Studies on hydrogenase activity and chlorobenzene respiration in <Emphasis Type="Italic">Dehalococcoides</Emphasis> sp. strain CBDB1

Hydrogen oxidation and electron transport were studied in the chlorobenzene-utilizing anaerobe Dehalococcoides sp. strain CBDB1. While Cu²⁺ and Hg²⁺ ions irreversibly inhibited hydrogenase activity in intact cells, Ni²⁺ ions inhibited reversibly. About 80% of the initial hydrogenase activity was inactivated within 30 s when the cells were exposed to air. In contrast, hydrogenase was active at a redox potential of +10 mV when this redox potential was established anoxically with a redox indicator. Viologen dyes served both as electron acceptor for hydrogenase and electron donor for the dehalogenase. A menaquinone analogue, 2,3-dimethyl 1,4-naphthoquinone, served neither as electron acceptor for the hydrogenase nor as electron donor for the dehalogenase. In addition, the menaquinone antagonist 2-n-heptyl-4-hydroxyquinoline-N-oxide had no effect on dechlorination catalyzed by cell suspensions or isolated membranes with hydrogen as electron donor, lending further support to the notion that menaquinone is not involved in electron transport. The ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenylhydrazone did not inhibit dechlorination by cell suspensions, indicating that strain CBDB1 does not require reverse electron transport. The ATP-synthase inhibitor N,N-dicyclohexylcarbodiimide inhibited the dechlorination reaction with cell suspensions; however, the latter effect was partially relieved by the addition of tetrachlorosalicylanilide. 1,2,3,4-Tetrachlorobenzene strongly inhibited dechlorination of other chlorobenzenes by cell suspensions with hydrogen as electron donor, but it did not interfere with either hydrogenase or dehalogenase activity.     

Cell membrane isolation from plasmodium ofPhysarum polycephalum

Plasmodia were fractionated to isolate a cell membrane rich fraction by sucrose density-gradient centrifugation. The fractions were identified by electron microscopic observation, PTA-chromic acid staining and assays of marker enzymes, applying the methods for cell membranes of higher plants. The cell membranes were recovered on the density of 1.13 g·cm⁻³.     

Electron spectroscopic imaging (ESI) applied to the detection of potential Ca2+-binding sites in plant cells

The applicability of the electron spectroscopic imaging technique for detection of the intracellular distribution of calcium in plant cells was tested with calyptra cells ofZea mays and with pollen tubes ofLilium longiflorum. After fixation in enhanced Ca²⁺ levels and embedding in resin, ultrathin sections were analyzed for the elemental distribution. Calcium and phosphorus were enriched in cell wall, plasma membrane, endoplasmic reticulum, mitochondria, and Golgi vesicles, mainly in granular or globular deposits appearing electron dense in transmission electron microscopy. The results demonstrated that the ESI-technique allows exact localization of calcium enrichment relative to specific cell organelles.     

Direct cathodic electron uptake coupled to sulfate reduction by Desulfovibrio ferrophilus IS5 biofilms

Direct electron uptake is emerging as a key process for electron transfer in anaerobic microbial communities, both between species and from extracellular sources, such as zero-valent iron (Fe⁰) or cathodic surfaces. In this study, we investigated cathodic electron uptake by Fe⁰-corroding Desulfovibrio ferrophilus IS5 and showed that electron uptake is dependent on direct cell contact via a biofilm on the cathode surface rather than through secreted intermediates. Induction of cathodic electron uptake by lactate-starved D. ferrophilus IS5 cells resulted in the expression of all components necessary for electron uptake; however, protein synthesis was required for full biofilm formation. Notably, proteinase K treatment uncoupled electron uptake from biofilm formation, likely through proteolytic degradation of proteinaceous components of the electron uptake machinery. We also showed that cathodic electron uptake is dependent on SO₄²⁻ reduction. The insensitivity of Fe⁰ corrosion to proteinase K treatment suggests that electron uptake from a cathode might involve different mechanism(s) than those involved in Fe⁰ corrosion.     

Detection of mycoplasma contamination in cultured human fibroblasts : Comparison of biochemical and microbiological techniques

Eighteen human fibroblast strains were tested for mycoplasma contamination by polyacrylamide gel electrophoresis of ³H-uridine labeled RNA and by standard microbiological culture techniques. Despite negative culture results, prominent 23S and 16S RNA peaks were detected in 11 of these cell strains. The mycoplasma origin of this RNA was indicated by electron microscopic demonstration of these organisms. Another indication of contamination was the decreased specific radioactivity of whole cell RNA extracted from cell strains infected with mycoplasma.     

Uptake of sulfate,sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfuricans

pH changes and sulfide production upon addition of sulfate, sulfite or thiosulfate to non-buffered H₂-saturated cell suspensions of Desulfovibrio desulfuricans were studied by means of electrodes. The addition of these electron acceptors resulted in a rapid alkalinization of the suspension which was accompanied by sulfide production. At-2° C, alkalinization without immediate sulfide production could be obtained. After addition of ³⁵S-labelled sulfate at-2° C, the label was found to be concentrated 7,500-fold in the cells, while 2 protons per sulfate molecule had disappeared from the outer bulk phase. Alkalinization and sulfide production from micromolar electron acceptor additions depended on the transmembraneous proton gradient ( pH), and were reversibly inhibited in alkaline solution (pH>8.0) or by the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Protonophore-inhibited sulfide production from sulfite or thiosulfate could be restored if the cell membranes were permeabilized by the detergent cetyltrimethylammonium bromide (CTAB), or if downhill transport was made possible by the addition of electron acceptors at millimolar concentrations. Sulfate was not reduced under these conditions, presumably because the cells did not contain ATP for its activation. K⁺-and Na⁺-ionophores such as nigericin, valinomycin or monensin appeared to be of limited efficiency in D. desulfuricans. In most experiments, sulfate reduction was inhibited by the K⁺–H⁺ antiporter nigericin in the presence of K⁺, but not by the thiocyanate anion or the K⁺-transporter valinomycin. The results indicate that sulfate, sulfite and thiosulfate are taken up by proton-anion symport, presumably as undissociated acids with an electroneutral mechanism, driven by the transmembraneous pH gradient ( pH) or by a solute gradient. Kinetics of alkalinization and sulfide production in cells grown with different electron acceptors revealed that D. desulfuricans has different specific uptake systems for sulfate and thiosulfate, and obviously also for sulfite. It is proposed that the electron acceptor transport finally will not consume net energy during growth in buffered medium: The protons taken up during active electron acceptor transport leave the cell with the reduced end-product by simple passive diffusion of H₂S.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - CTAB cethyltrimethylammonium bromide     

Selective loss of S-phase cells when making cell suspensions from lymphoid tissue.

The relative yield of S-phase cells when making cell suspensions from lymph nodes was determined by two different methods: by estimating the proportions of S-phase cells in sections and smears from lymph nodes undergoing a local graft-versus-host reaction, and by measuring the [³H]thymidine activity relative to DNA content in intact tissue and cell suspensions from normal lymph nodes. Both methods showed a large selective loss of S-phase cells in the process of making cell suspensions. The cell types preparing for division in the GVH nodes were then determined by light microscopic autoradiography combined with electron microscopy of neighboring ultrathin sections. The majority of dividing cells were lymphoid; some of these showed advanced signs of cell death.     

Insulin uptake by rat liver endothelium studied in fractionated liver cell suspensions

Summary Cellular distribution of insulin receptors was studied in fractionated rat liver cell suspensions using ¹²⁵1-insulin and a visual probe consisting of latex beads covalently linked to insulin (minibeads). Fractionation was done on metrizamide gradients which yielded two cellular fractions. The large cell fraction consisted mostly of hepatocytes and the small cell fraction consisted of 37% endothelial cells as well as Kupffer cells. The magnitude of insulin uptake by the endothelium-rich small cell fraction was at least double that of the uptake by the hepatocyte-rich fraction. The minibead technique demonstrated that in the small cell fraction only endothelial cells, and not Kupffer cells, were responsible for the insulin uptake. Our findings suggest that liver endothelium may be responsible for the uptake of circulating insulin and its transport to hepatocyte. This emphasizes the presence of a tissue-blood barrier in the liver.Abbreviations PRS phosphate-buffered saline - SEM scanning electron microscopy - TEM transmission electron microscopy     

Stimulation of monovalent cation fluxes by electron donors in the human red cell membrane

When human red cells are incubated at 37°C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb⁺ (K⁺) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb⁺ (K⁺) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca²⁺ (Gardos effect). The electron donors require the presence of intracellular Ca²⁺ to be effective, but at levels that do not stimulate by themselves the fluxes of K⁺. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca²⁺ alone; antimycin, uncouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect.The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca²⁺ more effective to elicit rapid K⁺ movements. Plasma membrane oxidoreductase activities could be engaged in this change.     

Adhesive interactions between CD34<Superscript>+</Superscript>-derived dendritic cell precursors and dermal microvascular endothelial cells studied by scanning electron microscopy

Dendritic cells are migratory cells. Before they extravasate from the circulation into the skin across capillary blood vessel walls, they have to interact with endothelial cells. Using a fluorimetric adhesion assay, we have recently shown that CD34⁺-derived dendritic cell precursors are able to bind to resting and stimulated dermal microvascular endothelial cells. In the present study, we attempted to visualize this process at an ultrastructural level. CD34⁺ progenitor cells were purified from human cord blood samples by means of immunomagnetic beads, and dendritic cells were generated by culture in the presence of GM-CSF, TNF- and hSCF for 5 days. Immature CD83^– CD86^low dendritic cells were added to human dermal microvascular endothelial cells grown to confluence on membrane chambers. After 2 h, unbound dendritic cell precursors were removed, and bound cells were prepared for routine scanning electron microscopy. We found that (1) dendritic cell precursors firmly adhere to microvascular endothelial cells, enveloping them with their surface processes; (2) dendritic cell precursors are extremely deformable as they squeeze through the dense network of microvascular endothelial cells; (3) microvascular endothelial cells form, in part, a multi-layered network rather than the typical cobblestone pattern as seen by phase-contrast microscopy. The morphology of dendritic cell precursors and of human dermal microvascular endothelial cells was examined here, for the first time, by scanning electron microscopy. These data further emphasize that CD34⁺-derived dendritic cells efficiently adhere to dermal microvascular endothelial cells.     

New approach of monitoring changes in chlorophyll a fluorescence of single guard cells and protoplasts in response to physiological stimuli

A new type of microfluorometer was applied to assess photosynthesis at the single-cell level by chlorophyll fluorescence using the saturation pulse method. A microscopy–pulse amplitude modulation (PAM) chlorophyll fluorometer was combined with a Zeiss Axiovert 25 inverted epifluorescence microscope for high-resolution measurements on single mesophyll and guard cells and the respective protoplasts. Available information includes effective quantum yield of photosystem II, relative electron transport rate and energization of the thylakoid membrane due to the transthylakoidal proton gradient. Dark–light induction curves of guard cell (GCPs) and mesophyll cell protoplasts (MCPs) displayed very similar characteristics, indicating similar functional organization of thylakoid membranes in both types of chloroplasts. Light response curves, however, revealed much earlier saturation of photosynthetic electron flow in GCPs than in MCPs. Under anaerobiosis, photosynthetic electron flow and membrane energization were severely suppressed. A similar effect was observed in guard cells when epidermal peels were incubated with the fungal toxin fusicoccin which activates the plasma membrane H⁺-ATPase and causes irreversible opening of stomata. The drop in electron transport rate was prevented by blocking ATP consumption of the H⁺ pump or by glucose addition. These results show that chlorophyll fluorescence quenching analysis allows profound insights into stomatal physiology.     

Identification of a potential artifact in the use of electron microscope autoradiography to localize saturated phospholipids in cells

The suitability of electron microscope autoradiography for studying the uptake and intracellular localization of lipid vesicles (liposomes) containing radiolabeled saturated phospholipids has been examined. Data are presented showing that preparation of specimens for electron microscope autoradiography by conventional methods is accompanied by significant translocation and intercellular redistribution of radiolabeled saturated lipids, causing spurious labeling patterns. Intercellular redistribution of radiolabeled lipid was demonstrated by mixing glutaraldehyde-fixed mouse L1210 cells that had been incubated with sonicated lipid vesicles containing [³H]dipalmitoyl phosphatidylcholine with an indicator cell population (fixed avian erythrocytes) which had not been exposed to vesicles and showing that after electron microscope processing radiolabeled grains were present in both cell types. The same redistribution artifact also probably affects the intracellular localization of radiolabeled lipids. This artifact is discussed in relation to previous work in which autoradiographic methods have been used for ultrastructural localization of saturated phospholipids in cells and tissues.     

Subcellular distribution of chromium in accumulating plant Leersia hexandra Swartz

The subcellular distribution of chromium in Leersia hexandra Swartz, a Cr-accumulating plant found in China, was studied by differential centrifugation, transmission electron microscope and energy dispersive analysis of X-ray. Subcellular fractionation of Cr-containing tissues showed that most of the accumulated Cr was isolated to the cell walls in roots and the vacuoles in leaves. When the plant was grown in a nutrient solution containing 60 mg L^?1 Cr, 83.2% of the root Cr was localized in the cell wall fraction, while 57.5% of leaf Cr was localized in the vacuole and cytoplasm fraction. Transmission electron microscopic analysis revealed that those cell compartments contained especially electron dense areas. Energy dispersive X-ray spectra showed the electron dense areas contained high Cr. However, the dark electron precipitates were never observed in the plant cells without Cr treatment. In all treatment groups (5, 30 and 60 mg L^?1), the fraction containing the lowest level of Cr was the organelle fraction in roots as well as leaves. These results indicated that Cr accumulated in the L. hexandra was preferentially stored in the cell walls of roots and the vacuoles of leaves. This phenomenon diverted Cr ions from metabolically active compartment (chloroplast, mitochondria), resulting in a reduction of Cr toxicity in the plant cell.     

Isolation of membrane-bound rat mast cell granules

A technique for obtaining membrane-bound rat peritoneal mast cell granules in high yield is described. Mast cells purified by centrifugation into 38% BSA gradients were sonicated in Ca²⁺, Mg²⁺-free Tyrode's-gelatin buffer, incubated in EDTA for 15 min at 37 °C, and differentially centrifuged through a 0.34 M sucrose cushion to yield a granular preparation with >80% of the granules bound by perigranular membranes. The perigranular membranes were demonstrated morphologically by light and electron microscopy and functionally by histamine distribution.     

Ultrastructural and ultracytochemical studies of the gill epithelium in the larvae of Salamandra salamandra (Amphibia,Urodela)

Summary The morphology of Salamandra salamandra (Linné, 1758) larva gills is described by scanning and transmission electron microscopy. Three main cell types comprising the surface of the gill epithelium are described: (a) pavement cell, (b) ciliary cell and (c) mitochondria-rich cell (MRC). Two subtypes of MRC were distinguished by their ultrastructural characteristics: a fibrillar cell and a tubulovesicular cell. K^ü-dependent-nitrophenyl-phosphatase (K-NPPase) activity, indicative of Na^ü-K^ü-ATPase activity was confined to the basolateral membranes of the pavement cells. MRC were devoid of such activity. Possible relationships between structure and function of the different cell types are discussed.     

Genetic Ablation of the ClC-2 Cl- Channel Disrupts Mouse Gastric Parietal Cell Acid Secretion

The present studies were designed to examine the effects of ClC-2 ablation on cellular morphology, parietal cell abundance, H/K ATPase expression, parietal cell ultrastructure and acid secretion using WT and ClC-2^-/- mouse stomachs. Cellular histology, morphology and proteins were examined using imaging techniques, electron microscopy and western blot. The effect of histamine on the pH of gastric contents was measured. Acid secretion was also measured using methods and secretagogues previously established to give maximal acid secretion and morphological change. Compared to WT, ClC-2^-/- gastric mucosal histological organization appeared disrupted, including dilation of gastric glands, shortening of the gastric gland region and disorganization of all cell layers. Parietal cell numbers and H/K ATPase expression were significantly reduced by 34% (P<0.05) and 53% (P<0.001) respectively and cytoplasmic tubulovesicles appeared markedly reduced on electron microscopic evaluation without evidence of canalicular expansion. In WT parietal cells, ClC-2 was apparent in a similar cellular location as the H/K ATPase by immunofluorescence and appeared associated with tubulovesicles by immunogold electron microscopy. Histamine-stimulated [H⁺] of the gastric contents was significantly (P<0.025) lower by 9.4 fold (89%) in the ClC-2^-/- mouse compared to WT. Histamine/carbachol stimulated gastric acid secretion was significantly reduced (range 84–95%, P<0.005) in ClC-2^-/- compared to WT, while pepsinogen secretion was unaffected. Genetic ablation of ClC-2 resulted in reduced gastric gland region, reduced parietal cell number, reduced H/K ATPase, reduced tubulovesicles and reduced stimulated acid secretion.     

Aquaporin 1-positive stromal niche-like cells directly interact with N-cadherin-positive clusters in the basal limbal epithelium

Stem cells have a specialized microenvironment for maintaining self-renewal and multipotent capacities. It is believed that a cornea epithelial stem cell niche exists in the limbus. To characterize the niche of limbal epithelial stem cells, we observed the limbal basal epithelial layer by histological analysis.Cell clusters or cell suspensions from limbal tissue were prepared with collagenase or dispase II and fixed for cytospin sections. Adhesion assays were done to quantitate calcium-dependent cell adhesion. Limbal tissue and cytospin sections were analyzed by immunohistochemistry, transmission electron microscopy and confocal microscopy.AQP1 positive (AQP1⁺) cells were observed as non-epithelial cells in the subepithelial stroma. AQP1 expression did not co-localize with CD31, podoplanin, MART-1 positive cells, but were observed in vimentin positive stromal cells. When we made a thorough search of limbal basal cells by confocal microscopy, AQP1⁺ were observed in the proximity of N-cad, K15 and p63 positive limbal basal epithelial cells. Furthermore, electron microscope revealed stromal cells penetrating the epithelial basal membrane and forming calcium-dependent cellular adhesions with N-cad⁺ limbal basal epithelial cells.Although we could not clearly detect the expression of N-cad in the AQP1⁺ cells, AQP1⁺ cells immediately beneath the epithelial basement membrane may be stromal niche-like cells that directly interact with N-cad⁺ limbal basal epithelial progenitor cells.     

New approaches to the study of sphingolipid enriched membrane domains: The use of microscopic autoradiography to reveal metabolically tritium labeled sphingolipids in cell cultures

This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture.To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3×10^–7 M [1-³H]sphingosine. [1-³H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-³H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy.Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis.     

Isolation and Characterization of a Mo6+ -Reducing Bacterium

A Mo⁶⁺ -reducing bacterium (strain 48), which grew on medium supplemented with 200 mM Mo⁶⁺, was isolated from stream water obtained from Chengkau, Malaysia. The chemical properties of strain 48 conform to the characteristics of Enterobacter cloacae. Under anaerobic conditions in the glucose-yeast extract medium containing phosphate ion (2.9 mM) and Mo⁶⁺ (10 mM), the bacterium reduced Mo⁶⁺ to form molybdenum blue. Approximately 27% of Mo⁶⁺ added to the medium was reduced after 28 h of cultivation. The reduction of Mo⁶⁺ with glucose as an electron donor was strongly inhibited by iodoacetic acid, sodium fluoride, and sodium cyanide, suggesting an involvement of the glycolytic pathway and electron transport in Mo⁶⁺ reduction. NADH and N,N,N′,N′ -tetramethyl-p-phenylenediamine served as electron donors for Mo⁶⁺ reduction. When NADH was used as an electron donor, at first cytochrome b in the cell extract was reduced, and then molybdenum blue was formed. Sodium cyanide strongly inhibited Mo⁶⁺ reduction by NADH (5 mM) but not the reduction of cytochrome b in the cell extract, suggesting that the reduced component of the electron transport system after cytochrome b serves as an electron donor for Mo⁶⁺ reduction. Both ferric and stannous ions strongly enhanced the activity of Mo⁶⁺ reduction by NADH.     

Understanding the Anodic Mechanism of a Seafloor Fuel Cell: Interactions Between Geochemistry and Microbial Activity

Seafloor fuel cells made with graphite electrodes generate electricity by promoting electron transfer in response to a natural voltage difference (−0.7 to −0.8 V) between anoxic sediments and overlying oxic seawater. Geochemical impacts of a seafloor fuel cell on sediment solids and porewaters were examined to identify the anodic mechanisms and substrates available for current production. In an estuarine environment with little dissolved sulfide, solid-phase acid volatile sulfide and Cr²⁺-reducible sulfur minerals decreased significantly toward the anode after 7 months of nearly continuous energy harvesting. Porewater iron and sulfate increased by millimolar amounts. Scanning electron microscope images showed a biofilm overcoating the anode, and electron microprobe analyses revealed accumulations of sulfur, iron, silicon and phosphorus at the electrode surface. Sulfur deposition was also observed on a laboratory fuel cell anode used to generate electricity with only dissolved sulfide as an electron donor. Moreover, current densities and voltages displayed by these purely chemical cells were similar to the values measured with field devices. These results indicate that electron transfer to seafloor fuel cells can readily result in the oxidation of dissolved and solid-phase forms of reduced sulfur producing mainly S⁰ which deposits at the electrode surface. This oxidation product is consistent with the observed enrichment of bacteria most closely related to Desulfobulbus/Desulfocapsa genera within the anode biofilm, and its presence is proposed to promote a localized biogeochemical cycle whereby biofilm bacteria regenerate sulfate and sulfide. This electron-shuttling mechanism may co-occur while these or other bacteria use the anode directly as a terminal electron acceptor.