Apparent Indispensability of Bacteria in Foraminiferan Nutrition

SYNOPSIS. Bacteria were required for the sustained reproduction of 4 species of foraminifera in gnotobiotic culture. None of the species of algae tested, singly or in combination, supported continuous reproduction of the foraminifera in bacteria-free gnotobiotic culture. It is inferred that bacteria have some nutritional factor required by the foraminifera that is either unavailable or unavailable in sufficient quantity in an exclusively algal diet.
Gnotobiotic clones of Quinqueloculina lata, Spiroloculina hyalina, Rosalina leei , and Allogromia laticollaris were established on bacteria + algae (usually 1 or 2 species). In balanced gnotobiotic cultures neither light nor foraminiferan density (organisms/ml) were limiting. As cultures aged, pH shifted and limited growth. When waste products were removed by washing, reproductive rates were higher.     

FLOW CYTOMETRIC APPLICABILITY OF FLUORESCENT VITALITY PROBES ON PHYTOPLANKTON1

The applicability of six fluorescent probes (four esterase probes: acetoxymethyl ester of Calcein [Calcein‐AM], 5‐chloromethylfluorescein diacetate [CMFDA], fluorescein diacetate [FDA], and 2′,7′‐dichlorofluorescein diacetate [H₂DCFDA]; and two membrane probes: bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and SYTOX‐Green) as vitality stains was tested on live and killed cells of 40 phytoplankton strains in exponential and stationary growth phases, belonging to 12 classes and consisting of four cold‐water, 26 temperate, and four warm‐water species. The combined live/dead ratios of all six probes indicated significant differences between the 12 plankton classes (P < 0.01) and between individual species (P < 0.05). No specific differences were observed among strains of one species, among species or strains from different origin, nor between cells in exponential and stationary growth phase except for FDA. FDA showed a significant (P < 0.05) drop of <20% in fluorescence intensity in stationary cells. Of the four esterase probes, the live/dead ratios of FDA and CMFDA were not significantly different from each other, and both performed better than Calcein‐AM and H₂DCFDA (P < 0.001). Of the two membrane probes, DIBAC₄(3) stained rhodophytes and euglenophytes much better than SYTOX‐Green. The 13 algal strains best stainable (high live/dead ratios) among all six probes belonged to nine genera from six classes of phytoplankton. In conclusion, FDA, CMFDA, DIBAC₄(3), and SYTOX‐Green represent a wide choice of vitality probes in the study of phytoplankton ecology, applicable in many species from different algal classes, originating from different regions and at different stages of growth.     

Molecular evidence that Reticulomyxa filosa is a freshwater naked foraminifer

Reticulomyxa filosa is a freshwater protist possessing fine granular, branching and anastomosing pseudopodia and therefore traditionally placed in the class Granuloreticulosea, order Athalamida, as a sister group to the order Foraminiferida. Recent studies have revealed remarkable similarities in pseudopodial motility and ultrastructure between R. filosa and foraminifera (e.g. Allogromia laticollaris), prompting us to conduct a molecular phylogenetic analysis of these seemingly disparate organisms. We sequenced the complete small-subunit of the ribosomal DNA of the cultured strain of R. filosa and compared it to the corresponding sequences of other protists including 12 species of foraminifera. We also sequenced and analyzed the actin coding genes from R. filosa and two species of foraminifera, Allogromia sp. and Ammonia sp. The analysis of both data sets clearly shows that R. filosa branches within the clade of foraminifera, suggesting that R. filosa is in fact a freshwater naked foraminiferan.     

An increase in model lipid membrane fluidity as a result of local anesthetic action

The effect of local anesthetics on the permeability of phospholipid liposomes of different composition for calcein has been investigated. The local anesthetics tested included amides (lidocaine, prilocaine, mepivacaine, and bupivacaine) and esters (benzocaine, procaine, and tetracaine). The permeability of large monolamellar liposomes was assessed by monitoring the fluorescence of calcein leaking from the phospholipid vesicles. All tested amide anesthetics exerted negligible effects on the permeability of dioleylphosphocholine (DOPC) liposomes for the fluorescent marker. The most efficient in this group was did bupivacaine. Amides had a more pronounced effect on membranes in which 20 mol % of DOPC was replaced by tetraoleoylcardiolipin (TOCL). Benzocaine and procaine at concentration up to 100 mM did not affect the permeability of DOPC liposomes. Membrane permeability of DOPC liposomes was not affected by the addition of tetracaine to the final concentration of 2 mM, while the increase of anesthetic concentration up to 50 mM was accompanied by an increase in the intensity of fluorescence of calcein released from the vesicles, and addition of the anesthetic to the concentration of 100 mM caused by complete release of the marker incorporated by the liposomes. The threshold concentration of tetracaine initiating calcein leakage from vesicles that contained 20 mol % TOCL was 7 mM, and the concentration corresponding to 100% calcein leakage was 20 mM. Confocal fluorescence microscopy of giant monolamellar liposomes formed from an equimolar mixture of DOPC and tetramiristoylcardiolipin demonstrated the destruction of solid ordered domains at the presence of anesthetics, and its destructive capacity increasing in the following order: procaine ≈ mepivacaine < bupivacaine ? tetracaine. Variability of the depth of anesthetic incorporation into the membrane may account for the dissimilar effects of local anesthetics on liposomes.     

Trophic Dynamics and Niches of Salt Marsh Foraminifera

Energetic considerations of the growth of three species of littoralbenthic foraminifera, Allogromia laticollaris, Rosalina leei,and Spiroloculina hyalina, have been made on laboratory-grownpopulations. Under optimum laboratory conditions A. laticollarishas the greatest intrinsic rate of increase (r = 2.533 org/day);S. hyalina (r = 1.472 org/day), and R. leei (r = 0.272 org/day)being less fecund. The respiration rates of the three specieswere similar (0.5–4.5 µ1/mg body wt/hr) within thetemperature range (15–35 C) tested. The species studiedare selective feeders. Only 4-5 of 28 species of algae testedwere consumed in significant quantities (40-150 x 10⁸ g/foram/day).Although great numbers of bacteria were eaten, their biomasswas negligible when compared to the algae. The ecological growthefficiency (E_e) of the three species tested is highest in freshcultures (5-20%) and declines rapidly. Evidence suggests thatthe species studied are well adapted for the rapid changes inthe microbial community structure which take place throughoutthe summer, and that community stability and high rate of productivityare achieved through diversity.     

Interaction of melittin with a model membrane in the presence of antibodies]

The method of fluorescence spectrofluorimetry has been applied to study the interaction of melittin at high and low ionic strength with the phosphatidylcholine model membrane in the presence of monospecific polyclonal and monoclonal antibodies against this peptide. The formation of antigen-antibody complex with the excess of the antigen is shown to decrease the leakage of calcein, a fluorescence dye. With the excess of antibodies the dye leakage is completely suppressed. This effect does not depend on the state of peptide aggregation before binding to the membrane. It is suggested that melittin antigenic determinants are located on the sites of peptide molecule which are necessary for its interaction with the membrane.     

A new model of reticulopodial motility and shape: evidence for a microtubule-based motor and an actin skeleton

Cytoskeletal inhibitors were used as probes to test the involvement of microtubules and actin microfilaments in the development, motility, and shape maintenance of the pseudopodial networks (i e, reticulopodia) of the foraminifers Allogromia sp strain NF and Allogromia laticollaris. Agents that disassemble cytoplasmic microtubules (cold, colchicine, and nocodazole) arrest all movement but have variable effects on reticulopodial shape. Electron microscopy reveals a granulofibrillar matrix but few, if any, microtubules in these motility-arrested reticulopods. Allogromiids treated with cytochalasin B or D lose substrate adhesion and undergo dramatic changes in shape and motile behavior, highlighted by the coalescence of reticulopodial cytoplasm into irregularly shaped bodies with chaotic motility. Serial semithick sections of such preparations, viewed by high-voltage electron microscopy, document a striking rearrangement of microtubules within these cytochalasin-induced bodies. All aspects of cytochalasin-altered motility are completely inhibited by colchicine. Actin is present in reticulopodia, as determined by staining with rhodamine-phalloidin; this staining is not observed in cytochalasin-treated organisms. These data provide compelling evidence that microtubules are required for reticulopodial motility. An actin-based cytoskeleton is thought to play a role in maintaining shape, mediating pseudopod/substrate adhesion, and coordinating the various microtubule-dependent processes.     

Bactericidal Activity of Curcumin I Is Associated with Damaging of Bacterial Membrane

Curcumin, an important constituent of turmeric, is known for various biological activities, primarily due to its antioxidant mechanism. The present study focused on the antibacterial activity of curcumin I, a significant component of commercial curcumin, against four genera of bacteria, including those that are Gram-positive (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa). These represent prominent human pathogens, particularly in hospital settings. Our study shows the strong antibacterial potential of curcumin I against all the tested bacteria from Gram-positive as well as Gram-negative groups. The integrity of the bacterial membrane was checked using two differential permeabilization indicating fluorescent probes, namely, propidium iodide and calcein. Both the membrane permeabilization assays confirmed membrane leakage in Gram-negative and Gram-positive bacteria on exposure to curcumin I. In addition, scanning electron microscopy and fluorescence microscopy were employed to confirm the membrane damages in bacterial cells on exposure to curcumin I. The present study confirms the broad-spectrum antibacterial nature of curcumin I, and its membrane damaging property. Findings from this study could provide impetus for further research on curcumin I regarding its antibiotic potential against rapidly emerging bacterial pathogens.     

Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies.

Calcein is a fluorescent probe that is widely used in studies of cell viability and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves the generation of reactive oxygen species (ROS). The photooxidation properties of calcein in solution were studied in the presence of histidine and tryptophan as oxidizable substrates. The photodegradation of histidine was mainly mediated by singlet oxygen (1O2), as shown by the inhibitory effect of sodium azide, a specific 1O2 scavenger. On the other hand, mixed photosensitization mechanisms were present when tryptophan was used as the target of the calcein-stimulated photoprocess. In addition to 1O2, hydroxyl radicals and hydrogen peroxide were involved as reactive species, as shown by using mannitol and catalase as scavengers. The calcein-photosensitized alterations of mitochondria as a potential source of artifacts in confocal microscopy studies of cells were considered. Irradiation of isolated mitochondria with visible light (500-600 nm) in the presence of calcein induced opening of the permeability transition (PT) pore. The extent of the mitochondrial membrane photodamage, however, was modulated by the nature of the calcein environment. Thus, pore opening was triggered at short irradiation times and low dye concentrations when calcein was dissolved in the bulk medium. On the contrary, calcein concentrated in the matrix space was rather inefficient as photosensitizer even at concentrations 10 times higher than those present in the external medium.     

Identification of Dekkera bruxellensis (Brettanomyces) from wine by fluorescence in situ hybridization using peptide nucleic acid probes

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.     

Cytological Observations of the Large Symbiotic Foraminifer Amphisorus kudakajimensis Using Calcein Acetoxymethyl Ester

Large benthic foraminifera are unicellular calcifying reef organisms that can form symbiotic relationships with a range of different microalgae. However, the cellular functions, such as symbiosis and calcification, and other aspects of cellular physiology in large benthic foraminifera are not fully understood. Amphisorus kudakajimensis was used as a model to determine the detailed cellular characteristics of large benthic foraminifera. We used calcein acetoxymethyl ester (calcein AM) as a fluorescent indicator for live confocal imaging. We demonstrated that calcein AM is a useful fluorescent indicator to stain the fine network of reticulopodia and the cytoplasm in living A. kudakajimensis. We showed that at least two types of reticulopodia exist in A. kudakajimensis: the straight bundle of reticulopodia that spreads from the aperture and the fine reticulopodia along the surface of the aperture and chamber walls. The cytoplasm in outer chambers was highly branched and contained a few dinoflagellates. In contrast, the inner chamberlets contained condensed cytoplasm and many dinoflagellates, suggesting that the cytoplasm of A. kudakajimensis performs different functions based on its location within the large test. Our confocal detailed image analysis provides real-time cellular morphology and cell physiology of living foraminifera.     

Development of a molecular bioswitch using fluorescence lifetime imaging: Incremental activation of fluorescein diacetate

Molecular bioswitches offer an invaluable asset in the shift from systemic to targeted treatments. Within the growing arsenal of switches are imaging probes that functionalize only in given locations or situations. Acetate esters are a common fluorescent example, known to activate upon interaction with esterases. Fluorescein diacetate (FDA) is one such fluorophore used in cell viability assays. These assays rely on the fact that the compound begins colorless and with no fluorescent signature whatsoever, and only after internalization into cells it is possible to detect a fluorescence signal. In this study, using fluorescence intensity (FI) and fluorescence lifetime (FLT) imaging, FDA is shown to be fluorescent even when unactivated. Furthermore, the FLT is shown to change with pH. Finally, the ability to image FDA in different environments simulated by tissue‐imitating phantoms is explored. Altogether, the ability of FDA to serve as a bioswitch when measured using FLT imaging microscopy (FLIM) is assessed. The combination of a spectrum of FDA activation and FLIM serves as a bioswitch, where biologically relevant stimulation can generate detectable and incremental variations.      

Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5‐chloromethylfluorescein diacetate

Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5‐chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat‐killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per‐cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live‐dead classification was essentially error free. But overlap between the frequency distributions of living and heat‐killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat‐killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8–10 of 24 species (i.e., 33%–42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone.     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.     

The induction of apoptosis by methotrexate in activated lymphocytes as indicated by fluorescence hyperpolarization: a possible model for predicting methotrexate therapy for rheumatoid arthritis patients

The objectives of this study were to test the in vitro response of healthy non-activated, activated, and rheumatoid arthritis (RA) lymphocytes to methotrexate (MTX), and design an in vitro model for predicting the efficiency of MTX treatment for RA patients. Considering the RA profile of clonal-expanded CD4(+) T cells, phytohemagglutinin-activated mononuclear cells taken from healthy donors were incubated with different concentrations of MTX. The MTX-immunosuppressive effect was tested by fluorescence intensity measurements, including PI assay and annexin V assay. For simple detection, we used the Individual Cell Scanner (IC-S), which enables the measurement of early events in individual cells. Healthy mononuclear cells (MNC), and MNC derived from RA patients, were tested by the IC-S while utilizing fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) as an established marker of activation or suppression. In healthy activated MNC, we found that MTX, through its early incubation period, interferes with the activation signal obtained by PHA and exerts an apoptotic signal, which is noted by increases in the FP. Comparing our model to six long-standing RA patients and five newly-diagnosed patients revealed significant differences in the FP measurements, including fluorescence depolarization as an early established measurement of lymphocyte activation, and hyperpolarization as a measurement of an early immunosuppressive effect. We conclude that MTX, an effective therapy for RA patients, could easily be tested by fluorescence polarization measurements of FDA before (or during) clinical use in order to predict its efficiency on a specific RA patient. Moreover, the FP measurements can be used for the diagnosis, and making timing and dosage decisions.     

DBD dyes as fluorescent probes for sensing lipophilic environments

Small fluorescent organic molecules based on [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) could be used as probes for lipophillic microenvironments in aqueous solutions by indicating the critical micelles concentration of detergents and staining cell organelles. Their fluorescence lifetime decreases drastically by the amount of water in their direct environment. Therefore they are potential probes for fluorescence lifetime imaging microscopy (FLIM).     

Analysis of antiphotobleaching reagents for use with FluoroNanogold in correlative microscopy.

Correlative microscopy is an important approach for bridging the resolution gap between fluorescence and electron microscopy. We have employed FluoroNanogold (FNG) as the detection system in these types of studies. This immunoprobe consists of a gold cluster compound to which a fluorochrome-labeled antibody is covalently linked. In these preparations, the fluorescence signal from FNG is first recorded then the gold cluster compound is subjected to a silver enhancement reaction before examination by electron microscopy. Potential complications are those associated with photochemical reactions that occur during fluorescence microscopy. We have evaluated this and some anti-photobleaching agents (i.e., 1,4-diazabicyclo[2.2.2]octane [DABCO],p-phenylenediamine [PPD], and N-propyl gallate [NPG]) for their utility with FNG in correlative microscopy. When DABCO was employed, the gold signal from FNG was dramatically diminished but the fluorescence signal was unaffected. The gold signal of DABCO-treated samples decreased to approximately 30% of that of the other samples. On the other hand, PPD and NPG did not adversely affect the FNG labeling. We recommend that either PPD or NPG be used and that DABCO be avoided as an antiphotobleaching reagent for this technique.     

Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123.

To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium. This work tests the hypothesis that dihydrofluorescein diacetate is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. To investigate this, dihydrofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO2], peroxynitrite, nitric oxide, horseradish peroxidase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and compared with the three other popular probes. Though oxidized dihydrofluorescein has higher molar fluorescence, comparison of the reactions of dihydrofluorescein with these other three probes in a cell-free system indicates that dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very complex. Based on the results reported here, it is no longer appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2, but rather as detectors of a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. Cell-loading studies indicate that dihydrofluorescein achieves higher intracellular concentrations than the second brightest intracellular probe, 2',7'-dichlorodihydrofluorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies.     

Evaluation of alkaline phosphatase- and digoxigenin-labelled probes for detection of the thermolabile hemolysin (tlh) gene of Vibrio parahaemolyticus

The biochemical identification and enumeration of Vibrio parahaemolyticus as described in the FDA Bacteriological Analytical Manual is expensive and labour-intensive. To reduce the time and effort necessary to verify the identity of V. parahaemolyticus, the use of a thermolabile haemolysin (tlh) gene probe is proposed. An alkaline phosphatase (AP)-labelled probe was evaluated for specificity against 26 strains of V. parahaemolyticus, 88 strains of other Vibrio species and 10 strains of non-vibrio species. Of the 124 isolates tested, the probe hybridized only with the 26 strains of V. parahaemolyticus, indicating species specificity. Two hundred and six suspect V. parahaemolyticus isolates from oysters were tested by this probe and API-20E diagnostic strips; there was 97% agreement between results. A digoxigenin (DIG)-labelled probe for detection of the tlh gene fragment was prepared by PCR and compared with the AP-labelled probe. When tested on 584 suspect V. parahaemolyticus isolates, results obtained with the AP- and DIG-labelled probes were in 98% agreement. These results suggest that the probes are equivalent for detection of the V. parahaemolyticus tlh gene.     

A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation

Global fitting algorithms have been shown to improve effectively the accuracy and precision of the analysis of fluorescence lifetime imaging microscopy data. Global analysis performs better than unconstrained data fitting when prior information exists, such as the spatial invariance of the lifetimes of individual fluorescent species. The highly coupled nature of global analysis often results in a significantly slower convergence of the data fitting algorithm as compared with unconstrained analysis. Convergence speed can be greatly accelerated by providing appropriate initial guesses. Realizing that the image morphology often correlates with fluorophore distribution, a global fitting algorithm has been developed to assign initial guesses throughout an image based on a segmentation analysis. This algorithm was tested on both simulated data sets and time-domain lifetime measurements. We have successfully measured fluorophore distribution in fibroblasts stained with Hoechst and calcein. This method further allows second harmonic generation from collagen and elastin autofluorescence to be differentiated in fluorescence lifetime imaging microscopy images of ex vivo human skin. On our experimental measurement, this algorithm increased convergence speed by over two orders of magnitude and achieved significantly better fits.