Epifluorescence Microscopy Imaging Research Articles

High cellular organization of pyoverdine biosynthesis in Pseudomonas aeruginosa: clustering of PvdA at the old cell pole

Pyoverdine I (PVDI) is the major siderophore produced by Pseudomonas aeruginosa PAO1 to import iron. Its biosynthesis requires the coordinated action of cytoplasmic, periplasmic and membrane proteins. The individual enzymatic activities of these proteins are well known. However, their subcellular distribution in particular areas of the cytoplasm, periplasm, or within the membrane has never been investigated. We used chromosomal replacement to generate P.aeruginosa strains producing fluorescent fusions with PvdA, one of the initial enzymes in the biosynthetic pathway of PVDI in the cytoplasm, and PvdQ, involved in the maturation of PVDI in the periplasm. Cellular fractionation indicated that a substantial amount of PvdA-YFP was located in the membrane fraction. Epifluorescence microscopy imaging showed that PvdA-YFP was mainly clustered at the old cell pole of bacteria, indicating a polar segregation of the protein. Epifluorescence and TIRF imaging on cells expressing labelled PvdQ showed that this enzyme was uniformly distributed in the periplasm, in contrast with PvdA-YFP. The description of the intracellular distribution of these enzymes contributes to the understanding of the PVDI biosynthetic pathway.

Microfluidic centrifuge based on a counterflow configuration

We present a microfluidic centrifuge with no moving parts, relying on a vortex formed between two counterflowing liquid streams. The centrifuge is driven by streams with a speed of 0.6–2.6 m/s, resulting in accelerations applied to samples between 50 and 2,000 g. The liquid flow in the centrifugation chamber and the transport of microparticles are visualized using epi-fluorescence microscopy and bright-field imaging with a high-speed camera. It is found that small particles follow the streamlines of the flow, whereas larger particles show a cross-stream migration. The size separation of different particles is demonstrated, and the experiments clearly indicate that as the flow speed increases, the particles in the vortex are increasingly driven outwards. Per construction, the centrifuge is ideally suited for handling small sample amounts and can be integrated with lab-on-a-chip systems.

Cardiolipin microdomains localize to negatively curved regions of Escherichia coli membranes.

Many proteins reside at the cell poles in rod-shaped bacteria. Several hypotheses have drawn a connection between protein localization and the large cell-wall curvature at the poles. One hypothesis has centered on the formation of microdomains of the lipid cardiolipin (CL), its localization to regions of high membrane curvature, and its interaction with membrane-associated proteins. A lack of experimental techniques has left this hypothesis unanswered. This paper describes a microtechnology-based technique for manipulating bacterial membrane curvature and quantitatively measuring its effect on the localization of CL and proteins in cells. We confined Escherichia coli spheroplasts in microchambers with defined shapes that were embossed into a layer of polymer and observed that the shape of the membrane deformed predictably to accommodate the walls of the microchambers. Combining this technique with epifluorescence microscopy and quantitative image analyses, we characterized the localization of CL microdomains in response to E. coli membrane curvature. CL microdomains localized to regions of high intrinsic negative curvature imposed by microchambers. We expressed a chimera of yellow fluorescent protein fused to the N-terminal region of MinD--a spatial determinant of E. coli division plane assembly--in spheroplasts and observed its colocalization with CL to regions of large, negative membrane curvature. Interestingly, the distribution of MinD was similar in spheroplasts derived from a CL synthase knockout strain. These studies demonstrate the curvature dependence of CL in membranes and test whether these structures participate in the localization of MinD to regions of negative curvature in cells.

Galactose modified iNKT cell agonists stabilised by a novel structural modification of CD1d lead to marked Th1 polarisation in vivo

Background and objectivesInvariant natural killer T (iNKT) cells are a subset of highly conserved regulatory cells with features of innate and adaptive immunity. They possess a wide range of effector...

Irradiation Leads to Disturbed Excitation-Contraction Coupling in Cardiac Myocytes Through ROS-Dependent CaMKII Activation

Introduction: Chest radiotherapy is part of therapeutic concepts for malignant diseases. However, radiation-induced cardiotoxic effects have become clinically relevant. The underlying pathomechanisms are poorly understood.Hypothesis: We investigated whether excitation-contraction coupling in cardiac myocytes, which depends on intact intracellular Ca cycling, may be directly affected by irradiation (IR).Methods: Isolated ventricular mouse myocytes were exposed to graded IR (0 Gy sham IR, 4 Gy and 20 Gy). Ca and Na handling properties and intracellular reactive oxygen species (ROS) levels were measured using epifluorescence microscopy (Fura-2, SBFI and CMH2-DCFDA, respectively) and confocal Ca (Fluo-4) imaging. Trypan blue staining indicated cellular injury. Western Blots revealed protein phosphorylation levels.Results: IR increased systolic sarcoplasmic reticulum (SR) Ca release leading to an acute positive-inotropic effect with Ca transients of 0.21±0.01 ratio units (r.u., F340nm/F380nm) at 20 Gy (N=89) vs. 0.15±0.01 r.u. in untreated sham control myocytes (N=98, P<0.05 using one-way ANOVA). Although SR Ca content was unaltered, an increased diastolic SR Ca leak measured as Ca sparks (752±113 Ca sparks∗pL−1∗s−1 at 20 Gy, N=32 vs. 208±34 sparks∗pL−1∗s−1 at 0 Gy, N=12, P<0.05 using one-way ANOVA) was accompanied by diastolic Ca overload and increased cellular injury. Furthermore, a rise in intracellular Na from 0.76± 0.01 r.u. (N=27) at 0 Gy to 0.79± 0.01 (N=39, P<0.05 using one-way ANOVA) at 20 Gy was observed after IR.IR-dependent elevation of ROS levels (by ∼667% at 20 Gy) contributed to Ca/calmodulin-dependent protein kinase II (CaMKII) activation. CaMKII-inhibition and ROS-scavenging prevented IR-dependent Ca overload, cellular dysfunction, and cell death.Conclusions: IR severely disturbs cardiac Ca handling and decreases myocyte viability. As underlying pathomechanism, a ROS/CaMKII signaling pathway was identified.

An image-based 384-well high-throughput screening method for the discovery of biofilm inhibitors in Vibrio cholerae

Bacterial biofilms are assemblages of bacterial cells and extracellular matrix that result in the creation of surface-associated macrocolony formation. Most bacteria are capable of forming biofilms under suitable conditions. Biofilm formation by pathogenic bacteria on medical implant devices has been linked to implant rejection in up to 10% of cases, due to biofilm-related secondary infections. In addition, biofilm formation has been implicated in both bacterial persistence and antibiotic resistance. In this study, a method has been developed for the discovery of small molecule inhibitors of biofilm formation in Vibrio cholerae, through the use of high-throughput epifluorescence microscopy imaging. Adaptation of a strategy for the growth of bacterial biofilms in wellplates, and the subsequent quantification of biofilm coverage within these wells, provides the first example of an image-based 384-well format system for the evaluation of biofilm inhibition in V. cholerae. Application of this method to the high-throughput screening of small molecule libraries has lead to the discovery of 29 biofilm lead structures, many of which eliminate biofilm formation without altering bacterial cell viability.

38 DNA METHYLATION IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

DNA de- and re-methylation are crucial for reprogramming of the differentiated parental/somatic genome in the ooplasm. The presented research was aimed at analysis of the DNA methylation dynamics in porcine preimplantation embryos developed in vivo (IV) and produced in vitro by IVF, somatic cell nuclear transfer (SCNT), and parthenogenetic activation (PA). Embryos of early and late 1-cell, 2-, 4-, and 8-cell, and early and late blastocysts stages obtained by the mentioned methods were fixed in 4% paraformaldehyde and subjected to immunocytochemistry using anti-5MetC (Mouse monoclonal, Abcam, Cambridge, MA, USA) antibody. DNA was labelled using Hoechst 33258 (Sigma, Copenhagen, Denmark). Epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) images were subjected to NIH imageJ software to measure the DNA methylation/DNA content signal by manually outlining the nuclei (n = 2003) of the embryos. The data were analysed using PROC-GLM statistical procedure in SAS 9.1 (SAS Institute Inc., Cary, NC, USA), least square means were compared and P-values were used to decide the significant differences within and between different groups of embryos. The 1-cell stages lacked active demethylation of paternal genome in IV and IVF embryos. Embryos produced under in vitro conditions presented higher levels of DNA methylation than IV. A lineage specific DNA methylation (hypermethylation of inner cell mass and hypomethylation of trophectoderm) observed in porcine IV late blastocysts was absent in PA and SCNT blastocysts despite the occurrence of de novo methylation in early blastocysts. SCNT early (50%) and late (14%) blastocysts presented DNA methylation pattern similar to IV early and late blastocysts, respectively. Concluding, DNA methylation patterns are strongly impaired under in vitro conditions in porcine preimplantation embryos.

A novel sucrose porogen‐based solid freeform fabrication system for bone scaffold manufacturing

PurposeMusculoskeletal conditions are a major health concern in the USA because of a large aging population and increased occurrence of sport‐related injuries. Bone tissue engineering may offer a less painful alternative to traditional bone grafts with lower risk of infection. The purpose of this paper is to present a novel porogen‐based fabrication system for tissue engineering scaffolds using sucrose (C12H22O11) as the porogen building material.Design/methodology/approachA new solid freeform fabrication system has been developed and tested, which uses pressurized extrusion to print highly biocompatible and water soluble sucrose bone scaffold porogens (or negtives). Polycaprolactone (PCL) scaffolds are manufactured by injecting molten polymer into the porogens, and the porogens are subsequently dissolved with water. The resultant scaffolds demonstrate the defined porous structure designed into the sucrose porogen manufacturing computer‐aided design model.FindingsTo optimize the porogen manufacturing process, the viscosity of sucrose mixtures is measured. Design of experiments is used to plan and analyze the relationships between the porogen characteristics and the process parameters. Reservoir pressure and print head speed are identified as the dominant factors affecting sucrose flow rate and porogen strut diameter, respectively. The biocompatibility of the new system is assessed by in vitro cell culture testing. Endothelial hybridoma cells (EAhy 926) and osteoblasts (7F2) seeded on the fabricated PCL scaffolds adhered to the scaffold and proliferated over four to six days. Epifluorescence and scanning electron microscopy images reveal cell spreading and multiple layers of cells on the scaffold surface. The results demonstrated the potential of the structured sucrose porogen‐based fabrication method in manufacturing bone tissue scaffolds.Originality/valueThis paper describes the first time use of biomaterials‐sucrose to make scaffold porogens and how an injection molded biopolymer scaffold can then be received.

Tracking Giant Folds in a Monolayer

The collapse dynamics of giant folds in a catanionic monolayer at the air-water interface are examined. A monolayer of dioctadecyldimethylammonium bromide (DODAB) and sodium dodecyl sulfate (SDS) in a 1:1 ratio is the system of study that previously was found to fold upon compression in a Langmuir trough. Carboxylate-coated polystyrene beads (1 microm diameter) are deposited and bound to the monolayer. Displacement of the beads is measured with epifluorescence microscopy and particle image velocimetry, yielding a measurement of the velocity of the monolayer around the fold. Reversibility is confirmed by measuring the amount of monolayer material entering and leaving the fold. Material near folds are found to have a maximum relative velocity on the order of 0.1 mm/s, and fold depths are found to be on the order of 1 mm. The folds exhibit regular unfolding behavior, which can be explained qualitatively by a simple mechanical model.

Photosynthetic picoplankton in Lake Tanganyika: biomass distribution patterns with depth, season and basin

Photosynthetic picoplankton (PPP), particularly picocyanobacteria (PCya), are known to be a major component of phytoplankton in oligotrophic waters. We investigated the dynamics of PPP and heterotrophic bacteria (HBact) in Lake Tanganyika during the dry and rainy seasons of 2004 to 2007, in the two basins of this large lake. Flow cytometry analyses showed that PPP was mainly composed by PCya of the phycoerythrin-rich Synechococcus type, with maximal abundances (2.3 × 10 4 -8.2 × 10 5 cells mL -1 ) found in the mixolimnion (10-20 m depth). PPP biomass integrated over the water column depth ranged between 0. 41 and 3. 09 g C m - 2 , with maximal values in the south basin during the dry season (2.28 ± 0. 62 g C m -2 on average). The contribution of PPP to total phytoplankton biomass ranged from 41 to 99%, with highest values in the south basin in the dry season. Cellular measurements by image analysis of epifluorescence microscopy images showed a significant increase of the cell volume of the PCya during this period. Flow cytometry also allowed enumeration of photosynthetic picoeukaryotes. Assuming a conversion factor of 530 fg C cell - 1 , they contributed on average to 6% to PPP biomass, except during the dry season in the south basin, where their contribution increased to up to 20% of PPP biomass. Integrated over a 100-m water column depth, PCya biomass was on average 1.4 times higher than HBact biomass. This study establishes reference values for the biomass contribution of this part of the microbial food web, covering for the first time, spatial (different sites), vertical (profiles at different depths) and seasonal variations on a multi-year basis. The results strengthen the view of a major role of PPP in the pelagic food web of large tropical Lake Tanganyika.

Lateral Distribution of a Poly(ethylene glycol)-Grafted Phospholipid in Phosphocholine Monolayers Studied by Epifluorescence Microscopy

Mixed monolayers of distearoylphosphatidylcholine (DSPC) and a poly(ethylene glycol)-(PEG)-grafted distearoylphosphatidylethanolamine with a PEG molecular weight of 2000, DSPE-PEG2000, spread on phosphate-buffered saline (PBS) were used as models of bio-non-fouling membrane-mimetic surfaces in order to visualize the lateral distribution of PEG2000-phospholipid in the host phospholipid matrix. Epifluorescence microscopy (EFM) was used to locate DSPE-PEG2000 molecules in the DSPC matrix by detecting the fluorescence from a fluorescein fluorophore attached to the distal end of the PEG2000 chain. Comparative analysis of surface pressure-area isotherms and EFM images revealed that DSPE-PEG2000 mixes nonideally with DSPC in monolayers on a PBS subphase. A transition from a phase-separated monolayer to a homogeneous mixture was observed with increasing surface pressure and PEG content. The effect of nonideal mixing behavior of DSPE-PEG2000 on its lateral distribution in the DSPC matrix was interpreted in terms of excluded volume interactions between the PEG2000 chains and a mismatch in the tilt of aliphatic chains on DSPC and DSPE-PEG2000 molecules.

New tools for resolving phylogenies: a systematic revision of the Convolutidae (Acoelomorpha, Acoela)

Molecular sequence data, morphological characters of spermatozoa, and newly obtained morphological characters of penis musculature are used here to revise the systematics of the family Convolutidae (Acoela). Species having isodiametric penes with non-anastomosing longitudinal muscles are transferred to the family Isodiametridae fam. nov. Species with longitudinal penis muscle fibres that anastomose or cross-over each other remain in the Convolutidae. Some species of the genera Convoluta and Conaperta (Convolutidae) are transferred to the genus Isodiametra gen. nov. (Isodiametridae fam. nov.). The genus Stomatricha (Otocelididae) is transferred to the family Convolutidae. Convoluta opisthandropora (Convolutidae) is transferred to the genus Pseudohaplogonaria (Haploposthiidae). Aphanostoma sanguineum (Convolutidae) is transferred to the genus Pseudactinoposthia (Actinoposthiidae). Zusammenfassung Molekulare Sequenzdaten, Spermien-Morphologie und erstmals ermittelte morphologische Merkmale der Penismuskulatur werden herangezogen, um die Verwandtschaftsverhaltnisse innerhalb der Familie Convolutidae (Acoela) neu zu beleuchten. Dabei werden Arten mit isodiametrischen Penes mit nicht anastomosierender Langsmuskulatur in die neue Familie Isodiametridae gestellt, wahrend Arten mit longitudinalen Penis-Muskelfasern, die sich verbunden oder einanderuberkreuzen, innerhalb der Convolutidae belassen. Einige Arten der Gattungen Convoluta und Conaperta (Convolutidae) werden in die neue Gattung Isodiametra gen. nov. (Isodametridae fam. nov.) gestellt. Die Gattung Stomatricha (Otocelididae) wird in die Familie Convolutidae uberfuhrt. Convoluta opisthandropora (Convolutidae) wird in die Gattung Pseudohaplogonaria (Haploposthiidae) gestellt und Aphanostoma sanguineum (Convolutidae) in die Gattung Pseudactinoposthia (Actinoposthiidae).

Fluorene-based fluorescent probes with high two-photon action cross-sections for biological multiphoton imaging applications

Two-photon fluorescence microscopy is a powerful tool for the study of dynamic cellular processes and live-cell imaging. Many commercially available fluorescent probes have been used in multiphoton-based imaging studies despite exhibiting relatively low two-photon absorption cross-section values in the tunability range of ultrafast Ti:sapphire lasers commonly used in multiphoton microscopy imaging. Furthermore, available fluorophores may be plagued with low fluorescence quantum yield and/or photoinstability (i.e., photobleaching) on exposure to the high peak power and photon density provided by the ultrafast laser source. To address the demand for better performing dyes, we prepare fluorophores tailored for multiphoton imaging. These fluorophores are based on the fluorene ring system, known to exhibit high fluorescence quantum yield (>0.7) and high photostability. Furthermore, an amine-reactive fluorescent probe for the covalent attachment onto amine-containing biomolecules is also prepared. Epi-fluorescence and two-photon fluorescence microscopy images of H9c2 rat cardiomyoblasts stained with an efficient two-photon absorbing fluorene fluorophore is demonstrated. Additionally, single-photon spectral characteristics of the amine-reactive fluorophore, as well as the two-photon absorption cross sections of its model adduct in solution, and spectral characterization of a bovine serum albumin (BSA) as a model bioconjugate are presented.

Protective Effect of Superoxide Dismutase Against Hair Graying in a Mouse Model¶

AbstractOxygen free radicals play a role in the aging process, and the protective effect of various antioxidants has been intensively studied, in particular for cutaneous aging. Besides hereditary factors, free radical‐mediated damage to melanocytes of the hair follicle has been considered as a mechanism for aging of the hair. It was the aim of this study to evaluate the role of photosensitization reactions for hair graying and to demonstrate potential protective effects of superoxide dismutase (SOD). Mice with black hair were depilated with the fingertips on a surface of 6 × 2.5 cm on both sides of the dorsum. The right side received five applications of a SOD‐containing gel before exposure to psoralen (concentration 0.5 mg/mL) plus UV‐A (365 nm, 4 J/cm2). The left side was pretreated in the same way with a gel free of SOD. When the hair started growing again, the SOD‐protected side was covered with black hair, whereas the hair on the vehicle‐treated side was gray or white in 27 of the 30 animals studied. The 0.01% SOD concentration was as protective as the 0.1% concentration. Heat‐inactivated SOD, applied in another five animals, was not protective. Using fluorescent labeling of the SOD with fluorescein isothiocyanate, epifluorescence microscopy and digital imaging processing, we show that SOD applied to the skin surface penetrates through the follicular appendages, as well as through the unbroken stratum corneum. Our findings suggest that superoxide radicals, generated by interaction of UV‐A light with the sensitizer, initiated the formation of secondary products with well‐known DNA‐damaging effects, such as lipid peroxidation products and tumor necrosis factor alpha. SOD prevented the damage to melanocyte DNA by dismutating superoxide. Photosensitization may be another mechanism for hair graying, which can be influenced by antioxidants. Given the large number of exogenous and endogenous sensitizers, this mechanism deserves further study for human hair graying.

Protective Effect of Superoxide Dismutase Against Hair Graying in a Mouse Model¶

Oxygen free radicals play a role in the aging process, and the protective effect of various antioxidants has been intensively studied, in particular for cutaneous aging. Besides hereditary factors, free radical-mediated damage to melanocytes of the hair follicle has been considered as a mechanism for aging of the hair. It was the aim of this study to evaluate the role of photosensitization reactions for hair graying and to demonstrate potential protective effects of superoxide dismutase (SOD). Mice with black hair were depilated with the fingertips on a surface of 6 x 2.5 cm on both sides of the dorsum. The right side received five applications of a SOD-containing gel before exposure to psoralen (concentration 0.5 mg/mL) plus UV-A (365 nm, 4 J/cm2). The left side was pretreated in the same way with a gel free of SOD. When the hair started growing again, the SOD-protected side was covered with black hair, whereas the hair on the vehicle-treated side was gray or white in 27 of the 30 animals studied. The 0.01% SOD concentration was as protective as the 0.1% concentration. Heat-inactivated SOD, applied in another five animals, was not protective. Using fluorescent labeling of the SOD with fluorescein isothiocyanate, epifluorescence microscopy and digital imaging processing, we show that SOD applied to the skin surface penetrates through the follicular appendages, as well as through the unbroken stratum corneum. Our findings suggest that superoxide radicals, generated by interaction of UV-A light with the sensitizer, initiated the formation of secondary products with well-known DNA-damaging effects, such as lipid peroxidation products and tumor necrosis factor alpha. SOD prevented the damage to melanocyte DNA by dismutating superoxide. Photosensitization may be another mechanism for hair graying, which can be influenced by antioxidants. Given the large number of exogenous and endogenous sensitizers, this mechanism deserves further study for human hair graying.

Layer-by-Layer Biosensor Assembly Incorporating Functionalized Quantum Dots

Layer-by-layer (LbL) assembly has been utilized to fabricate an ultrathin film of polyelectrolytes. The architecture was composed of chitosan and organophosphorus hydrolase polycations along with thioglycolic acid-capped CdSe quantum dots (QDs) as the polyanion. The topography of the films was studied using epifluorescence microscopy imaging. The photoluminescence property of the functionalized QDs improved when sandwiched between the polycation layers. The enhanced optical property of QDs allowed easy monitoring of LbL growth and detection of paraoxon with high sensitivity. The presence of organophosphorus compounds was confirmed through UV−vis and emission spectroscopies.

Quantification and localization of trace metals in natural plankton cells using a synchrotron X-ray fluorescence microprobe

The accumulation of trace metals by planktonic protists influences the growth of primary producers, metal biogeochemical cycling, and metal bioaccumulation in aquatic food chains. Despite their importance, unequivocal measurements of trace element concentrations in individual plankton cells have not been possible to date. We have used the 2-ID-E side-branch hard x-ray microprobe at the Advanced Photon Source to measure trace elements in individual marine plankton cells. This microprobe employs zoneplate optics to produce the sub-micron spatial resolution and low background fluorescence required to produce trace element maps of planktonic protist cells ranging in size from 3 to >50 {micro}m. We have developed preservation, rinsing, and mounting protocols that remove most of the salt from our marine samples, thus simplifying the identification of unknown cells and reducing high Cl-related background fluorescence. We have also developed spectral modeling techniques that account for the frequent overlap of adjacent fluorescence peaks and non-uniform detector response. Finally, we have used parallel soft x-ray transmission and epifluorescence microscopy images to estimate C normalized trace element concentrations, identify functional cell types (e.g., photosynthetic vs. non-photosynthetic), and correlate cell structures with spatial patterns in trace element fluorescence.

Increase In Intracellular Sodium Evoked by Glutamate Transporter Activation In Cortical Astrocytes: A Fluorescence Microscopy Analysis

Primary cultures of mouse cortical astrocytes were used to investigate the changes in intracellular Na+ concentration ([Na+]i) following exposure to the excitatory neurotransmitter glutamate. The fluorescent probe sodium-binding benzofuran isophthalate (SBFI) was used to measure [Na+]i by epifluorescence microscopy imaging. Detection of the low light level fluorescent signal was accomplished using a Gen III+ intensified CCD camera. Fluorescence excitation ratio images of dye-loaded cells were obtained after sequential illumination at 340nnm and 380nm, with an emission observed at &gt;520nm. Ratio images were proportional in intensity to [Na+]i. In situ calibration of the fluorescent signals was obtained for each experiment and each cell under study by equilibrating [Na+]i with external Na+ after treatment with the cation ionophores monensin and gramicidin, and with ouabain, a specific inhibitor of the Na+/K+ ATPase. Cells on glass coverslips were perfused at 35°C in a closed microscope chamber using solutions buffered with 25 mM bicarbonate/5%CO2.

The critical pressures of gas vesicles in Planktorhrix rubescens in relation tothe depth of winter mixing in Lake Zürich, Switzerland

The vertical distribution of the cyanobacterium Planktothrix (Oscillatoria) rubescens in Lake Zurich was investigated from March 1993 to June 1995 by collecting filaments on filters and measuring them by epifluorescence microscopy and computer image analysis. The initial population, which began to stratify in April, decreased by up to 99% by June. During the summer, the population peaked at depths of 8—15 m; it reached a maximum areal filament-volume concentration of -60 cm3 m~2 of lake surface in early September and was then entrained in the deepening surface layer. It became mixed progressively deeper, to the lake bottom in the cold winter of 1993-94, but less completely in the milder winter of 1994-95. Most of the filaments remained viable during the winter. At the end of the mild winter of 1994-5, 70% of filaments in the water column retained buoyancy, but after the cold winter of 1996-7 only 22% were buoyant. Few remained buoyant below 80 m, where the hydrostatic pressure caused gas vesicle collapse. The proportion that remain buoyant decreases with the depth and duration of winter mixing, and increases with the critical collapse pressure (pc) of the gas vesicles, which provide buoyancy. Strains of P.rubescens isolated from Lake Zurich differed in mean pc of their gas vesicles, from 0.9 to 1.1 MPa, the highest values in freshwater cyanobacteria. Allowing for a turgor pressure of 0.2 MPa, these strains would remain buoyant at depths down to 70 and 90 m, respectively. Natural selection for gas vesicles of high pc will operate by increasing the proportion of filaments that remain buoyant in the upper parts of the water column after circulation to various depths during the winter because only buoyant filaments will form the inoculum for the following season.

Distribution of glycoconjugates in the uterine tube (oviduct) of horses

Abstract Objective To examine glycoconjugates in the isthmic and ampullar regions of the uterine tube (oviduct) of horses during estrus, diestrus, and pregnancy. Sample Population Oviductal samples from 17 mares. Procedure Oviducts were collected during estrus (n = 3), diestrus (n = 3), or pregnancy (n = 3), embedded, and snap frozen in liquid nitrogen. Frozen sections (5 to 6 μm in thickness) were stained with 100 μg/ml of fluorescein-isothiocyanate-conjugated lectin (30 min at 38.5 C) and were evaluated by use of epifluorescence microscopy and video image analysis. Specificity of lectins was established by blocking with the corresponding carbohydrate. Dolichos biflorus agglutinin (DBA)-affinity studies on western blots of oviductal lavage fluid, oviductal explant conditioned media, and apical membrane proteins from isthmic and ampullar regions of oviducts were used to identify glycoproteins with galactosyl residues. Results Use of 4 lectins resulted in differential labeling of the luminal surface of the oviductal epithelium. Both DBA and soybean agglutinin labeled the apical epithelium of the isthmus, but not the ampullar oviduct. Soybean agglutinin resulted in more-intensely labeled epithelium in the isthmic region of oviducts during estrus and pregnancy than during diestrus. The DBA labeled a number of glycoproteins in conditioned media from both regions of the oviduct. These glycoproteins ranged from 14 to 200 kd, with major glycoproteins identified at 31 and 57 kd. Conclusions The predominant glycoconjugates in the oviduct of horses are galactosyl residues. There are regional differences in the distribution of these galactosyl glycoconjugates in the isthmic and ampullar oviduct. (Am J Vet Res 1997;58:816–822)