% of the total counts of microbial cells were estimated to be Bacteria cells that hybridized to EUB338, and few cells were identified by the archaeal probe ARCH915. Almost all Bacteria cells were hard to identify with the known group-specific probes, such as ALF19, GAM42a, and CF319, while 88 to 90% of the Bacteria cells hybridized with SUP05-187 and >98% of them were considered members of the SUP05 and SUP01 populations. In a low-temperature vent fluid emitted from a bivalve-colonized mound, the SUP05 cells accounted for >99% of the Bacteria cells, suggesting that a portion of the plume cells originated on the surface of the seafloor at a depth of about 1,380 m. From further analysis of cell morphology (i.e., cell size and cell elongation index) we inferred that the SUP05 cells were active in the plume layer at a depth of 1,050 to 1,200 m compared to the activity in a near-bottom layer, while many elongated cells were found between these layers. These findings suggest that the morphology and distribution of SUP05 cells have complex relationships with hydrothermal activities and water circulation. Although growth and production rates remain to be defined, we concluded that this Suiyo Seamount caldera has functioned as a natural continuous incubator for these two phylotypes of Bacteria in an aphotic deep-sea environment.     

Genetically encoded fluorescent redox sensors

Background

Life is a constant flow of electrons via redox couples. Redox reactions determine many if not all major cellular functions. Until recently, redox processes remained hidden from direct observation in living systems due to the lack of adequate methodology. Over the last years, imaging tools including small molecule probes and genetically encoded sensors appeared, which provided, for the first time, an opportunity to visualize and, in some cases, quantify redox reactions in live cells. Genetically encoded fluorescent redox probes, such as HyPer, rxYFP and roGFPs, have been used in several models, ranging from cultured cells to transgenic animals, and now enough information has been collected to highlight advantages and pitfalls of these probes.

Scope of review

In this review, we describe the main types of genetically encoded redox probes, their essential properties, advantages and disadvantages. We also provide an overview of the most important, in our opinion, results obtained using these probes. Finally, we discuss redox-dependent photoconversions of GFP and other prospective directions in redox probe development.

Major conclusions

Fluorescent protein-based redox probes have important advantages such as high specificity, possibility of transgenesis and fine subcellular targeting. For proper selection of a redox sensor for a particular model, it is important to understand that HyPer and roGFP2-Orp1 are the probes for H₂O₂, whereas roGFP1/2, rxYFP and roGFP2-Grx1 are the probes for GSH/GSSG redox state. Possible pH changes should be carefully controlled in experiments with HyPer and rxYFP.

General significance

Genetically encoded redox probes are the only instruments allowing real-time monitoring of reactive oxygen species and thiol redox state in living cells and tissues. We believe that in the near future the palette of FP-based redox probes will be expanded to red and far-red parts of the spectrum and to other important reactive species such as NO, O₂ and superoxide. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.