2003

mardi 21 avril 2009
par   G. Grégori

Adams SL, Hessian PA, Mladenov PV (2003) Flow cytometric evaluation of mitochondrial function and membrane integrity of marine invertebrate sperm. Invertebrate Reproduction & Development 44 :45-51

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A method was developed to evaluate the mitochondrial function and membrane integrity of marine invertebrate sperm using flow cytometry in combination with rhodamine 123 (R123) and propidium iodide (PI) staining. The method was evaluated with sperm of sea urchins (Evechinus chloroticus), mussels (Perna canaliculus) and abalone (Haliotis iris). Using R123 and PI simultaneously, it was possible to distinguish live sperm with functioning mitochondria and intact membranes from dying or dead sperm. The mitochondrial inhibitor rotenone was used to confirm that R123 was only accumulated by sperm with functioning mitochondria. The stain combination was further validated using known ratios of fresh to killed sperm. Among individual males evaluated, the percentage of sperm identified as live varied considerably, irrespective of the species studied. Among samples from the same male, however, the percentage of sperm identified as live was similar. The method developed has application for assessing the quality of sperm used in cryopreservation and bioassays.


Andrade L, Gonzalez AM, Araujo FV, Paranhos R (2003) Flow cytometry assessment of bacterioplankton in tropical marine environments. J Microbiol Methods 55 :841-850

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Flow cytometry was used to characterize bacterioplankton from two tropical environments in Brazil : the eutrophic Guanabara Bay and the oligotrophic southwest Atlantic Ocean. Bacterial abundance was evaluated by flow cytometry, and cells were stained with SYTO 13, allowing demonstration of differences in nucleic acid content. Bacterial production was also evaluated by means of 3H-leucine incorporation. Bacterial numbers were different for both sites. In Atlantic Ocean samples, we found a maximum of 5.50 x 10(5) cells ml(-1), and low nucleic acid content organisms predominated. In Guanabara Bay, bacterial numbers were one order of magnitude higher than in the ocean, and they varied from outer bay (1.01 x 10(6) cells ml(-1)) to inner bay (6.90 x 10(6) cells ml(-1)). Bacterial activity in ocean samples varied from 4.6 to 126 ng C l(-1) h(-1), while in the bay, mean values ranged from 1.95 microg C l(-1) h(-1) (outer bay) to 7.35 microg C l(-1) h(-1) (inner bay). Values found for both parameters are characteristic of different trophic situations. These results illustrate the utility of cytometric analyses of bacterioplankton populations in characterizing their large spatial and temporal scales of distribution in aquatic ecosystems.


Beardsley C, Pernthaler J, Wosniok W, Amann R (2003) Are readily culturable bacteria in coastal North Sea waters suppressed by selective grazing mortality ? Applied and Environmental Microbiology 69 :2624-2630

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We studied the growth of six culturable bacterial lineages from coastal North Sea picoplankton in environmental samples under different incubation conditions. The grazing pressure of heterotrophic nanoflagellates (HNF) was reduced either by double prefiltration through 0.8-mum-pore-size filters or by 10-fold dilutions with 0.2-mum (pore-size) prefiltered seawater. We hypothesized that those gamma-proteobacterial genera that are rapidly enriched would also be most strongly affected by HNF regrowth. In the absence of HNF, the mean protein content per bacterial cell increased in both treatments compared to environmental samples, whereas the opposite trend was found in incubations of unaltered seawater. Significant responses to the experimental manipulations were observed in Alteromonas, Pseudoalteromonas, and Vibrio populations. No treatment-specific effects could be detected for members of the Roseobacter group, the Cytophaga latercula-C marinoflava lineage, or the NOR5 clade. Statistical analysis confirmed a transient increase in the proportions of Alteromonas, Pseudoalteromonas, and Vibrio cells at reduced HNF densities only, followed by an overproportional decline during the phase of HNF regrowth. Cells from these genera were significantly larger than the community average in the dilution treatments, and changes in their relative abundances were negatively correlated with HNF densities. Our findings suggest that bacteria affiliated with frequently isolated genera such as Alteromonas, Pseudoalteromonas, and Vibrio might be rare in coastal North Sea picoplankton because their rapid growth response to changing environmental conditions is counterbalanced by a higher grazing mortality.


Behrens S, Ruhland C, Inacio J, Huber H, Fonseca A, Spencer-Martins I, Fuchs BM, Amann R (2003) In situ accessibility of small-subunit rRNA of members of the domains Bacteria, Archaea, and Eucarya to Cy3-labeled oligonucleotide probes. Appl Environ Microbiol 69 :1748-1758

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Low accessibility of the rRNA is together with cell wall impermeability and low cellular ribosome content a frequent reason for failure of whole-cell fluorescence hybridization with fluorescently labeled oligonucleotide probes. In this study we compare accessibility data for the 16S rRNA of Escherichia coli (gamma Proteobacteria, Bacteria) with the phylogenetically distantly related organisms Pirellula sp. strain 1 (Planctomycetes, Bacteria) and Metallosphaera sedula (Crenarchaeota, Archaea) and the 18S rRNA accessibility of Saccharomyces cerevisiae (Eucarya). For a total of 537 Cy3-labeled probes, the signal intensities of hybridized cells were quantified under standardized conditions by flow cytometry. The relative probe-conferred fluorescence intensities are shown on color-coded small-subunit rRNA secondary-structure models. For Pirellula sp., most of the probes belong to class II and III (72% of the whole data set), whereas most of the probes targeting sites on M. sedula were grouped into class V and VI (46% of the whole data set). For E. coli, 45% of all probes of the data set belong to class III and IV. A consensus model for the accessibility of the small-subunit rRNA to oligonucleotide probes is proposed which uses 60 homolog target sites of the three prokaryotic 16S rRNA molecules. In general, open regions were localized around helices 13 and 14 including target positions 285 to 338, whereas helix 22 (positions 585 to 656) and the 3’ half of helix 47 (positions 1320 to 1345) were generally inaccessible. Finally, the 16S rRNA consensus model was compared to data on the in situ accessibility of the 18S rRNA of S. cerevisiae.


Bertilsson S, Berglund O, Karl DM, Chisholm SW (2003) Elemental composition of marine Prochlorococcus and Synechococcus : Implications for the ecological stoichiometry of the sea. Limnology and Oceanography 48 :1721-1731

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The elemental composition of marine cyanobacteria is an important determinant of the ecological stoichiometry in low-latitude marine biomes. We analyzed the cellular carbon (C), nitrogen (N), and phosphorus (P) contents of Prochlorococcus (MED4) and Synechococcus (WH8103 and WH8012) under nutrient-replete and P-starved conditions. Under nutrient-replete conditions, C, N, and P quotas (femtogram cell(-1)) of the three strains were 46 +/- 4, 9.4 +/- 0.9, and 1.0 +/- 0.2 for MED4 ; 92 +/- 13, 20 +/- 3, and 1.8 +/- 0.1 for WH8012 ; and 213 +/- 7, 50 +/- 2, 3.3 +/- 10.5 for WH8103. In P-limited cultures, they were 61 +/- 2, 9.6 +/- 0.1, and 0.3 +/- 0.1 for MED4 ; 132 +/- 6, 21 +/- 2, and 0.5 +/- 0.2 for WH8012 ; and 244 +/- 21, 40 +/- 4, and 0.8 +/- 0.01 for WH8103. P limitation had no effect on the N cell quota of MED4 and WH8012 but reduced the N content of WH8103. The cellular C quota was consistently higher in P-limited than in nutrient-replete cultures. All three strains had higher C : P and N : P ratios than the Redfield ratio under both nutrient-replete and P-limited conditions. The C:N molar ratios ranged 5-5.7 in replete cultures and 7.1-7.5 in P-limited cultures ; C : P ranged 121-165 in the replete cultures and 464-779 under P limitation ; N:P ranged 21-33 in the replete cultures and 59-109 under P limitation. Our results suggest that Prochlorococcus and Syneehococcus may have relatively low P requirements in the field, and thus the particulate organic matter they produce would differ from the Redfield ratio (106C : 16N : 1P) often assumed for the production of new particulate organic matter in the sea.


Biegala IC, Not F, Vaulot D, Simon N (2003) Quantitative assessment of picoeukaryotes in the natural environment by using taxon-specific oligonucleotide probes in association with tyramide signal amplification-fluorescence in situ hybridization and flow cytometry. Appl Environ Microbiol 69 :5519-5529

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12957941

Picoeukaryotes (cells of <3 micro m in diameter) contribute significantly to marine plankton biomass and productivity, and recently molecular studies have brought to light their wide diversity. Among the methods that have been used so far to quantify aquatic microorganisms, fluorescence in situ hybridization of oligonucleotide probes combined with flow cytometry offers the advantages of both high resolution for taxonomic identification and automated cell counting. However, cell losses, cell clumps, and low signal-to-background ratio have often been mentioned as major problems for routine application of this combination of techniques. We developed a new protocol associating tyramide signal amplification-fluorescence in situ hybridization and flow cytometry, which allows the detection of picoeukaryotes in cultures during both the exponential and stationary phases. The use of surfactant and sonication proved to be essential for the detection and quantification of picoeukaryotes from the natural environment, with as little as a few tenths of a milliliter of 3- micro m-pore-size prefiltered sea water. The routine application of the technique was tested along a coastal transect off Brittany (France), where the different groups of picoeukaryotes were investigated using already published specific probes and a newly designed probe that targets the order Mamiellales (Prasinophyceae, Chlorophyta). Among the picoeukaryotes, Mamiellales outnumbered by 1 order of magnitude both the cyanobacteria and the non-Chlorophyta, which were represented mainly by the Pelagophyceae class. Picoeukaryote abundance increased from open toward more estuarine water, probably following changes in water temperature and stability.


Bihari N, Micic M, Batel R, Zahn RK (2003) Flow cytometric detection of DNA cell cycle alterations in hemocytes of mussels (Mytilus galloprovincialis) off the Adriatic coast, Croatia. Aquat Toxicol 64 :121-129

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Studies were carried out to determine the alteration in DNA cell cycle characteristics of hemocytes of the mussel Mytilus galloprovincialis collected at 17 different locations (146 individuals) along the Adriatic coast, Croatia. In order to connect possible genomic manifestation to urban and/or industrial waste flow cytometry was used. We studied incidence of altered DNA profile reflective of chromosomal fragmentation phenomena or aneuploid mosaicism, coefficient of variation (CV) in DNA fluorescence as a measure of intraindividual genome size variability and DNA index (DI) as a measure of ploidy. The different classes of DNA cell cycle alterations found in this study mirror either acute or cumulative genotoxic effects of the surrounding environment on mussel hemocyte DNA. These are intraindividual genome size variability (CV>8, seven individuals from four sites), aneuploidy (altered DNA profile and DI<0.9, 45 individuals from 14 sites) and accidental apoptotic processes (altered DNA profile and presence of apoptotic cells, two individuals from two sites). Normal cell cycle DNA profiles were obtained for 89 (60.9%) individuals from all 17 sites and for 146 examined samples polyploids were absent. Flow cytometry proved to be a powerful technique for the determination of alterations in cell cycle characteristics in mussel hemocyte DNA. Therefore, it may be used in pollution control measurements to distinguish affected or vulnerable populations from healthy populations living in the presence of a wide variety of marine environmental contaminants.


Brookes JD, Regel RH, Ganf GG, Burch MD (2003) Applications of flow cytometry to phytoplankton research. International Association of Theoretical and Applied Limnology, Vol 28 Pt 3, Proceedings 28 :1311-1316
1482

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Cao J, Zhao P, Zhao LJ, Wu SM, Zhu SY, Qi ZT (2003) Identification and expression of human CD81 gene on murine NIH/3T3 cell membrane. Journal of Microbiological Methods 54 :81-85

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The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). In this study, eukaryotic expression vector pCDM8-hCD81 containing hCD81 cDNA and pSV2neo helper plasmid was used to cotransfect with lipofectamine into murine fibroblast cell line NIH/3T3 to establish an hCD81-expressing cell line. Resistant cell clones were obtained 20 days after the selection with neomycin (600 mug/ml) and then cultured as monoclones. The expression of the transfected hCD81 gene in the cells was verified by RT-PCR and flow cytometry analyses. One of the selected cell clones showed obvious expression of hCD81 and was named NIH/3T3-hCD81. Competitive inhibition tests indicated that the binding of monoclonal anti-hCD81 (JS-81) to NIH/3T3-hCD81 cells was inhibited by recombinant HCV E2 protein, suggesting that the expressed hCD81 molecules on NIH/3T3-hCD81 cells maintain natural conformation of binding to HCV E2. The transfected NIH/3T3-hCD81 cells should be of great potential value in studies on HCV attachment and onset of infection. (C) 2003 Elsevier Science B.V. All rights reserved.


Caruso G, Mancuso M, Crisafi E (2003) Combined fluorescent antibody assay and viability staining for the assessment of the physiological states of Escherichia coli in seawaters. Journal of Applied Microbiology 95 :225-233

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Aims : A comparison of methods that combine the use of immune sera with specific fluorescent probes for testing viability at single cell level was performed in order to estimate different living attributes of Escherichia coli in natural seawater samples.
Methods and Results : Cell culturability was assayed by plate method, respiratory activity and membrane integrity were determined by an indirect fluorescent antibody assay, combined with 5-cyano-2, 3 ditolyl tetrazolium chloride and propidium iodide, respectively. Results showed the coexistence of different physiological states within the E. coli population, of which a large fraction (46%) of cells was actively respiring.
Conclusions : The methodological approach used offer interesting perspectives in water pollution monitoring, particularly when the differentiation between dead and living E. coli cells is required for a more precise assessment of the bacteriological quality of seawaters.
Significance and Impact of the Study : The study suggests the importance of knowledge of the viability status of faecal bacteria in aquatic environments as a fundamental issue for the preservation of public health ; the availability of rapid analytical procedures for this purpose may find significant applications in the evaluation of the sanitary risk consequent to water use.


Casotti R, Landolfi A, Brunet C, D’Ortenzio F, Mangoni O, d’Alcala MR, Denis M (2003) Composition and dynamics of the phytoplankton of the Ionian Sea (eastern Mediterranean). Journal of Geophysical Research-Oceans 108
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[1] The relationship between phytoplankton distribution and dynamics, and the resident water masses in the Ionian Sea was investigated in the spring of 1999 by flow cytometry, HPLC, microscopy, C-14 incorporation, and serial dilutions. More than 50% of total biomass was represented by ultraphytoplankton, with proportions increasing with depth (up to 80%) and eastward (up to 60%). Synechococcus sp. cyanobacteria dominated ultraphytoplankton numbers and biomass. Hydrological stability explained the relative vertical distribution of Synechococcus sp. and Prochlorococcus sp. cyanobacteria and their degree of photoacclimation. The northwestern area ("Italian Side’’) was in a transition from bloom to oligotrophic conditions, reflected in high biological instability in terms of phytoplankton composition, photoacclimative properties, and photosynthetic efficiency (P/B of 11.40 mg C mg chl(-1) h(-1)). The influence of the Adriatic Surface Water, carrying a trace of the spring bloom was visible from satellite imagery (SeaWiFS). The eastern part of the sampled area ("Greek Side,’’ GS) was hydrologically more stable (low estimated vertical velocities), resulting in higher photosynthetic efficiency (P/B of 20.47 mg C mg chl(-1) h(-1)) and phytoplankton photoacclimative properties. The Atlantic Water (AW) was the most oligotrophic in terms of nutrient concentration, chla, and productivity (208.0 mg C m(-2) d(-1)), but also the most variable in terms of mesoscale features. Growth of Synechococcus sp. and Prochlorococcus sp. was slow (0.36 d(-1) at max), while picoeukaryotes grew well in the deep chlorophyll maximum (DCM) of the GS (0.67 d(-1)) Picoeukaryote growth was tightly coupled with grazing (0.80 d(-1)), indicating efficient biomass recycling. Biological characterization and DCM dynamics in the three areas are discussed in relation to physical features present at the time of sampling.


Collier JL, Palenik B (2003) Phycoerythrin-containing picoplankton in the Southern California Bight. Deep-Sea Research Part Ii-Topical Studies in Oceanography 50 :2405-2422

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Flow cytometry was used to examine the distribution of phycoerythrin-rich picophytoplankton. referred to here as Synechococcus, off the Southern California coast during six California Cooperative oceanic Fisheries Investigations (CalCOFI) cruises. Depth profiles revealed that Synechococcus was most abundant in the surface mixed layer, gradually disappearing with depth below the thermocline. Within the surface mixed layer, Synechococcus abundance was generally greater and more variable at stations shoreward of the California Current than at stations offshore of it. In waters associated with the California Current not impacted by upwelling, Synechococcus abundance increased with increasing bulk chlorophyll. In contrast, Synechococcus abundance declined with increasing bulk chlorophyll at stations that were impacted by upwelling. Synechococcus at stations impacted by upwelling also had more phycoerythrin per cell than at non-upwelling stations. Offshore of the California Current, Synechococcus cells in waters intruding from the Central North Pacific displayed higher side-scatter relative to forward scatter than did Synechococcus cells elsewhere in the region. Flow cytometrically distinct Synechococcus cell types were also detected below the thermocline at most of the stations where depth profiles were analyzed. These patterns in Synechococcus abundance and cellular characteristics might reflect physiological and/or genetic differences among Synechococcus associated with the various water masses that comprise the CalCOFI region. The data presented here provide a framework from which to launch more detailed and mechanistic studies examining the role of Synechococcus in the CalCOFI ecosystem. (C) 2003 Elsevier Ltd. All rights reserved.


Corporeau C, Auffret M (2003) In situ hybridisation for flow cytometry : a molecular method for monitoring stress-gene expression in hemolymph cells of oysters. Aquatic Toxicology 64 :427-435

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In the present study, we developed a molecular method for flow cytometry to detect the effects of environmental factors on the stress-response in immunocompetent cells of the European flat oyster, Ostrea edulis. Stress-generating conditions were applied to individuals acclimated in the laboratory to environmental salinity and temperature. Oligonucleotidic probes were applied to quantify the expression of HSP/C70 or metallothionein genes. After a heatstress, a response was reflected in hemocytes by an increased amount of HSP/C70 mRNA and quantitative changes in HSP/C70 protein expression. The technique of in situ hybridisation described here also allowed to quantify the expression of metallothionein mRNA in oysters exposed to a heavy metal-exposure. The detection of stress protein markers by using such quantitative methods could be applied in contamination studies in oysters and other bivalves where monitoring the status of hemolymph cells is required. (C) 2003 Elsevier Science B.V. All rights reserved.


Crosbie ND, Pockl M, Weisse T (2003) Rapid establishment of clonal isolates of freshwater autotrophic picoplankton by single-cell and single-colony sorting. Journal of Microbiological Methods 55 :361-370

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We describe single-cell and single-colony sorting protocols which allowed for rapid establishment of a diverse culture collection of clonal autotrophic picoplankton (APP) isolates originating from oligotrophic and oligo-mesotrophic subalpine lakes. Overall sort recoveries, expressed as the percentage of sorted microwells exhibiting APP growth, ranged from 5% to 17% depending on the type of APP, but the growth success varied greatly (from 0% to 68%) depending on the origin of the sorted sample. We applied two direct sequencing and two denaturing gradient gel electrophoresis (DGGE) protocols to identify and characterize the genetic purity of 21 of our picocyanobacteria cultures, namely, direct sequencing of the 16S rRNA gene and cpcBA-IGS region, and DGGE analyses involving a 194-bp fragment of the internal transcribed spacer (ITS) and a ca. 500-bp fragment of the phycocyanin (PC) operon (cpcBA-IGS, novel protocol described herein). Of those 21 picocyanobacteria cultures obtained by single-cell/single-colony sorting and subsequently characterized genetically/screened for genetic purity, only one culture was composed of multiple picocyanobacterial strains. (C) 2003 Elsevier B.V. All rights reserved.


Culverhouse PF, Williams R, Reguera B, Herry V, Gonzalez-Gil S (2003) Do experts make mistakes ? A comparison of human and machine identification of dinoflagellates. Marine Ecology-Progress Series 247 :17-25

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The authors present evidence of the difficulties facing human taxonomists/ecologists in identifying marine dinoflagellates. This is especially important for work on harmful algal blooms in marine aquaculture. It is shown that it is difficult for people to categorise specimens from species with significant morphological variation, perhaps with morphologies overlapping with those of other species. Trained personnel can be expected to achieve 67 to 83% self-consistency and 43% consensus between people in an expert taxonomic labelling task. Experts who are routinely engaged in particular discriminations can return accuracies in the range of 84 to 95%. In general, neither human nor machine can be expected to give highly accurate or repeatable labelling of specimens. It is also shown that automation methods can perform as well as humans on these complex categorisations.


Dai X, Boll J (2003) Evaluation of attachment of Cryptosporidium parvum and Giardia lamblia to soil particles. J Environ Qual 32 :296-304

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Transport of Cryptosporidium parvum oocysts and Giardia lamblia cysts in the aquatic environment is poorly understood. Information about their transport is essential for actual risk assessment and development of effective control practices. Several studies have suggested that attachment to soil particles is not likely to occur, but the hypothesis has not been well tested, partly because enumeration of C. parvum oocysts or G. lamblia cysts [written as (oo)cysts] in the presence of soil has been difficult. In this paper, a combination of flow cytometry, and epifluorescence and confocal microscopy was successfully used to enumerate (oo)cysts in the presence of soil and determine whether (oo)cysts travel freely in water or attached to soil particles. The maximum soil concentration in water samples for reliable enumeration of (oo)cysts was 2 mg/L. Particle attachment experiments detected attached pairs of oppositely charged beads and (oo)cysts, while no attachment was observed between like charged beads, (oo)cysts, and soil particles. These results support the hypothesis that electrostatic forces govern the interaction between (oo)cysts and soil particles. Batch experiments further confirmed the null hypothesis (p > 0.05) that (oo)cysts do not attach to natural soil particles. These findings suggest that, when (oo)cysts have been entrained in overland flow (i.e., runoff), they will travel freely in the water and not as part of the particulate sediment load.


de la Jara A, Mendoza H, Martel A, Molina C, Nordstron L, de la Rosa V, Diaz R (2003) Flow cytometric determination of lipid content in a marine dinoflagellate, Crypthecodinium cohnii. Journal of Applied Phycology 15 :433-438

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Since flow cytometry allows rapid, simultaneous and quantitative measurements related to cell morphology and physiologicy, the lipid-specific fluorescent dye, Nile Red, was employed for the in vivo lipid quantification of Crypthecodinium cohnii, a heterotrophic marine dinoflagellate rich in polyunsaturated long chain fatty acids. The fluorescence signal was linearly correlated with the neutral and polar lipid content as determined by gravimetric techniques. A significant correlation of NR signal was also observed between the polar to neutral lipid ratio and docohexaenoic acid per cell. The results demonstrate a method for rapid lipid quantification that can be used in the selection, isolation and culture control of C cohnii clones with high lipid and DHA content.


DiTullio GR, Geesey ME, Jones DR, Daly KL, Campbell L, Smith WO (2003) Phytoplankton assemblage structure and primary productivity along 170 degrees W in the South Pacific Ocean. Marine Ecology-Progress Series 255 :55-80

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Phytoplankton pigments were measured using HPLC during non-ENSO conditions in mid-summer along a South Pacific transect from 67degreesS to the equator along 170degreesW. Highest concentrations of chlorophyll a (chl a) occurred in the Polar and the Subtropical Fronts (PF and STF, respectively) with concentrations exceeding 500 ng l(-1). In the STF, there was a distinct subsurface chl a maximum (SCM) at 40 m, which gradually deepened northward to 120 m in the Subtropical Convergence Zone. Northwards, the SCM shoaled to about 30 m in the Equatorial Zone (EZ). Relatively high concentrations of fucoxanthin and 19’-butanoyloxyfucoxanthin occurred in the nutrient-rich waters south of the Subantarctic Front, and CHEMTAX analyses indicated that diatoms, chrysophytes, pelagophytes, and haptophytes dominated the phytoplankton assemblage. Northward of the PF to the STF, where silicate concentrations were <1 muM, pelagophytes and coccolithophorids dominated the water column ; diatoms were virtually absent, and Phaeocystis, prasinophytes, cryptophytes, and chlorophytes contributed significantly to the total algal biomass. Phaeocystis populations were dominant at or below the 1% light level. In the South Pacific Gyre (SPG), Synechococcus (Syn) and Prochlorococcus (Pro) were major components of the phytoplankton assemblage with Pro dominant as indicated by both flow cytometry and by the ratio of divinyl chl a:total chl a (0.43 +/- 0.07). Photoacclimation by Pro in the SPG was pronounced, with a higher average divinyl chl a per cell ratio in the SCM (1.1) relative to values (0.1) in the upper waters (0 to 100 m). Primary production rates exceeding 1 mug C l(-1) h(-1) occurred at the surface in the STF. Surface primary production rates were generally <0.4 mug C l(-1) h(-1) across the SPG, but exceeded 1.4 mug C l(-1) h(-1) at the equator. In the EZ, Pro dominated the phytoplankton assemblage, but Phaeocystis and prasinophytes were also major constituents of the assemblage.


Faucet J, Maurice M, Gagnaire B, Renault T, Burgeot T (2003) Isolation and primary culture of gill and digestive gland cells from the common mussel Mytilus edulis. Methods Cell Sci 25 :177-184

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As the marine mussel Mytilus edulis is commonly used as a sentinel species, it would be useful to develop a primary culture of the target organs most often in contact with the marine environment. This study reports an improved method for dissociating the digestive gland and gills of M. edulis and considers the effect of mussel storage on cell viability and functionality before culture initiation. Viability and enzymatic activities such as those of esterase and peroxidase were monitored by flow cytometry, a sensitive, objective technique allowing large volumes of cells to be counted within a short time. A primary culture of digestive gland showed more than 75% viability after 72 h. Mussels were maintained in an aquarium containing clean, oxygenated seawater at 12 degrees C for two days before culture initiation, and dissociation was performed mechanically and chemically with Ca-Mg-free saline to obtain digestive gland cells. Application of non-specific esterase activity, using fluorescein diacetate (FDA test) coupled with flow cytometry, characterised the functionality of digestive gland and gill cells in culture.


Fki L, Masmoudi R, Drira N, Rival A (2003) An optimised protocol for plant regeneration from embryogenic suspension cultures of date palm, Phoenix dactylifera L., cv. Deglet Nour. Plant Cell Reports 21 :517-524

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An improved protocol is described for the large-scale micropropagation of an elite date palm (Phoenix dactylifera L.) cultivar, Deglet Nour. Clonal plants were regenerated from somatic embryos derived from highly proliferating suspension cultures. Friable embryogenic calli were initiated from both leaf and inflorescence explants. Suspension cultures consisting of pro-embryonic masses were established from calli showing a high competency for somatic embryogenesis. The subculture of suspensions in liquid medium enriched with low amounts of plant growth regulators (1 mg l(-1) 2,4-dichlorophenoxyacetic acid with 300 mg l(-1) charcoal) resulted in the differentiation of large numbers of somatic embryos. The productivity of the cultures increased 20-fold (from 10 to 200 embryos per month per 100 mg fresh weight of embryogenic callus) when embryogenic suspensions were used instead of standard cultures on solid media. The overall production of somatic embryos reached 10,000 units per litre per month. Partial desiccation of the mature somatic embryos, corresponding to a decrease in water content from 90% to 75%, significantly improved germination rates (from 25% to 80%). The cutting back of the cotyledonary leaf was also found to stimulate embryo germination. Flow cytometric analysis showed that the micropropagation protocol followed here did not affect the ploidy level of somatic embryo-derived plantlets.


Fu Y, O’Kelly C, Sieracki M, Distel DL (2003) Protistan grazing analysis by flow cytometry using prey labeled by in vivo expression of fluorescent proteins. Appl Environ Microbiol 69 :6848-6855

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Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined ; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.


Fu YT, O’Kelly C, Sieracki M, Distel DL (2003) Protistan grazing analysis by flow cytometry using prey labeled by in vivo expression of fluorescent proteins. Applied and Environmental Microbiology 69 :6848-6855

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Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan How cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein) -labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined ; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5- (4,6-dichloro-triazin-2-yI) -amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.


Fuller NJ, Marie D, Partensky F, Vaulot D, Post AF, Scanlan DJ (2003) Clade-specific 16S ribosomal DNA oligonucleotides reveal the predominance of a single marine Synechococcus clade throughout a stratified water column in the Red Sea. Applied and Environmental Microbiology 69 :2430-2443

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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 VVH 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.


Furuya K, Hayashi M, Yabushita Y, Ishikawa A (2003) Phytoplankton dynamics in the East China Sea in spring and summer as revealed by HPLC-derived pigment signatures. Deep-Sea Research Part Ii-Topical Studies in Oceanography 50 :367-387

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The community structure of phytoplankton was investigated from pigment composition in the East China Sea during an early spring bloom and a well-stratified summer season. Pigment concentration determined by high-performance liquid chromatography was interpreted using a matrix factorization program "CHEMTAX" (Mackey et al., 1996) to obtain the chlorophyll alpha biomass of phytoplankton groups at class level. There was a distinct horizontal heterogeneity in phytoplankton composition during spring. The spring bloom of diatoms was observed on the shelf, where weak stratification produced by overlying less saline surface water supported the bloom. In contrast, off the shelf where Kuroshio water prevailed, phytoplankton abundance was low and the community was composed mainly of prochlorophytes, chrysophytes, prymnesiophytes and chlorophytes. Patchy high concentrations of fucoxanthin were observed off the shelf, indicating transport of diatom-rich water from the shelf. In a clear contrast to the spring season, a "two-layer" distribution of phytoplankton prevailed both off and on the shelf in summer. There was a well-developed subsurface chlorophyll maximum at all the stations except near Changjiang estuary. In the upper mixed layer where nutrient salts were depleted, the phytoplankton community was characterized by high contribution of cyanobacteria followed by prochlorophytes, whereas in the chlorophyll maximum, prochlorophytes, chrysophytes and prymnesiophytes were most abundant. In both seasons, total amount of chlorophyll alpha was primarily determined by that of diatoms, and the chlorophyll alpha of procaryotes and eucaryotic flagellates did not exceed 0.4 and 1 mug 1(-1), respectively, suggesting herbivorous control. Active grazing of macrozooplankton also was indicated in the diatom patch by the presence of degradation products of chlorophyll a. (C) 2002 Elsevier Science Ltd. All rights reserved.


Gagnaire B, Renault T, Bouilly K, Lapegue S, Thomas-Guyon H (2003) Study of atrazine effects on Pacific oyster, Crassostrea gigas, haemocytes. Curr Pharm Des 9 :193-199

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Shellfish farming is an important economic activity around the world. This activity often takes place in areas subjected to various recurring pollutions. The recrudescent use of herbicides in agriculture including atrazine implies pollutant transfer towards aquatic environment in estuarine areas. Harmful effects of such substances on animals in marine environment, particularly on cultured bivalves, are poorly documented. Bivalve molluscs such as mussels and oysters have been postulated as ideal indicator organisms because of their way of life. They filter large volumes of seawater and may therefore accumulate and concentrate contaminants within their tissues. Moreover, development of techniques allowing effect analysis of such compounds on bivalve biology may lead to the development of diagnosis tools adapted to analyze pollutant transfer towards estuarine areas. In this context, influence of atrazine on defence mechanisms was analyzed in Pacific oysters, Crassostrea gigas. Atrazine was tested in vitro and in vivo on oyster haemocytes, and its effects were analyzed by flow cytometry. Haemocyte viability, cell cycle and cellular activities were monitored. Atrazine induced no significant effect in oyster under tested conditions except for peroxidase activity.


Geng MY, Li FC, Xin XL, Li J, Yan ZW, Guan HS (2003) The potential molecular targets of marine sulfated polymannuroguluronate interfering with HIV-1 entry - Interaction between SPMG and HIV-1 rgp120 and CD4 molecule. Antiviral Research 59 :127-135

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The potential targets of marine sulfated polymannuroguluronate (SPMG) involved in inhibition of HIV-1 entry were investigated by surface plasmon resonance and flow cytometry. Results indicated that binding of SPMG either to soluble oligomeric rgp120 or to complexed rgp120-sCD4 mainly resided in V3 loop region. In addition, SPMG was shown to be less accessible for sCD4 when sCD4 had pre-interacted with rgp120, though SPMG per se multivalently bound to sCD4 with relatively low affinity. While the pre-incubation of SPMG with rgp120 caused a partial blockade of rgp120 binding to sCD4, suggesting that SPMG either shared common binding sites on gp120 with sCD4 or masked the docking sites of gp120 for sCD4. Taken together, V3 domain was demonstrated to be the major site mediating interaction of SPMG with complexed rgp120-sCD4. It seems likely that SPMG binds to both rgp120 and sCD4, but has less accessibility for sCD4 when sCD4 has already bound to rgp120. Nevertheless, addition of SPMG either prior to or after the interaction of rgp120 with sCD4 may suppress rgp120 binding to sCD4. The exact pattern of this trimolecular complex formation at the cell membrane-anchored virus level requires further clarification. (C) 2003 Elsevier Science B.V. All rights reserved.


Green RE, Sosik HM, Olson RJ, DuRand MD (2003) Flow cytometric determination of size and complex refractive index for marine particles : comparison with independent and bulk estimates. Appl Opt 42 :526-541

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We advance a method to determine the diameter D and the complex refractive index (n + n’i) of marine particles from flow cytometric measurements of forward scattering, side scattering, and chlorophyll fluorescence combined with Mie theory. To understand better the application of Mie theory with its assumptions to flow cytometry (FCM) measurements of phytoplankton cells, we evaluate our flow cytometric-Mie (FCM-Mie) method by comparing results from a variety of phytoplankton cultures with independent estimates of cell D and with estimates of n and n’ from the inversion of bulk measurements. Cell D initially estimated from the FCM-Mie method is lower than independent estimates, and n and n’ are generally higher than bulk estimates. These differences reflect lower forward scattering and higher side scattering for single-cell measurements than predicted by Mie theory. The application of empirical scattering corrections improves FCM-Mie estimates of cell size, n, and n’ ; notably size is determined accurately for cells grown in both high- and low-light conditions, and n’ is correlated with intracellular chlorophyll concentration. A comparison of results for phytoplankton and mineral particles suggests that differences in n between these particle types can be determined from FCM measurements. In application to natural mixtures of particles, eukaryotic pico/nanophytoplankton and Synechococcus have minimum mean values of n’ in surface waters, and nonphytoplankton particles have higher values of n than phytoplankton at all depths.


Gregori G, Denis M, Lefevre D, Romano JC (2003) [Viability of heterotrophic bacteria in the Bay of Marseilles]. C R Biol 326 :739-750

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Marine microorganism activities are commonly assessed by bulk methods and assigned to the total cell count. The presence in significant amounts of ghost, dead, and damaged cells makes such as assignment a non-correct one. A Nucleic Acid Double Staining protocol (NADS) of fresh water bacteria (Barbesti et al., Cytometry 40 (2000) 214-218) has been adapted to resolve viable, damaged and dead cells in marine environments (Gregori et al., Appl. Environ. Microbiol. 67 (2001) 4662-4670). The present reports the first in situ application of this approach, conducted in the Bay of Marseilles in winter and spring periods at two sites with contrasted features.


Gregori G, Denis M, Seorbati S, Citterio S (2003) Resolution of viable and membrane-compromised free bacteria in aquatic environments by flow cytometry. Curr Protoc Cytom Chapter 11 :Unit 11 15

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In aquatic environments, free heterotrophic bacteria play an extremely important role because of their high biomass, wide panel of metabolisms, and ubiquity, as well as the toxicity of certain species. This unit presents a nucleic-acid double-staining protocol (NADS) for flow cytometry that can distinguish the fractions of viable, damaged, or membrane-compromised cells within the free-bacterial community. The NADS protocol is based on the simultaneous utilization of two nucleic acid stains, membrane-permeant SYBR Green and membrane-impermeant PI. The efficiency of the double staining is magnified by the FRET from SYBR Green to PI when both are bound to the nucleic acids. Full quenching of SYBR Green fluorescence by PI will identify cells with a compromised membrane, partial quenching will indicate cells with a slightly damaged membrane, and lack of quenching will characterize cells with an intact membrane. Samples do not require any pretreatment and this protocol can be performed almost anywhere.


Gruden CL, Khijniak A, Adriaens P (2003) Activity assessment of microorganisms eluted from sediments using 5-cyano-2,3-ditolyl tetrazolium chloride : a quantitative comparison of flow cytometry to epifluorescent microscopy. Journal of Microbiological Methods 55 :865-874

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Enhanced natural recovery may be successfully implemented at contaminated sediment sites, which are often characterized by large volumes of sediments with low to moderate levels of contamination to cost-effectively reduce human and ecological risks. In order to evaluate the potential for microbial contribution to remediation strategies, physiological assessment of indigenous microorganisms is essential. We report here a method for rapid and accurate assessment of metabolically (5-cyano2,3-ditolyl tetrazolium chloride [CTC]) active microorganisms eluted from sediment, based on flow cytometry (FCM). Microorganisms eluted from sediment and suspended in estuarine medium were stained with CTC and counterstained with the DNA stain Picogreen (PG). Optimal stain concentrations and incubation times were employed. FCM quantification of the dual-stained microorganisms was not statistically different (paired t test ; alpha = 0.05 ; df= 10) from enumeration (total or active numbers) by an established method (fluorescent microscopy) over two orders of magnitude (approximately 10(4)10(6)/ml). This research suggests that FCM, which allows the collection and analysis of multiple parameters (light scatter and fluorescence emission), is a good candidate for microbial characterization in complex environmental matrices, such as sediments, across a broad range of activity levels (- 2% to 84% of total). Potential applications for this FCM-based method include the rapid assessment of changes in sediment microbial activity in response to enhanced bioremediation strategies. (C) 2003 Elsevier B.V. All rights reserved.


Gunnes G, Valheim M, Press CM, Tverdal A, Storset A (2003) Comparison of flow cytometry and image morphometry in the quantitative analysis of cell population markers in the lymph node of sheep. Vet Immunol Immunopathol 94 :177-183

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Two approaches to the quantitative analysis of cell population markers in tissues are flow cytometry and image morphometry. To compare these methods, sheep lymph nodes were collected and analysed for CD8+ and CD21+ cell populations, which were selected to represent dispersed and concentrated cell populations, respectively. These two populations were measured as a percentage of total cell count (flow) or total tissue area (morphometry). The two populations were also measured as a percentage of respective base populations (CD2+ cells for CD8 and MHC II+ cells for CD21). A simple linear regression analysis showed that when the cell populations were assessed as a percentage of total cell count or total area, measurements obtained with flow and morphometry only correlated significantly with the dispersed CD8+ population and not with the highly concentrated CD21+ population. However, when the cell populations were assessed as a percentage of their base population, measurements obtained with flow and morphometry showed a significant correlation for both the dispersed and concentrated cell populations. This study demonstrates that measurements of lymph node cell populations obtained with the two methods are comparable, but that tissue distribution of cell populations should be considered, when the unit of measurement is chosen.


Hautefort I, Proenca MJ, Hinton JCD (2003) Single-copy green fluorescent protein gene fusions allow accurate measurement of Salmonella gene expression in vitro and during infection of mammalian cells. Applied and Environmental Microbiology 69 :7480-7491

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We developed a reliable and flexible green fluorescent protein (GFP)-based system for measuring gene expression in individual bacterial cells. Until now, most systems have relied upon plasmid-borne gfp gene fusions, risking problems associated with plasmid instability. We show that a recently developed GFP variant, GFP(+), is suitable for assessing bacterial gene expression. Various gfp(+) transcriptional fusions were constructed and integrated as single copies into the chromosome of Salmonella enterica serovar Typhimurium. A comparison of the expression levels of proU-lacZ and proU-gfp(+) fusions showed that GFP(+) reported proU activity in individual Salmonella cells as accurately as beta-galactosidase reported activity for entire populations. The single-copy gfp(+) fusions were ideal for monitoring up- and downregulation of Salmonella virulence genes. We discovered that in vitro induction of the SPI1 gene prgH occurs only in a portion of the population and that the proportion varies with the growth phase. We determined the level of expression of the SPI2 gene ssaG in bacteria released from marine macrophages. Our results demonstrate for the first time that single-copy GFP(+) fusions reliably report gene expression in simple and complex environments. This approach promises to allow accurate measurement of gene expression in individual bacteria during animal infection.


Hegaret H, Wikfors GH, Soudant P (2003) Flow cytometric analysis of haemocytes from eastern oysters, Crassostrea virginica, subjected to a sudden temperature elevation II. Haemocyte functions : aggregation, viability, phagocytosis, and respiratory burst. Journal of Experimental Marine Biology and Ecology 293 :249-265

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The capability of an oyster to respond to environmental stresses, such as periodically high summer temperatures, as well as disease or parasite infections, depends, in large measure, upon the viability and functional capability of haemocytes. Eastern oysters (Crassostrea virginica) were subjected to a sudden increase in temperature from 20 to 28 degreesC for 1 week, and several haemocyte functions were determined before and after the temperature elevation using the flow cytometer. Previously, we described the characterization of different haemocyte types using new and modified flow cytometric methods. In this report, we provide detailed protocols for flow cytometric methods to : (1) determine haemocyte aggregation using paired samples with or without an antiaggregant solution ; (2) assess haemocyte viability using propidium iodide (PI) ; (3) quantify haemocyte phagocytosis with fluorescent microbeads ; and (4) measure the respiratory burst response of individual haemocytes using 2,7’-dichlorofluorescein diacetate (DCFH-DA) and zymosan to activate the release of reactive oxygen species (ROS).
The temperature increase caused no significant change in haemocyte aggregation, although there was a trend of increasing aggregation in granulocytes and small granulocytes, but a slight decrease in hyalinocyte aggregation. Phagocytosis of all haemocyte types decreased after the temperature increase. Significantly higher percentages of dead haemocytes in all haemocyte types (attributable to a large increase in mortality of hyalinocytes, the most numerous cells) were found after the temperature increase, suggesting generally less capable immune function. Numbers of dead small granulocytes and granulocytes tended to decrease, but this was not statistically significant. Effects of temperature elevation upon respiratory burst were not statistically significant ; however, a trend of increased ROS production after temperature elevation was consistent for all haemocyte types. Granulocytes, hyalinocytes, and small granulocytes showed increased production of ROS in the presence of zymosan ; granulocytes showed the highest induced fluorescence. (C) 2003 Elsevier B.V. All rights reserved.


Hidemura A, Saito H, Fukatsu K, Matsuda T, Kitayama J, Ikeda S, Kang W, Nagawa H (2003) Oral administration of Bifidobacterium longum culture condensate in a diet-restricted murine peritonitis model enhances polymorphonuclear neutrophil recruitment into the local inflammatory site. Nutrition 19 :270-274

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OBJECTIVE : Dietary restriction impairs polymorphonuclear neutrophil (PMN) recruitment into the local inflammatory site, resulting in susceptibility to infection. Probiotics enhance host immunity via conditioning host intestinal microflora. Oral administration of Bifidobacterium longum culture condensate (BCC) in a diet-restricted marine peritonitis model may enhance PMN recruitment into the inflammatory site.
METHODS : Male ICR mice (n = 40) were assigned in equal numbers to control or BCC groups and subjected to 75% restricted food intake for 7 d. During dietary restriction, controls received only standard mouse chow, whereas the BCC group received standard mouse chow containing 1% BCC. Mice were killed before (0 h) or after (2 or 4 h) intraperitoneal glycogen injection. Peritoneal lavage fluid and exudative cells were recovered by, peritoneal lavage. Peritoneal exudative cell number was counted. Taper necrosis factor-alpha, interleukin-6, macrophage inflammatory protein-2, and interleukin-10 concentrations in peritoneal lavage-fluid were determined by enzyme-linked immuosorbent assay. CD11b, CD18 ; CD31, and CD62L expressions on circulating PMNs were measured by flow cytometry.
RESULTS : Oral BCC administration unregulated PMN recruitment into the peritoneal cavity and increased peritoneal fluid cytokine concentrations as well as CD18 and CD62L expressions on circulating PMNs during glycogen-induced peritonitis.
CONCLUSIONS : Oral BCC administration in a diet-restricted marine peritonitis model augmented P recruitment into the inflammatory site by upregulating cytokine concentrations in the local inflammatory site and adhesion molecule expression on circulating PMNs. Oral BCC administration may be a favorable modality for improving dietary restriction-induced host immunosuppression. (C)Elsevier Science Inc. 2003.


Huynh-Delerme C, Fessard V, Kiefer-Biasizzo H, Puiseux-Dao S (2003) Characteristics of okadaic acid—induced cytotoxic effects in CHO K1 cells. Environ Toxicol 18 :383-394

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This article reports the results of investigations into the process of cell death induced in the Chinese hamster ovary cell K1 subclone (CHO K1) by okadaic acid (OA), a hydrophobic polyether produced by marine dinoflagellates. The IC50 was about 13 nM OA after 24 h of treatment, as determined using neutral red. With the MTT assay, the IC50 was 25 nM, although in this case 25% of the initial staining was still observed at 100 nM. Hoechst staining showed that mitotic figures accumulated at 12 nM OA after a 24- or 48-h treatment. In experiments limited to a 3-day treatment without changing the medium, CHO K1 cells were engaged in the death process at 50 nM OA after about 20 h and at 10 nM OA after 48 h. In many cells nuclear fragmentation that resulted in the apparent appearance of vesicles correlated with increasing cellular volume. But additional cell fragmentation was not observed with any treatment, and the chromatin material seemed to progressively disappear inside the cells. DNA fragmentation was analyzed by electrophoresis and with the TUNEL technique. With both techniques, the DNA was fragmented by 48 h in both 25 and 50 nM OA. Electrophoresis showed that both adherent and nonadherent cells were affected. Annexin-positive/ propidium iodide (PI)-negative cells were rarely observed after OA treatment. Some were seen under the scanning cytometer after 20 h at 50 nM OA or after 48 h at 10 nM OA, but they were never detected by flow cytometry. Most of the time scanning cytometry showed either unstained cells or PI-positive (annexin-positive or -negative) cells (48 h, 50 nM, or 72 h, 10 nM). Flow cytometry cytograms showed two cell subpopulations : one composed of a majority of smaller cells, the other of larger cells. The larger cells markedly decreased with time and OA treatment (50 and 100 nM). Stained-cell counting showed that all cells that stained were both annexin- and PI positive and that most PI-positive cells were smaller. Ki67 antigen labeling showed the proliferative activity of CHO K1 cultures but also demonstrated the loss of this activity in smaller cells treated with 50 nM OA for 48 h. We concluded that in our culture conditions the main OA target within CHO K1 cultures was dividing cells. Our results suggest that cells with disturbed metaphase-anaphase enter apoptosis, leading to necrotic daughter cells.


Jacquet S, Bratbak G (2003) Effects of ultraviolet radiation on marine virus-phytoplankton interactions. Fems Microbiology Ecology 44 :279-289

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Ambient ultraviolet radiation (UVR) is harmful to many biological systems and increased UVR, due to a reduced ozone layer, may have many unforeseen consequences. Viruses are the most abundant biological particles in the sea and are thought to play an important role in the structure and functioning of aquatic ecosystems. Although an increasing number of studies have been published during the last 15 years, aquatic viral ecology is still in its infancy and little is known about the effect of environmental factors on virus life cycle and host-virus interactions. Using flow cytometry, we have investigated the effect of UVR (UVB intensity : 0.22 W m(-2) and UVA/UVB ratio similar to 30) on five different cultured marine phytoplankton host-virus systems (CeV-Chrysoehromulina ericina, EhV-Emiliania huxleyi, MpV-Micromonas pusilla, PpV-Phaeocystis pouchetti and PoV-Pyramimonas orientalis). Viruses appear to be susceptible to UV, but also they might provide some protection to their hosts. It is shown that (i) some of the investigated microalgae that have been co-cultured with viruses are less sensitive (e.g. P. pouchetii, M. pusilla) to UVB stress compared to susceptible microalgae (i.e. virus-free cultures), (ii) different viruses have different sensitivities to UVB in terms of both their abundance patterns (no effect for most of them except EhV) and infectivity (from no effect for PoV, to complete inactivation for PpV), (iii) UVA has no effect on host-virus interactions. Our results show UVB to be a potentially important factor in the regulation of virus-host interactions in surface waters. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.


Joachimsthal EL, Ivanov V, Tay JH, Tay ST (2003) Flow cytometry and conventional enumeration of microorganisms in ships’ ballast water and marine samples. Mar Pollut Bull 46 :308-313

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Conventional methods for bacteriological testing of water quality take long periods of time to complete. This makes them inappropriate for a shipping industry that is attempting to comply with the International Maritime Organization’s anticipated regulations for ballast water discharge. Flow cytometry for the analysis of marine and ship’s ballast water is a comparatively fast and accurate method. Compared to a 5% standard error for flow cytometry analysis the standard methods of culturing and epifluorescence analysis have errors of 2-58% and 10-30%, respectively. Also, unlike culturing methods, flow cytometry is capable of detecting both non-viable and viable but non-culturable microorganisms which can still pose health risks. The great variability in both cell concentrations and microbial content for the samples tested is an indication of the difficulties facing microbial monitoring programmes. The concentration of microorganisms in the ballast tank was generally lower than in local seawater. The proportion of aerobic, microaerophilic, and facultative anaerobic microorganisms present appeared to be influenced by conditions in the ballast tank. The gradual creation of anaerobic conditions in a ballast tank could lead to the accumulation of facultative anaerobic microorganisms, which might represent a potential source of pathogenic species.


Joachimsthal EL, Ivanov V, Tay JH, Tay STL (2003) Flow cytometry and conventional enumeration of microorganisms in ships’ ballast water and marine samples. Marine Pollution Bulletin 46 :308-313

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Conventional methods for bacteriological testing of water quality take long periods of time to complete. This makes them inappropriate for a shipping industry that is attempting to comply with the International Maritime Organization’s anticipated regulations for ballast water discharge. Flow cytometry for the analysis of marine and ship’s ballast water is a comparatively fast and accurate method. Compared to a 5% standard error for flow cytometry analysis the standard methods of culturing and epifluorescence analysis have errors of 2-58% and 10-30%, respectively. Also, unlike culturing methods, flow cytometry is capable of detecting both non-viable and viable but non-culturable microorganisms which can still pose health risks. The great variability in both cell concentrations and microbial content for the samples tested is an indication of the difficulties facing microbial monitoring programmes. The concentration of microorganisms in the ballast tank was generally lower than in local seawater. The proportion of aerobic, microaerophilic, and facultative anaerobic microorganisms present appeared to be influenced by conditions in the ballast tank. The gradual creation of anaerobic conditions in a ballast tank could lead to the accumulation of facultative anaerobic microorganisms, which might represent a potential source of pathogenic species. (C) 2002 Elsevier Science Ltd. All rights reserved.


Jochem FJ (2003) Photo- and heterotrophic pico- and nanoplankton in the Mississippi River plume : distribution and grazing activity. Journal of Plankton Research 25 :1201-1214

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The abundance of pico- and nanophytoplankton, bacteria and heterotrophic nanoflagellates, and grazing rates on phototrophic pico- and nanoplankton and bacterioplankton were assessed along a salinity gradient (0.2-34.4) in the Mississippi River plume in May 2000. Grazing rates were established by serial dilution experiments, and analysis by flow cytometry allowed differentiation of grazing rates for different phytoplankton subpopulations (eukaryotes, Synechococcus spp., Prochlorococcus spp.). Grazing rates on phytoplankton tended to increase along the salinity gradient and often approached or exceeded 1 day(-1). Phytoplankton net growth rates (growth-grazing) were mostly negative, except for positive values for eukaryotic nanoplankton in the low-salinity, high-chlorophyll region. Grazing pressure on bacteria was moderate (similar to0.5 day(-1)) and bacteria gained positive net growth rates of similar to0.3 day(-1). Eukaryotic nanophytoplankton were the major phototrophic biomass and protozoan food source, contributing 30-80% of the total consumed carbon. Bacteria were the second most important food source at 9-48% of the total consumed carbon. Synechococcus spp. and Prochlorococcus spp. remained an insignificant Portion of protozoan carbon consumption, probably due to their low contribution to the total pico- and nanoplankton biomass. Group-specific grazing losses relative to standing stocks suggest protozoan prey preference for eukaryotes over bacteria. Protozoan grazers exerted a major grazing pressure on pico- and nanophytoplankton, but less so on bacteria.


Koce JD, Vilhar B, Bohanec B, Dermastia M (2003) Genome size of Adriatic seagrasses. Aquatic Botany 77 :17-25

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Genome size (C-value) was measured in four species of Adriatic seagrasses with interphase-peak DNA image cytometry. The estimated 2C-value was 1.5 pg DNA for Zostera noltii (2n = 12), 1.2 pg for Zostera marina (2n = 12), 1.1 pg for Cymodocea nodosa (2n = 28) and 6.2 pg for Posidonia oceanica, using Pisuin sativum (2C-value = 8.84 pg) as the calibration standard. Seagrass leaves were fixed in 4% buffered formaldehyde to mitigate stoichiometric error due to tannins and post-fixed in 3:1 methanol:acetic acid (MAA). DNA was stained with the Feulgen reaction after hydrolysis in 5 M HCl for 90 min at 20 degreesC. Comparison of genome size of seagrasses with the data for other species of Alismatidae indicated that the ancestral genome of Alismatidae was relatively small. (C) 2003 Elsevier B.V. All rights reserved.


Lambert C, Soudant P, Choquet G, Paillard C (2003) Measurement of Crassostrea gigas hemocyte oxidative metabolism by flow cytometry and the inhibiting capacity of pathogenic vibrios. Fish & Shellfish Immunology 15 :225-240

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A flow cytometric method to measure the production of oxidative metabolism products was adapted for use with Crassostrea gigas hemocytes. The method is based upon the oxidation, by hydrogen peroxide (H2O2), of intracellular 2’,7’-dichlorofluorescin (DCFH) to green-fluorescent dichlorofluorescein. Activation of the respiratory burst (RB) was tested using phorbol myristate acetate with no success. By contrast, activation by zymosan particles increased oxidation of DCFH in C gigas hemocytes, mainly granulocytes, and optimization tests showed a good response with 20 zymosan particles per hemocyte. Anti-aggregant solution, used to prevent hemocytes from clumping during bleeding, inhibited the RB activity measured by DCFH oxidation. The flow cytometric method developed during this work was used to evaluate the DCFH oxidation-inhibiting capacity of four strains of vibrio bacteria, known or suspected to be pathogenic for bivalves. (C) 2003 Elsevier Ltd. All rights reserved.


Li FH, Xiang JH, Zhou LH, Wu CG, Zhang XJ (2003) Optimization of triploid induction by heat shock in Chinese shrimp Fenneropenaeus chinensis. Aquaculture 219 :221-231

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Chromosome manipulation for commercially valuable marine animals plays an important role in aquaculture. The special reproductive characteristics of shrimp make it difficult to control fertilization and synchronize egg development, so research on chromosome manipulation in shrimp has proceeded very slowly. In the present study, triploid shrimp Fenneropenaeus chinensis were induced by heat shocks and the optimal-inducing condition was screened at different spawning temperatures. Level of triploid induction for each treatment was evaluated by flow cytometry at nauplius stage. The highest level of triploid induction reached to more than 90%. Starting time for each treatment was very crucial for triploid induction in shrimp. One optimal treatment condition for triploid induction was heat shock (29-32 degreesC), starting at 18-20 min for duration of 10 min. These conditions varied depending on the temperature at spawning. Triploid level at embryo stage and nauplius stage was not different, suggesting the same hatching rate between diploids and triploids. Heat shock is a very effective way to induce triploids in this species, and can be easily used on large scale without any harmful effect on the environment as compared with chemical treatment. (C) 2003 Elsevier Science B.V. All rights reserved.


Lin YS, Tsai YJ, Tsay JS, Lin JK (2003) Factors affecting the levels of tea polyphenols and caffeine in tea leaves. Journal of Agricultural and Food Chemistry 51 :1864-1873

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An isocratic HPLC procedure was developed for the simultaneous determination of caffeine and six catechins in tea samples. When 31 commercial teas extracted by boiling water or 75% ethanol were analyzed by HPLC, the levels of (-)-epigallocatechin 3-gallate (EGCG), and total catechins in teas were in the order green tea (old leaves) > green tea (young leaves) and oolong tea > black tea and pu-erh tea. Tea samples extracted by 75% ethanol could yield higher levels of EGCG and total catechins. The contents of caffeine and catechins also have been measured in fresh tea leaves from the Tea Experiment Station in Wen-Shan or Taitung ; the old tea leaves contain less caffeine but more EGCG and total catechins than young ones. To compare caffeine and catechins in the same tea but manufactured by different fermentation processes, the level of caffeine in different manufactured teas was in the order black tea > oolong tea > green tea > fresh tea leaf, but the levels of EGCG and total catechins were in the order green tea > oolong tea > fresh tea leaf > black tea. In addition, six commercial tea extracts were used to test the biological functions including hydroxyl radical scavenging, nitric oxide suppressing, and apoptotic effects. The pu-erh tea extracts protected the plasmid DNA from damage by the Fenton reaction as well as the control at a concentration of 100 mug/mL. The nitric oxide suppressing effect of tea extracts was in the order pu-erh tea greater than or equal to black tea greater than or equal to green tea > oolong tea. The induction of apoptosis by tea extract has been demonstrated by DNA fragmentation ladder and flow cytometry. It appeared that the ability of tea extracts to induce HL-60 cells apoptosis was in the order green tea > oolong > black tea > pu-erh tea. All tea extracts extracted by 75% ethanol have stronger biological functions than those extracted by boiling water.


Meiyu G, Fuchuan L, Xianliang X, Jing L, Zuowei Y, Huashi G (2003) The potential molecular targets of marine sulfated polymannuroguluronate interfering with HIV-1 entry. Interaction between SPMG and HIV-1 rgp120 and CD4 molecule. Antiviral Res 59 :127-135

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The potential targets of marine sulfated polymannuroguluronate (SPMG) involved in inhibition of HIV-1 entry were investigated by surface plasmon resonance and flow cytometry. Results indicated that binding of SPMG either to soluble oligomeric rgp120 or to complexed rgp120-sCD4 mainly resided in V3 loop region. In addition, SPMG was shown to be less accessible for sCD4 when sCD4 had pre-interacted with rgp120, though SPMG per se multivalently bound to sCD4 with relatively low affinity. While the pre-incubation of SPMG with rgp120 caused a partial blockade of rgp120 binding to sCD4, suggesting that SPMG either shared common binding sites on gp120 with sCD4 or masked the docking sites of gp120 for sCD4. Taken together, V3 domain was demonstrated to be the major site mediating interaction of SPMG with complexed rgp120-sCD4. It seems likely that SPMG binds to both rgp120 and sCD4, but has less accessibility for sCD4 when sCD4 has already bound to rgp120. Nevertheless, addition of SPMG either prior to or after the interaction of rgp120 with sCD4 may suppress rgp120 binding to sCD4. The exact pattern of this trimolecular complex formation at the cell membrane-anchored virus level requires further clarification.


Moreno-Garrido I, Hampel M, Lubian LM, Blasco J (2003) Sediment toxicity tests using benthic marine microalgae Cylindrotheca closterium (Ehremberg) Lewin and Reimann (Bacillariophyceae). Ecotoxicology and Environmental Safety 54 :290-295

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A new method for sediment toxicity testing using marine benthic pennate noncolonial diatom (Cylindrotheca closterium, formerly Nitzschia closterium) has been developed. This microalgae showed a good growth rate during the experimental period, even when low enriched media were used. Sediment spiked with heavy metals [cadmium (Cd), copper (Cu), and lead (Pb)] was employed to determine the EC50 values, using microalgal growth inhibition as the endpoint. The obtained results were as follows : Three heavy metals (Cd, Cu, and Pb), previously spiked on experimental sediment, were separately assayed in toxicity tests. The EC50 values for these heavy metals in microalgal growth inhibition tests resulted to be 79 mg kg(-1) for Cd, 26 mg kg(-1) for Cu, and 29 mg kg(-1) for Pb (in experimental sediment). The influence of sediment granulometry on the growth of microalgal population was also studied, finding that the growth of the microalgal population on media containing sediment with a relation sand-size:silt size of 9:1 was not different from optimal growth (occurring in media containing 100% sand-sized sediment). The diatom C closterium proved to be a suitable organism for sediment toxicity tests, due to its sensitivity and fast growth even in poorly enriched media. (C) 2003 Elsevier Science (USA). All rights reserved.


Moritomo T, Serata K, Teshirogi K, Aikawa H, Inoue Y, Itou T, Nakanishi T (2003) Flow cytometric analysis of the neutrophil respiratory burst of ayu, Plecoglossus altivelis : comparison with other fresh water fish. Fish & Shellfish Immunology 15 :29-38

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Neutrophils of vertebrates undergo respiratory burst activity (RBA) as a defense mechanism against bacterial infections. We report here that ayu (Plecoglossus altivelis) have unusually high RBAs even when they are in a healthy condition. Kidney and blood leukocytes were obtained from ayu, rainbow trout (Oneorhynchus mykiss), carp (Cyprinus carpio), eel (Anguilla japonica), and pond smelt (Hypomesus nipponensis). Neutrophil RBA was measured by flow cytometry using dihydrorhodamine after stimulation with phorbol myristate acetate. The amount of RBA of neutrophils from both blood and kidney was significantly higher in ayu than in the other species (e.g. the fluorescence intensity of ayu blood neutrophils was about 3-7 times higher than that from trout and carp, and that of ayu kidney neutrophils was 2-19 times higher than that of rainbow trout, carp, eel, and pond smelt). This unique character of ayu neutrophils was invariable even at different ages, locations, and sex-maturation stages. (C) 2003 Published by Elsevier Science Ltd.


Nandakumar K, Obika H, Shinozaki T, Ooie T, Utsumi A, Yano T (2003) Laser impact assessment in a biofilm-forming bacterium Pseudoalteromonas carrageenovora using a flow cytometric system. Biotechnol Bioeng 82 :399-402

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Impact by pulsed laser irradiations from an Nd:YAG laser on the marine biofilm-forming bacterium Pseudoalteromonas carrageenovora has been studied using a flow cytometric system. The biofilm-forming bacteria in the planktonic state have been irradiated while flowing, and the mortality and bacterial attachment have been determined by exposing TiN coupons in the system. Coupons suspended in the non-irradiated bacterial flow were treated as the control. The fluence used in the study was 0.1 J/cm(2). Three flow rates (14, 28, and 42 cm/min) and two exposure durations (15 and 30 min) were tested. The results showed the increase in bacterial mortality with the decrease in flow rate. The maximum mortality of 27.5% was observed when the flow rate was 14 cm/min. The bacterial attachment increased with the increase in flow rate and exposure duration. The area of bacterial attachment on the experimental coupons exposed to the irradiated sample was significantly lesser than that for the nonirradiated sample. The results thus show in a flowing system, low power pulsed laser irradiations could reduce the bacterial attachment even though it did not cause significant mortality.


Nandakumar K, Obika H, Shinozaki T, Ooie T, Utsumi A, Yano T (2003) Laser impact on marine planktonic diatoms : an experimental study using a flow cytometry system. Biofouling 19 :133-138

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A flow cytometry system was used to evaluate the impact of pulsed laser irradiations from an Nd:YAG laser on two marine coastal water diatoms, Chaetoceros gracilis and Skeletonema costatum. Three flow speeds, i.e. 9, 18 and 27 ml min-1 and three laser fluences, i.e. 0.025, 0.05 and 0.1 J cm-2 pulse-1 were tested during this study. The reduction in cell density and chlorophyll a (chl a) concentrations were monitored by reference to non-irradiated samples as controls. Upon irradiation, the cell density and the chl a concentrations became reduced significantly compared to the control (one way ANOVA p < 0.001 for the cell density in both the species and p < 0.05 for chl a concentrations in both species). A maximum mortality of 0.77 log10 (about 83%) for C. gracilis and 0.68 log10 (about 78%) for S. costatum was observed at 9 ml min-1 flow speed and 0.1 J cm-2 laser fluence. The maximum reduction observed in the chl a concentration was about 26% (control 0.413 and sample 0.306 mg ml-1) for C. gracilis and 27% (control 0.222 and sample 0.16 mg ml-1) for S. costatum, when the flow rate was 9 ml min-1 and the fluence 0.1 J cm-2. In general, mortality increased with an increase in the laser fluence. The results thus show if the cooling water is laser-irradiated to mitigate biofouling, this could result in significant damage to the planktonic flora of the flowing seawater system, which in turn might reduce algal biofilm formation on industrially important structures. The reduction in the chl a concentration showed that the laser irradiations also could result in a significant reduction in the primary productivity of the cooling water.


Olson RJ, Shalapyonok A, Sosik HM (2003) An automated submersible flow cytometer for analyzing pico- and nanophytoplankton : FlowCytobot. Deep-Sea Research Part I-Oceanographic Research Papers 50 :301-315

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Flow cytometry is a valuable tool for the analysis of phytoplankton and other suspended particles because of its speed and quantitative measurements, but the method’s oceanographic application has been limited by the need to take discrete water samples for analysis on board ship or in the laboratory. For this reason, we have developed an automated flow cytometer (FlowCytobot) that can operate in situ and unattended. The new instrument utilizes a diode-pumped 532 nm laser and can measure light scattering and fluorescence of particles as small as Synechococcus cells. For operation at the LEO-15 mooring site off New Jersey, it is connected to shore by power and communications cables, and is controlled by a microcomputer whose programming can be loaded remotely. The sampling rate is adjustable ; we have used from 12.5 to 50 mul min(-1). Integrated signals from each particle (two light scattering angles and two fluorescence emission bands) are transmitted to a shore-based computer, where they are accessible by Internet and can be examined in real time. FlowCytobot was deployed at LEO-15 from late July to early October 200 1, where it operated continuously (aside from occasional power or communications interruptions at the node) without outside intervention. Even after 2 months of in situ operation, FlowCytobot’s measurements were similar to those of a conventional flow cytometer, as shown by analysis of a discrete water sample taken at the location of the sample inlet. In addition to documenting seasonal and event-scale changes in size distributions and population abundances in the pico- and nanophytoplankton, FlowCytobot will be useful for examining diel cycles in light scattering and pigment fluorescence of cells in situ that may allow estimation of rates of production by different phytoplankton groups. (C) 2003 Elsevier Science Ltd. All rights reserved.


Orellana MV, Verdugo P (2003) Ultraviolet radiation blocks the organic carbon exchange between the dissolved phase and the gel phase in the ocean. Limnology and Oceanography 48 :1618-1623

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Dissolved organic carbon (DOC) is one of the major reservoirs of active organic carbon on Earth. Although the bulk of the marine DOC pool is largely composed of small refractory polymeric material, new evidence suggests that similar to10% of the DOC pool (10(16) g C) can enter the microbial loop by forming microscopic gels that can eventually be colonized and degraded by bacteria. Marine microgels result from a spontaneous and reversible assembly/dispersion equilibrium of DOC polymers forming hydrated Ca-bonded tangled polymer networks. Here we test the hypothesis that ultraviolet (UV) photocleavage should strongly inhibit the formation of microgels, because the stability of tangled networks decreases exponentially with polymer length. Because of the loss of ozone shielding, the UV-B spectral component of solar radiation (lambda = 280-320 nm) has undergone a dramatic increase in the past few decades, particularly in the polar regions. We used dynamic laser-scattering spectroscopy and flow cytometry to investigate UV-induced DOC polymer cracking and the effect of UV on DOC assembly/dispersion equilibrium in 0.2 mum filtered seawater. Results indicate that exposure of seawater to UV-B fluxes equivalent to those found in Antarctica during summer solstice can cleave DOC polymers, inhibit their spontaneous assembly, and/or disperse assembled microgels. Our results agree with previous observations that indicated that fragmentation produced by UV photolysis increases exponentially with exposure time and suggested that UV could limit the supply of microbial substrate by hindering microgel formation. UV cleavage yields short-chain polymers that do not assemble and could eventually account for the old refractory DOC pool found in seawater.


Parrow MW, Burkholder JM (2003) Estuarine heterotrophic cryptoperidiniopsoids (Dinophyceae) : Life cycle and culture studies. Journal of Phycology 39 :678-696

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Cryptoperidiniopsoids are an unclassified group of delicately thecate heterotrophic dinoflagellates known to be common in eastern U.S. estuarine waters. Over the past 10 years cryptoperidiniopsoids were isolated from different geographical regions and cultured with cryptophyte algal prey. In the seven clonal isolates examined, reproduction was strongly linked to the availability of prey cells. The dinoflagellates phagocytized the contents of prey cells through a tube-like peduncle, similarly as close relatives of Pfiesteria spp. and several other heterotrophic species. Cell division occurred while encysted, most commonly yielding two biflagellated offspring. Abundant fusing gametes, phagotrophic planozygotes, and cysts with a pronounced nuclear cyclosis characterized persistent sexuality. Cysts with nuclear cyclosis produced two flagellated offspring cells. The resistance of reproductive cysts to antimicrobial treatments was examined, and a simple high-yield technique was developed for population synchronization while ridding the dinoflagellates of most contaminating vacuolar prey DNA and external contaminants. The DNA content and population DNA profiles of synchronously excysted cryptoperidiniopsoids from different isolates were measured using flow cytometry and were related to the life history of these and other dinoflagellates. Cryptophyte-fed cultures with versus without extracellular bacteria were compared, and bacteria apparently promoted cryptoperidiniopsoid feeding and growth. Externally bacteria-free dinoflagellates were cultured in media enriched with dissolved organic nutrients, and nutritional benefit may have occurred in some treatments. The potential for mixotrophic nutrition from maintenance of cryptophyte chloroplasts was examined using flow cytometrically sorted cells, but evidence of kleptoplastidy was not found in these isolates under the conditions imposed.


Pendleton A, Pope B, Weeds A, Koffer A (2003) Latrunculin B or ATP depletion induces cofilin-dependent translocation of actin into nuclei of mast cells. J Biol Chem 278 :14394-14400

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12566455

Increasing cellular G-actin, using latrunculin B, in either intact or permeabilized rat peritoneal mast cells, caused translocation of both actin and an actin regulatory protein, cofilin, into the nuclei. The effect was not associated with an increase in the proportion of apoptotic cells. The major part of the nuclear actin was not stained by rhodamine-phalloidin but could be visualized with an actin antibody, indicating its monomeric or a conformationally distinct state, e.g. cofilin-decorated filaments. Introduction of anti-cofilin into permeabilized cells inhibited nuclear actin accumulation, implying that an active, cofilin-dependent, import exists in this system. Nuclear actin was localized outside the ethidium bromide-stained region, in the extrachromosomal nuclear domain. In permeabilized cells, the appearance of nuclear actin and cofilin was not significantly affected by increasing [Ca(2+)] and/or adding guanosine 5’-O-(3-thiotriphosphate), but was greatly promoted when ATP was withdrawn. Similarly, ATP depletion in intact cells also induced nuclear actin accumulation. In contrast to the effects of latrunculin B, ATP depletion was associated with an increase in cortical F-actin. Our results suggest that the presence of actin in the nucleus may be required for certain stress-induced responses and that cofilin is essential for the nuclear import of actin.


Pettersen EF, Ulvenes M, Melingen GO, Wergeland HI (2003) Peripheral blood and head kidney leucocyte populations during out-of-season (0+) parr-smolt transformation and seawater transfer of Atlantic salmon (Salmo salar L.). Fish Shellfish Immunol 15 :373-385

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Using monoclonal antibodies (MAb) and flow cytometry, Atlantic salmon neutrophils and Ig+ cells in blood and head kidney were studied in under-yearling out-of-season (0+) smolts, and 2 and 4 weeks after transfer to seawater. The parr-smolt transformation was induced using a phase advanced simulated natural photoperiod regime, and sampling of four fish was performed at regular intervals, starting on the date of the photoperiod initiation. During the freshwater period the proportion of neutrophils in the head kidney leucocytes (HKL) remained quite stable and only gradual changes in Ig+ cells were observed. In the peripheral blood leucocytes (PBL), the proportion of neutrophils markedly increased during the last month prior to seawater transfer. The most notable changes in the proportions of MAb+ leucocytes were observed in PBL after seawater transfer, with a significant increase in Ig+ cells and a significant decrease in neutrophils after two weeks in seawater. In the freshwater samples, although there were fluctuations, a decrease in the numbers of total leucocytes per millilitre blood and per gram head kidney during parr-smolt transformation was observed. The number of MAb+ cells in blood appeared to be relatively stable, while the number in head kidney tended to decrease. Following seawater transfer, the numbers of total and MAb+ leucocytes in both blood and head kidney increased markedly. The results suggest that changes in both distribution and numbers of leucocytes in peripheral blood and head kidney take place during parr-smolt transformation, and that marked changes are associated with seawater transfer. Some mechanisms possibly involved are indicated.


Pile AJ, Grant A, Hinde R, Borowitzka MA (2003) Heterotrophy on ultraplankton communities is an important source of nitrogen for a sponge-rhodophyte symbiosis. Journal of Experimental Biology 206 :4533-4538

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Grazing on ultraplankton by the sponge partner of an invertebrate/algal symbiotic association can provide enough particulate organic nitrogen to support the nitrogen needs of both partners. The previously unknown natural diet of the sponge in the Haliclona-Ceratodictyon association consists of bacteria and protozoans, which are rich sources of nitrogen. Retention of ultraplankton varied with season and time of day. During the winter there was an order of magnitude more nitrogen taken up than in summer. Time of day during each season also affected the amount of ultraplankton retained. In summer retention was higher at night whereas the opposite was true during winter. Overall, the Haliclona-Ceratodietyon association is able to meet its metabolic nitrogen demands through grazing on the naturally occurring water column community.


Puddu A, Zoppini A, Fazi S, Rosati M, Amalfitano S, Magaletti E (2003) Bacterial uptake of DOM released from P-limited phytoplankton. Fems Microbiology Ecology 46 :257-268

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The growth and the structure of a coastal bacterioplankton community were monitored in short-term bottle experiments in order to investigate the bacterial uptake of extracellular organic carbon released by the diatom Cylindrotheca closterium grown under P-balanced and P-depleted conditions. Bacterial specific growth rates and carbon demand were significantly lower in the exudates from P-depleted algae (24%# and 30% reduction, respectively). The origin of the extracellular carbon appeared also to affect the taxonomic composition of the bacterioplankton assemblage, mainly reducing the development of gamma-Proteobacteria. This pattern of bacterial carbon uptake could contribute to a longer persistence of the exudates released in P-depleted conditions affecting the dynamics of the carbon cycle in marine environments. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.


Ragone Calvo LM, Dungan CF, Roberson BS, Burreson EM (2003) Systematic evaluation of factors controlling Perkinsus marinus transmission dynamics in lower Chesapeake Bay. Dis Aquat Organ 56 :75-86

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14524504

The transmission of Perkinsus marinus in eastern oysters Crassostrea virginica in relation to water temperature, host oyster mortality, and water-column abundance of anti-P. marinus antibody-labeled cells was systematically examined for 20 mo at a site in the lower York River, Virginia, USA. Uninfected sentinel oysters were naturally exposed to the parasite at 2 wk intervals throughout the course of the study to determine the periodicity and rates of parasite transmission. The timing and magnitude of disease-associated oyster mortalities in a local P. marinus-infected oyster population were estimated by monitoring a captive subset of the local oyster population. Flow cytometric immunodetection methods were employed to estimate the abundance of P. marinus cells in water samples collected 3 times each week. The acquisition of P. marinus infections by naive sentinel oysters occurred sporadically at all times of the year ; however, the highest incidence of infection occurred during the months of August and September. This window of maximum parasite transmission coincided with the death of infected hosts within the captive local oyster population. Counts of antibody-labeled cells ranged from 10 to 11900 cells l(-1), with the highest abundances in July and August coincident with maximum summer temperatures. A statistically significant relationship between water-column parasite abundance and infection-acquisition rate was not observed ; however, highest parasite-transmission rates in both years occurred during periods of elevated water-column abundance of parasite cells. These results support the prevailing model of P. marinus transmission dynamics by which maximum transmission rates are observed during periods of maximum P. marinus-associated host mortality. However, our results also indicate that transmission can occur when host mortality is low or absent, so alternative mortality-independent dissemination mechanisms are likely. The results also suggest that atypically early-summer oyster mortality from Haplosporidium nelsoni infection, at a time when infections of P. marinus are light, has a significant indirect influence on P. marinus transmission dynamics. Elimination of these hosts prior to late-summer P. marinus infection-intensification effectively reduces the overall number of P. marinus cells disseminated.


Ribes M, Coma R, Atkinson MJ, Kinzie RA (2003) Particle removal by coral reef communities : picoplankton is a major source of nitrogen. Marine Ecology-Progress Series 257 :13-23

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Removal and uptake of planktonic particulate organic matter by coral reef benthos is widely recognized as an important pathway for carbon and nutrients. We placed 3 natural assemblages of coral reef benthos, including 3 species of corals with associated sponges, ascidians, actinians, and bryozoans, in a long flume (24 x 0.4 x 0.3 m). Water was re-circulated at various speeds (5, 13, 22, and 32 cm s(-1)) over 6 h, and the disappearance of particles (pico-, nano-, microplankton and detrital particles) were measured using flow cytometry and microscopy. Control communities consisted of dead coral skeletons. Rates of removal of all particles were proportional to their concentrations. The first-order rate constant for the decrease in particle concentration ranged from 36 to 97 x 10(-6) m s(-1) (mean +/- SD = 63 +/- 16 x 10(-6) m s(-1)), with 71% of this variation explained by particle type. Water velocity had no significant effect on these rate constants. Living particles contributed 96% of the total nitrogen removal, with picoplankton (cells 0.2 to 2 mum) accounting for 92%. Overall, nitrogen removal from particles (8.8 to 10.3 mmol N m(-2) d(-1)) appears to be similar in magnitude to that of dissolved inorganic nitrogen ; thus, picoplankton is a major source of nitrogen for these coral reef assemblages.


Sachidanandham R, Gin KY (2003) Flow cytometric detection of beta-D-glucuronidase gene in wild-type bacterial cells using in-situ PCR. Biotechnol Bioeng 82 :127-133

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An in situ PCR-based flow cytometry method useful for monitoring the presence or absence of the beta-D-glucuronidase gene in Escherichia coli has been developed. A single-step fixation and permeabilization procedure, which maintained cell integrity at the elevated temperatures used during thermal cycling in the presence of PCR reagents, was demonstrated. We have chosen a shorter DNA sequence of length 147 bp for the PCR. Cells subjected to in situ PCR using fluorescein-12-dUTP as a label, showed the presence of uid both in epifluorescence microscopic examination and flow cytometric analysis. Multi-parametric analysis of flow cytometric profiles revealed that the efficiency of labeling was found to be high. The potential of in situ PCR for the detection of uid in intact coliform cells was then successfully tested with a fecal coliform isolated from the coastal waters of Singapore.


Saeij JPJ, Groeneveld A, Van Rooijen N, Haenen OLM, Wiegedjes GF (2003) Minor effect of depletion of resident macrophages from peritoneal cavity on resistance of common carp Cyprinus carpio to blood flagellates. Diseases of Aquatic Organisms 57 :67-75

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Carp Cyprinus carpio macrophages were depleted by intraperitoneal (i.p.) injection of clodronate-liposomes for the in vivo study of the effect of macrophage depletion on the resistance of carp to infection with blood flagellate parasites. Clodronate released inside the cell induces apoptosis of (murine) macrophages. Following i.p. injection of carp with liposomes alone, but not with Trypanoplasma borreli, neutrophilic granulocytes rapidly migrated from the head kidney to the peritoneal cavity. The majority of liposomes in the peritoneal cavity were not taken up by newly arrived neutrophilic granulocytes, however, but by resident macrophages. After 2 i.p. injections of clodronate-liposomes, the percentage of macrophages present in the peritoneal cavity was significantly reduced, as evaluated by flow cytometry. Macrophage-depleted carp that were infected i.p. with T. borreli suffered from high mortality. However, these fish did not show lethal parasitaemia but did show clear bacteraemia. Macrophage-depleted carp that were infected i.p. with Trypanosoma carassii showed a minor increase in parasitaemia. In addition, macrophage-depleted carp, immune to T. borreli as a result of having survived a prior infection, remained immune to i.p. reinfection with T. borreli. Succesful depletion of peritoneal macrophages seemed to have a minor effect on the resistance of carp against blood flagellates. However, carp macrophages are essential as a first line of defence against (bacterial) infection.


Selph KE, Landry MR, Laws EA (2003) Heterotrophic nanoflagellate enhancement of bacterial growth through nutrient remineralization in chemostat culture. Aquatic Microbial Ecology 32 :23-37

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Heterotrophic nanoflagellates are the principal consumers of picoplankton in the ocean. Their role as nutrient remineralizers is also well established. However, the coupled interactions between grazer consumption and prey growth are less well understood. In this work, we demonstrate a tight coupling among flagellate grazing, nitrogen remineralization, and prey growth, resulting in bacterial growth rates averaging 2- to 14-fold higher in the presence of flagellate grazers. These results were obtained using 2-stage, nitrogen-limited chemostats containing a mixed culture of heterotrophic bacteria enriched from seawater and Paraphysomonas bandaiensis, a chrysomonad flagellate. Abundance and biovolume of the flagellates were monitored on a daily basis, as was bacterial abundance. Grazing rates were measured using short-term tracer uptake experiments (fluorescently-labeled bacteria and beads), and these data were used to calculate gross bacterial growth rates in the presence of grazers. A mass balance approach was used to estimate reduced nitrogen regenerated by the protist and nitrogen demand of the heterotrophic bacteria. These independent methods of assessing grazer growth and feeding, coupled with estimates of flagellate gross growth efficiency, provided strong, internally consistent constraints on the estimates of bacterial growth rates in the presence of grazers. Under these culture conditions, P. bandaiensis had a carbon-based gross growth efficiency averaging 28%. This work shows that independently measured grazing rates are essential in protist culture work if system dynamics are to be understood. It also underscores the necessity of including protist remineralization pathways in models if realistic simulations are to be obtained.


Servais P, Casamayor EO, Courties C, Catala P, Parthuisot N, Lebaron P (2003) Activity and diversity of bacterial cells with high and low nucleic acid content. Aquatic Microbial Ecology 33 :41-51

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In most aquatic environments, at least 2 subpopulations of bacterial cells can be discriminated by flow cytometry based on their nucleic acid content. Recent investigations have shown that the cells with a high nucleic acid (HNA) content have a higher cell-specific activity (CSA) cell than those with a low nucleic acid (LNA) content. In this study, the CSA and biomass-specific activities (BSA) of HNA and LNA cells from different aquatic ecosystems, including marine, brackish and freshwater, were investigated using radioactive leucine incorporation and cell sorting by flow cytometry. The genetic diversity of natural assemblages, HNA and LNA cells was investigated using the SSCP (PCR single-strand conformation polymorphism) method. Data showed that both CSA and BSA of HNA cells were always significantly higher than CSA and BSA of LNA cells. In addition, HNA cells had a dominant contribution to the production of the total community (77 to 98%). For the different samples, the SSCP fingerprints from the natural assemblage and from the 2 sorted fractions were not significantly different. This clearly suggests that HNA and LNA subpopulations were composed by the same dominant species and, thus, confirms an important heterogeneity of physiological states within most natural populations.


Sliwinska E (2003) Cell cycle and germination of fresh, dried and deteriorated sugarbeet seeds as indicators of optimal harvest time. Seed Science Research 13 :131-138

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Seeds of sugar beet (Beta vulgaris L.) were collected at weekly intervals from 3 weeks before to 1 week after commercial harvest time, dried and stored at room temperature (18-22degreesC). Laboratory germination tests and flow cytometric analyses were performed immediately after harvest (fresh seeds) and five times at weekly intervals during storage (dry seeds). After 6 months of storage, seeds were exposed to a controlled deterioration treatment (CD). The proportion of G(2) nuclei in the embryo was constant in the fresh seeds, regardless of their maturity. It decreased, however, after drying and CD, especially in those seeds harvested before maturation drying had commenced. The proportion of endosperm cells in the seed decreased with maturation, and a further decrease was observed after drying and CD. These observations suggest that nuclei with a higher nuclear DNA content were more sensitive to water stress caused by premature desiccation and to deterioration than nuclei with a lower DNA content. Fresh seeds exhibited some germination, but this increased after drying, suggesting that desiccation induced a switch from the developmental to the germination mode. Germination percentages were the highest in dry seeds collected at the commercial harvest time and a week after. This high germinability coincided with the highest proportion of G(2) cells in the embryo. It is concluded that flow cytometry provides information about the status of sugarbeet seed maturation, seed quality and storage potential, and can be used for estimation of optimal harvest time.


Sun J, Liu DY (2003) Geometric models for calculating cell biovolume and surface area for phytoplankton. Journal of Plankton Research 25 :1331-1346

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Phytoplankton biovolume can be measured or calculated through the calculation of similar geometric models. A set of geometric models is suggested for calculating cell biovolume and surface area for 284 phytoplankton genera in China Sea waters. Thirty-one geometric shapes have been assigned to estimate the biovolume and surface area of phytoplankton cells. Reductions of error and microscopic effort are also discussed. The model has been verified by its application in the China Seas regions. The software to make these calculations is available at http://www.ouc.edu.cn/csmxy/sunjun/ biovolume.htm


Sunamura M, Maruyama A, Tsuji T, Kurane R (2003) Spectral imaging detection and counting of microbial cells in marine sediment. Journal of Microbiological Methods 53 :57-65

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Semiautomated detection and counting techniques for microbial cells in soil and marine sediment using miwcroscopic-spectral-imaging analysis were developed. Microbial cells in microscopic fields were selectively detected from other fluorescent particles by their fluorescent spectrum, based on the spectral shift between the conjunction and nonconjunction of DNA fluorochrome (SYBR Green II) with nucleic acids. Using this technique, microbial cells could be easily detected in soil and 30cm deep sediment samples from Tokyo Bay, both of which contain particles other than microbial cells. Total cell density was semiautomatically estimated at 1-6 X 10(9) cells cm(-3) of sediment sampled at different depths in Tokyo Bay, which corresponded to 65-106% (mean 88%) of visual direct counting. This technique may be useful for detecting microbial cells in soil and sediment samples from the deeper subsurface environment. (C) 2002 Elsevier Science B.V. All rights reserved.


Toledo G, Palenik B (2003) A Synechococcus serotype is found preferentially in surface marine waters. Limnology and Oceanography 48 :1744-1755

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In marine ecosystems, gradients of light, temperature, and nutrients occur horizontally (coastal to offshore) and vertically. The extent to which microorganisms acclimate or speciate in response to these gradients is under active investigation. Strain isolation data (e.g., site or depth), environmental DNA clone libraries, and preliminary physiology experiments have indicated that marine Synechococcus strain CC9605 might be adapted to the surface oligotrophic ocean. In the present work, we used an immunofluorescent approach to detect the CC9605 serotype in the California Current during September 1998. At two offshore stations, samples were collected along vertical profiles. The relative abundance of the CC9605 serotype was significantly higher in shallow depths within the mixed layer than in deeper depths at the two stations, with maximum values (+/- standard deviation) of 10.3% +/- 6.4 and 28.7% +/- 9.5. Surface samples along an offshore-inshore transect showed higher abundance in the most oligotrophic site (8% +/- 3), compared with almost 1% inshore, but one coastal site also had high relative abundance of the CC9605 serotype (7% +/- 0.5). These data indicate that Synechococcus strains are not uniformly distributed and that some strains, such as CC9605, are more abundant in the mixed layer of the euphotic zone than below the mixed layer.


Tsuji N, Hirayanagi N, Iwabe O, Namba T, Tagawa M, Miyamoto S, Miyasaka H, Takagi M, Hirata K, Miyamoto K (2003) Regulation of phytochelatin synthesis by zinc and cadmium in marine green alga, Dunaliella tertiolecta. Phytochemistry 62 :453-459

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12620358

Although Cd(2+) is a more effective inducer of phytochelatin (PC) synthesis than Zn(2+) in higher plants, we have observed greater induction of PC synthesis by Zn(2+) than Cd(2+) in the marine green alga, Dunaliella tertiolecta. To elucidate this unique regulation of PC synthesis by Zn(2+), we investigated the effects of Zn(2+) and Cd(2+) on the activities of both phytochelatin synthase (PC synthase) and enzymes in the GSH biosynthetic pathway. PC synthase was more strongly activated by Cd(2+) than by Zn(2+), but the difference was not very big. On the other hand, gamma-glutamylcysteine synthetase (gamma-ECS) and glutathione synthetase (GS) were activated by both heavy metals, but their activities were higher in Zn-treated cells than in Cd-treated cells. Dose-dependent stimulation of intracellular reactive oxygen species (ROS) production was observed with Zn(2+), but not Cd(2+) treatment. These results suggest that Zn(2+) strongly promotes the synthesis of GSH through indirect activation of gamma-ECS and GS by stimulating ROS generation. This acceleration of the flux rate for GSH synthesis might mainly contribute to high level PC synthesis.


Vedrine C, Leclerc JC, Durrieu C, Tran-Minh C (2003) Optical whole-cell biosensor using Chlorella vulgaris designed for monitoring herbicides. Biosens Bioelectron 18 :457-463

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12604263

An optical biosensor was designed for determination of herbicides as aquatic contaminants. Detection was obtained with immobilised Chlorella vulgaris microalgae entrapped on a quartz microfibre filter and placed in a five-membrane-home-made-flow cell. The algal chlorophyll fluorescence modified by the presence of herbicides was collected at the tip of an optical fibre bundle and sent to a fluorimeter. A continuous culture was set up to produce algal cells in reproducible conditions for measurement optimisation. Effects of flow rate, algal density, temperature, and pH on the biosensor response to atrazine were studied. Reversibility and detection limits were determined for DNOC and atrazine, simazine, isoproturon, diuron. Detection of photosystem II (PSII) herbicides was achieved at sub-ppb concentration level.


Wang L, Geng M, Li J, Guan H, Ding J (2003) Studies of marine sulfated polymannuroguluronate on endothelial cell proliferation and endothelial immunity and related mechanisms. J Pharmacol Sci 92 :367-373

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12939521

Anti-proliferation action and enhancement of endothelial cell immunity and related mechanisms by marine sulfated polymannuroguluronate (SPMG) were investigated in the present studies. Endothelial cell proliferation was evaluated by MTT assay. Intercellular adhesion molecule-1 (ICAM-1) expression was analyzed by flow cytometry. The interaction of SPMG with basic fibroblast growth factor (bFGF) was evaluated by surface plasmon resonance. Results showed that SPMG exhibited a significant inhibitory effect against proliferation in both normal human umbilical vein endothelial cells (HUVEC) and bFGF-treated HUVEC, the action of which was completely abrogated by bFGF antibody. SPMG exerted high affinity to bFGF in a multivalent pattern, characterized by one molecule SPMG binding to 3 - 4 molecules of bFGF. Moreover, SPMG enhanced ICAM-1 expression in HUVEC and prevented and restored bFGF-treated downregulation of ICAM-1 expression in HUVEC, the expression of which was not counteracted by bFGF antibody. In conclusion, this is the first report demonstrating that SPMG exerted an anti-proliferation effect dependent on the bFGF-regulated pathway and afforded upregulatory activity on ICAM-1 expression regardless of the involvement of bFGF.


Wang LC, Geng MY, Li J, Guan HS, Ding J (2003) Studies of marine sulfated polymannuroguluronate on endothelial cell proliferation and endothelial immunity and related mechanisms. Journal of Pharmacological Sciences 92 :367-373

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Anti-proliferation action and enhancement of endothelial cell immunity and related mechanisms by marine sulfated polymannuroguluronate (SPMG) were investigated in the present studies. Endothelial cell proliferation was evaluated by MTT assay. Intercellular adhesion molecule-1 (ICAM-1) expression was analyzed by flow cytometry. The interaction of SPMG with basic fibroblast growth factor (bFGF) was evaluated by surface plasmon resonance. Results showed that SPMG exhibited a significant inhibitory effect against proliferation in both normal human umbilical vein endothelial cells (HUVEC) and bFGF-treated HUVEC, the action of which was completely abrogated by bFGF antibody. SPMG exerted high affinity to bFGF in a multivalent pattern, characterized by one molecule SPMG binding to 3 - 4 molecules of bFGF. Moreover, SPMG enhanced ICAM-1 expression in HUVEC and prevented and restored bFGF-treated downregulation of ICAM-1 expression in HUVEC, the expression of which was not counteracted by bFGF antibody. In conclusion, this is the first report demonstrating that SPMG exerted an anti-proliferation effect dependent on the bFGF-regulated pathway and afforded upregulatory activity on ICAM-1 expression regardless of the involvement of bFGF.


Whiteley AS, Griffiths RI, Bailey MJ (2003) Analysis of the microbial functional diversity within water-stressed soil communities by flow cytometric analysis and CTC plus cell sorting. Journal of Microbiological Methods 54 :257-267

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Total and active cell counts within soil samples were determined by culture-independent methods using flow cytometry and preparative Nycodenz gradient centrifugation. Whole cells were purified from soil cores and total extractable cell counts assessed by SYBR Green 11 fluorescence, while active cell counts were determined by 5-cyano-2,3-ditolyl tetrazolium chloride reduction (CTC+ cells). Parallel microcosms, maintained at either field water capacity or subjected to drying, indicated that the total extractable cell count remained between 10(8) and 10(9) g(-1) (dry weight). In contrast, the CTC+ active count fell threefold in dried microcosms (6% of total cell count) when compared to wetted microcosms (18% of total cell count). Specifically, these data highlighted an overall deactivation of microbial biomass during water stress, with 16S rDNA analyses of flow-sorted CTC+ cells demonstrating shifts within the active diversity. Flow cytometry coupled with cell purification techniques represents a significant tool for operationally defining an active and redundant microbial component within soil communities and is demonstrated during water stress. (C) 2003 Elsevier Science B.V. All rights reserved.


Worden AZ, Binder BJ (2003) Growth regulation of rRNA content in Prochlorococcus and Synechococcus (marine cyanobacteria) measured by whole-cell hybridization of rRNA-targeted peptide nucleic acids. Journal of Phycology 39 :527-534

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The cyanobacteria Synechococcus and Prochlorococcus are important primary producers in marine ecosystems. Because currently available approaches for estimating microbial growth rates can be difficult to apply in the field, we have been exploring the feasibility of using quantitative rRNA measurements as the basis for making such estimates. In this study we examined the relationship between rRNA and growth rate in several Synechococcus and Prochlorococcus strains over a range of light-regulated growth rates. Whole-cell hybridization with fluorescently labeled peptide nucleic acid (PNA) probes was used in conjunction with flow cytometry to quantify rRNA on a per cell basis. This PNA probing technique allowed rRNA analysis in a phycoerythrin-containing Synechococcus strain (WH7803) and in a non-phycoerythrin-containing strain and in Prochlorococcus . All the strains showed a qualitatively similar tri-phasic relationship between rRNA.cell(-1) and growth rate, involving relatively little change in rRNA.cell(-1) at low growth rates, linear increase at intermediate growth rates, and a plateau and/or decrease at the highest growth rates. The onset of each phase was associated with the relative, rather than absolute, growth rate of each strain. In the Synechococcus strains, rRNA normalized to flow cytometrically measured forward angle light scatter (an indicator of size) was well-correlated with growth rate across strains. These findings support the idea that cellular rRNA may be useful as an indicator of in situ growth rate in natural Synechococcus and Prochlorococcus populations.


Zubkov MV, Fuchs BM, Tarran GA, Burkill PH, Amann R (2003) High rate of uptake of organic nitrogen compounds by Prochlorococcus cyanobacteria as a key to their dominance in oligotrophic oceanic waters. Appl Environ Microbiol 69 :1299-1304

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12571062

Direct evidence that marine cyanobacteria take up organic nitrogen compounds in situ at high rates is reported. About 33% of the total bacterioplankton turnover of amino acids, determined with a representative [(35)S]methionine precursor and flow sorting, can be assigned to Prochlorococcus spp. and 3% can be assigned to Synechococcus spp. in the oligotrophic and mesotrophic parts of the Arabian Sea, respectively. This finding may provide a mechanism for Prochlorococcus’ competitive dominance over both strictly autotrophic algae and other bacteria in oligotrophic regions sustained by nutrient remineralization via a microbial loop.


Zubkov MV, Lopez-Urrutia A (2003) Effect of appendicularians and copepods on bacterioplankton composition and growth in the English Channel. Aquatic Microbial Ecology 32 :39-46

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We compared the effects of the presence of the appendicularian Oikopleura dioica and the copepods Acartia clausii and Calanus helgolandicus on the coastal bacterioplankton community off Plymouth. Mesozooplankton were added to water samples and bacterioplankton growth was monitored by flow cytometry. Phylogenetic composition of bacterioplankton was analysed using fluorescence in situ hybridisation (FISH) with rRNA-targeted oligonucleotide probes. The bacterioplankton composition did not change in the presence of either appendicularians or copepods, and generally the same proportions of bacterioplankton groups were determined. In late spring, 15 +/- 2% of cells hybridised with a probe specific to the Kingdom Archaea. The majority of cells (88 +/- 2%) belonged to the Kingdom Bacteria, and 86% of cells were identified using group-specific probes. The Cytophage-Flavobacterium cluster dominated the community, comprising 64 +/- 0.5% of cells. The gamma-proteobacteria were the second abundant group, comprising 11 +/- 0.5% of cells, and the SAR86 cluster of gamma-proteobacteria accounted for 6 +/- 5%. The alpha-proteobacteria comprised 10 +/- 5% of bacterioplankton, and the Roseobacteria related cluster represented 9 +/- 3% of cells. The reduction of bacterioplankton growth caused by appendicularian bacterivory was 0.4 to 14% ind.(-1) l(-1), and the total appendicularian population could reduce bacterial growth in coastal waters in late spring-summer by up to 9%. In contrast to the appendicularians the copepods stimulated bacterial growth, and in summer the bacterioplankton growth may be increased by up to 13% by the combined effect of dominant copepod populations. Thus, the appendicularians and copepods had an opposite but moderate effect on the bacterioplankton growth and no effect on the bacterioplankton composition.


Zubkov MV, Quartly GD (2003) Ultraplankton distribution in surface waters of the Mozambique Channel - flow cytometry and satellite imagery. Aquatic Microbial Ecology 33 :155-161

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The composition of ultraplankton (UP) in near-surface samples collected underway every 1 to 6 h from a ship sailing from Durban to the Seychelles was determined by flow cytometry, using both autofluorescence pigments and fluorescence DNA staining. Prochlorococcus (Pro) (17 to 160 x 10(3) cells ml(-1)) numerically dominated the ultraphytoplankton (UPP), followed by Synechococcus (Syn) (4.5 to 57 x 10(3) cells ml(-1)) and eukaryotic algae (EA) (0.6 to 4.2 x 10(3) cells ml(-1)). The abundance of heterotrophic bacterioplankton (HB) was 0.4 to 1.3 x 10(6) cells ml(-1). A strong correlation (r = 0.8 to 0.97) was observed between sea-viewing wide field of view sensor (SeaWiFS) satellite estimates of total chlorophyll a (chl a) concentration and chl a concentration, abundance and biomass of EA as well as abundance and biomass of HB. This shows the potential for deducing spatial distributions of these 2 groups for ecosystem modelling using satellite data. Although the correlation between satellite chl a estimates and Syn chl a concentration was strong (r = 0.83 to 0.88), the correlation with its abundance and biomass was poor (r < 0.6) due to high variability (factor of 12) in cellular chl a content and to a lesser extent to diurnal cycles. The relationships were similar when either only daytime or all UP measurements were compared with the satellite data. No relationship was found between satellite data and Pro chl a concentration, abundance or biomass, even after correction for a pronounced diel cycle, suggesting that the SeaWiFS instrument might not detect Pro chl a.