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Low intensity (< 2 Vpp/cm (peak to peak voltage/cm)), high frequency (10-30 MHz), and 10 min alternating electric fields (sine wave with no DC component) induce non-contact and enzyme-free cell detachment of anchorage-dependent cells directly from commercially available cell culture flasks and stack plates. 0.25 Vpp/cm, 20 MHz alternating electric field for 10 min at room temperature (RT) induced maximum detachment and separated 99.5 ± 0.1% (mean ± SEM, n = 6) of CHO-K1 and 99.8 ± 0.2% of BALB/3T3 cells from the culture flasks. Both vertical and lateral alternating electric field applications for 10 min at RT detach the CHO-K1 cells from 25 cm2 culture flasks. The alternating electric field application induced cell detachment is almost noncytotoxic, and over 90% of the detached cells remained alive. The alternating electric field applied CHO-K1 cells for 90 min showed little or no lag phase and immediately enter exponential phase in cell growth. Combination of the 20 MHz alternating electric field and enzymatic treatment for 4 min at 37 °C showed synergetic effect and quickly detached human induced pluripotent stem cells from a laminin-coated culture flask compared with the only enzymatic treatment. These results indicate that the rapid cell detachment with both the electric field application and the enzymatic treatment could be applied to subcultures of cells that are susceptible to prolonged enzymatic digestion damage for mass culture of sustainable clinical use.

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A novel marine bacterial strain, designated JAM 119T, was isolated from a tubeworm trophosome in Kagoshima Bay, Japan. Cells were Gram-negative, rod-shaped, non-spore-forming aerobic chemoorganotrophs and motile by means of a single polar flagellum. The isolate grew optimally at 25-27 °C and in the presence of 3 % NaCl. The major respiratory quinone was Q-10. The predominant fatty acid was C18 : 1ω7c. Phosphatidylcholine, phosphatidylglycerol and an unidentified aminolipid were the major polar lipids. On the basis of 16S rRNA gene sequence analysis, the isolated strain was closely affiliated with members of the genus Planktotalea in the class Alphaproteobacteria, and the 16S rRNA gene sequence similarity of the new isolates with the closest related species, Planktotalea frisia SH6-1T, was 97.3 %. The DNA G+C content of the novel strain was 57.0 mol%. Based on differences in taxonomic characteristics, the isolated strain represents a novel species of the genus Planktotalea, for which the name Planktotalealamellibrachiae sp. nov. (type strain JAM 119T; JCM 31859T=DSMZ 104669T) is proposed.

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The purpose of this study was to develop a novel electrical retrieval method (ER method) for living sponge-associated microorganisms from marine sponges frozen at -80 °C. A -0.3-V vs. Ag/AgCl constant potential applied for 2 h at 9 °C induced the attachment of the sponge-associated microorganisms to an indium tin oxide/glass (ITO) or a gallium-doped zinc oxide/glass (GZO) working electrode. The electrically attached microorganisms from homogenized Spirastrella insignis tissues had intact cell membranes and showed intracellular dehydrogenase activity. Dead microorganisms were not attracted to the electrode when the homogenized tissues were autoclaved for 15 min at 121 °C before use. The electrically attached microorganisms included cultivable microorganisms retrieved after detachment from the electrode by application of a 9-MHz sine-wave potential. Using the ER method, we obtained 32 phyla and 72 classes of bacteria and 3 archaea of Crenarchaeota thermoprotei, Marine Group I, and Thaumarchaeota incertae sedis from marine sponges S. insignis and Callyspongia confoederata. Employment of the ER method for extraction and purification of the living microorganisms holds potential of single-cell cultivation for genome, transcriptome, proteome, and metabolome analyses of bioactive compounds producing sponge-associated microorganisms.

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The purpose of this study was to develop novel methods for attachment and cultivation of specifically positioned single yeast cells on a microelectrode surface with the application of a weak electrical potential. Saccharomyces cerevisiae diploid strains attached to an indium tin oxide/glass (ITO) electrode to which a negative potential between -0.2 and -0.4 V vs. Ag/AgCl was applied, while they did not adhere to a gallium-doped zinc oxide/glass electrode surface. The yeast cells attached to the negative potential-applied ITO electrodes showed normal cell proliferation. We found that the flocculin FLO10 gene-disrupted diploid BY4743 mutant strain (flo10Δ /flo10Δ) almost completely lost the ability to adhere to the negative potential-applied ITO electrode. Our results indicate that the mechanisms of diploid BY4743 S. cerevisiae adhesion involve interaction between the negative potential-applied ITO electrode and the Flo10 protein on the cell wall surface. A combination of micropatterning techniques of living single yeast cell on the ITO electrode and omics technologies holds potential of novel, highly parallelized, microchip-based single-cell analysis that will contribute to new screening concepts and applications.

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A novel Gram-stain-negative, aerobic, heterotrophic, stalked and capsulated bacterium with potential denitrification ability, designated strain TAR-002(T), was isolated from deep seafloor sediment in Japan. Colonies lacked lustre, and were viscous and translucent white. The ranges of temperature, pH and salt concentration for growth were 8-30 °C, pH 6.0-10.0 and 1-3% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain TAR-002(T) belongs to the genus Brevundimonas of the class Alphaproteobacteria. Levels of similarity between the 16S rRNA gene sequence of strain TAR-002(T) and those of the type strains of species of the genus Brevundimonas were 93.5-98.9%; the most closely related species was Brevundimonas basaltis. In DNA-DNA hybridization assays between strain TAR-002(T) and its phylogenetic neighbours, Brevundimonas lenta DS-18(T), B. basaltis J22(T), Brevundimonas subvibrioides ATCC 15264(T) and Brevundimonas alba DSM 4736(T), mean hybridization levels were 6.4-27.7%. The G+C content of strain TAR-002(T) was 70.3 mol%. Q-10 was the major respiratory isoprenoid quinone. The major fatty acids were C(18:1)ω7c and C(16:0), and the presence of 1,2-di-O-acyl-3-O-[D-glucopyranosyl-(1 → 4)-α-D-glucopyranuronosyl]glycerol (DGL) indicates the affiliation of strain TAR-002(T) with the genus Brevundimonas. On the basis of biological characteristics and 16S rRNA gene sequence comparisons, strain TAR-002(T) is considered to represent a novel species of the genus Brevundimonas, for which the name Brevundimonas denitrificans sp. nov. is proposed; the type strain is TAR-002(T) ( =NBRC 110107(T) =CECT 8537(T)).

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Hydrostatic pressure is the only one of a range of environmental parameters (water temperature, salinity, light availability, and so on) that increases in proportion with depth. Pressure tolerance is therefore essential to understand the foundation of populations and current diversity of faunal compositions at various depths. In the present study, we used a newly developed pressure chamber system to examine changes in larval activity of the salt-lake crustacean, Artemia franciscana, in response to a range of hydrostatic pressures. We showed that A. franciscana larvae were able to survive for a short period at pressures of ≤ 60 MPa (approximately equal to the pressure of 6000 m deep). At a pressure of > 20 MPa, larval motor ability was suppressed, but not lost. Meanwhile, at a pressure of > 40 MPa, some of the larval motor ability was lost without recovery after decompression. For all experiments, discordance of movement and timing between right and left appendages, was observed at pressures of > 20 MPa. Our results indicate that the limit of pressure for sustaining active behavior of A. franciscana larvae is ∼20 MPa, whereas the limit of pressure for survival is within the range 30-60 MPa. Thus, members of the genus Artemia possess the ability to resist a higher range of pressures than their natural habitat depth. Our findings demonstrated an example of an organism capable of invading deeper environment in terms of physical pressure tolerance, and indicate the need and importance of pressure study as an experimental method.

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We developed an electrical modulation method for attachment and detachment of microorganisms. Living microorganisms suspended in non-nutritive media such as PBS⁻ and artificial seawater were attracted by and selectively attached to indium tin oxide (ITO)/glass electrode regions to which a negative potential was applied. The microorganisms suspended in LB medium and glucose solution were not attracted to the ITO electrode. Dead microorganisms were not attracted to the ITO electrode. The living microorganisms were retrieved after detachment from the ITO electrode by application of a high-frequency triangular wave potential. When we applied this method to separate microorganisms from deep-sea sediment, bacteria belonging to 19 phyla and 23 classes were collected without undesirable high molecular weight contaminants such as humic acids. At the phylum and class level, respectively, 95 and 87 % of the phylotypes among electrically retrieved bacteria were common to the gene clones from the direct sediment DNA extraction. This technique is a novel useful method to prepare bacterial cells in a single population or a community for metagenomic analyses.

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The purpose of this study was to develop the modulation methods for the attachment and detachment of specifically positioned adhesive animal cells cultured on an electrode surface with the application of a weak electrical potential. A patterned indium tin oxide (ITO) optically transparent working electrode was placed on the bottom of a chamber slide with a counter-(Pt) and reference (Ag/AgCl) electrode. The ITO patterning was formed by a reticulate ITO region and arrayed square glass regions of varying size. Using the 3-electrode culture system, the author succeeded in modulation of the attachment and detachment of animal cells on the working electrode surface. Animal cells suspended in serum or sera containing medium were drawn to and attached on a reticulate ITO electrode region to which a +0.4-V vs. Ag/AgCl-positive potential was applied. Meanwhile, the cells were successfully placed on the square glass regions by -0.3-V vs. Ag/AgCl-negative potential application. Animal cells were detached not only from the ITO electrode but also from the square glass regions after the application of a ±10-mV vs. Ag/AgCl, 9-MHz [corrected] rectangular wave potential in PBS(-) for 30-60 min. Rectangular wave potential-induced cell detachment is almost completely noncytotoxic, and no statistical differences between trypsinization and the high frequency wave potential application were observed in HeLa cell growth. The electrical modulation of the specifically positioned cell attachment and detachment techniques holds potential for novel optical microscopic cell sorting analysis in lab-on-chip devices.

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We used DNA transfection and protein introduction techniques to investigate the pressure tolerance of cytoskeletal structures in pectoral fin cells derived from the deep-sea fish Simenchelys parasiticus (habitat depth, 366-2,630 m). The deep-sea fish cells have G418 resistance. The cell number increased until day 6 of cultivation and all cells had died by day 35 when cultured in 35-mm Petri dishes in medium containing G418. Enhanced yellow fluorescent protein-tagged human beta-actin (EYFP-actin) was stably expressed by 1 in 100,000 deep-sea fish cells. Because almost none of the EYFP-actin was incorporated into actin filaments of the cells, we replaced the relatively large EYFP tag with a chemical fluorescent compound and succeeded in incorporating fluorescently labeled rabbit actins into the deep-sea fish actin filaments. Most of the filament structure in the cells with rabbit actin inserted underwent depolymerization when subjected to pressure of 100 MPa for 20 min, in contrast to control cells. There were no differences in the tubulin filament structure between control cells and deep-sea fish cells with fluorescein-labeled bovine tubulin inserted after the application of pressure ranging from 40 to 100 MPa for 20 min.

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The effect of elevated ambient pressures in deep sea fish residing at certain bottom depths or even covering different depth levels during migration is poorly understood. Elevated pressures are known to influence membrane properties of various excitable tissues in many species. Reliable results on membrane properties require freshly isolated living cells and short decompression times. During a scientific cruise south of Japan, deep sea fish were sampled from depths up to 1.000 m by using the intelligent operative net sampling system IONESS. On-site electrophysiological recordings of resting membrane potentials were performed in freshly isolated skeletal muscles from Sigmops gracile. Experiments were conducted at various extracellular K+ concentrations to derive relative membrane ion permeabilities and estimate intracellular K+ concentrations [K+]i in the muscles studied. With increasing sampling depth, a tendency for depolarized resting membrane potentials was observed. This could be explained by an increase in relative Na+ over K+ resting membrane permeabilities. Fish samples from deeper sites also had larger [K+]i values compared with shallower sites. This study represents a first approach to perform sophisticated physiological live-cell experiments on board a fully operating ship. These data are expected to more realistically reflect the physiological state of biological preparations residing in the deep sea.

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Establishing tissue cultures derived from deep-sea multicellular organisms has been extremely difficult because of the serious damage they sustain upon decompression and exposure to the high temperature of surface seawater. We developed a novel pressure-stat aquarium system for the study of living deep-sea multicellular organisms under pressure. Using this system, we have succeeded in maintaining a variety of deep-sea multicellular organisms under pressure and atmospheric conditions after gradual, slow decompression. Furthermore, we successfully cultivated and freeze-stocked pectoral fin cells of the deep-sea eel Simenchelys parasiticus collected at a depth of 1,162 m under atmospheric pressure conditions. This review describes novel capture and maintenance devices for deep-sea organisms and cell culture studies of the organisms under atmospheric and pressure conditions.

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We investigated the pressure tolerance of deep-sea eel (Simenchelys parasiticus; habitat depth, 366-2,630 m) cells, conger eel (Conger myriaster) cells, and mouse 3T3-L1 cells. Although there were no living mouse 3T3-L1 and conger eel cells after 130 MPa (0.1 MPa = 1 bar) hydrostatic pressurization for 20 min, all deep-sea eel cells remained alive after being subjected to pressures up to 150 MPa for 20 min. Pressurization at 40 MPa for 20 min induced disruption of actin and tubulin filaments with profound cell-shape changes in the mouse and conger eel cells. In the deep-sea eel cells, microtubules and some actin filaments were disrupted after being subjected to hydrostatic pressure of 100 MPa and greater for 20 min. Conger eel cells were sensitive to pressure and did not grow at 10 MPa. Mouse 3T3-L1 cells grew faster under pressure of 5 MPa than at atmospheric pressure and stopped growing at 18 MPa. Deep-sea eel cells were capable of growth in pressures up to 25 MPa and stopped growing at 30 MPa. Deep-sea eel cells required 4 h at 20 MPa to finish the M phase, which was approximately fourfold the time required under atmospheric conditions.

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We report successful larval hatching of deep-sea shrimp after decompression to atmospheric pressure. Three specimens of deep-sea shrimp were collected from an ocean depth of 1157 m at cold-seep sites off Hatsushima Island in Sagami Bay, Japan, using a pressure-stat aquarium system. Phylogenetic analysis of Alvinocaris sp. based on cytochrome c oxidase subunit gene sequences confirmed that these species were a member of the genus Alvinocaris. All 3 specimens survived to reach atmospheric pressure conditions after stepwise 63-day decompression. Two of the specimens contained eggs, which hatched after 10 and 16 days, respectively, of full decompression. Although no molting of the shrimp larvae was observed during 74 days of rearing under atmospheric pressure, the larvae developed conventional dark-adapted eyes after 15 days.

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We successfully cultivated fin cells of the deep-sea eel Simenchelys parasiticus (collected at 1,162 m) in L-15 medium supplemented with fetal bovine serum (FBS) and additional NaCl. We found that the pectoral fin cells proliferated in L-15 medium enriched with 4 g/l of NaCl salt (pH 7.3) containing 10% FBS at 10 degrees C and 15 degrees C. No cells were attached to the plastic culture plates when Dulbecco's modified Eagle's medium (pH 7.8) or 0-2 g/l of NaCl was added to the medium or when incubation was carried out at 4 degrees C. The majority of the explant outgrowth cells were detached when temperature increased to higher than 15 degrees C. The rate of proliferation of the fin cells was extremely slow and was dependent on the FBS concentration. Cell growth was enhanced by approximately 2.2-fold, and doubling time decreased from 170 h to 77 h when the FBS concentration was increased from 10% to 20% (v/v). Our established deep-sea eel cells were passaged 16 times over a 1-year period under atmospheric pressure conditions.

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Normal human dermal fibroblasts were found to survive and to be active in producing interleukin (IL)-6 and IL-8 under extremely high hydrostatic pressure, up to 40 MPa (1 atm=0.101325 MPa=1.03323 kgf/cm(2)), for 20 min. An inhibitor of protein kinase C (PKC) reduced the amount of IL-6 production, whereas IL-8 production was increased following pressure application. The activation of PKC in response to exposure to the pressure stress was detected by using the PKC-specific probe Rim-1. These findings indicate that IL-6 production induced by hydrostatic pressure stresses was dependent on the PKC signaling pathway. In contrast, pressure-induced IL-8 production was inhibited by PKC activity.

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We found that extremely high hydrostatic pressure stresses induced a variety of cytokines production in normal human dermal fibroblasts. Normal human dermal fibroblasts were found to survive and were active in producing interleukin (IL) -6, -8, and monocyte chemoattractant protein-1 (MCP-1) under extremely high hydrostatic pressure, up to 70 MPa (=690.8 atm=713.8 kgf/cm2). 70 MPa pressure application extremely enhanced IL-6 and IL-8 secretions (about 130 folds) without transcriptional enhancement. Although induction of IL-1alpha, IL-1beta, and IL-12 mRNAs was appeared under high hydrostatic pressure condition, no translation of IL-1alpha, IL-1beta, and IL-12 proteins was found. Extracellular accumulation of constitutively produced MCP-1 was down regulated in the pressure-applied fibroblasts in the absence of transcriptional repression. These results indicated that hydrostatic pressure stresses triggered post-transcriptional regulation mechanisms that modulated the cytokines production.