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1.
Rafael A Vargas Lucía Botero Luisa Lagos Marcela Camacho 《Cellular physiology and biochemistry》2004,14(4-6):197-202
Oocytes from Xenopus laevis are commonly used as an expression system for ion channel proteins. The aim of this study was to determine whether oocytes from the Colombian native toad, Bufo marinus, could be used as an alternative expression system for ion channel protein expression and functional characterization using the two-microelectrode voltage clamp method. B. marinus oocytes and X. laevis were isolated and cultured in similar conditions. The mean resting membrane potential of B. marinus oocytes was similar to that of X. laevis oocytes as well as the whole-cell basal currents. The potassium ion channel Kv1.1 was successfully expressed in B. marinus oocytes and showed a typical outward rectifying current. Potassium channel blockers reduced these currents. The similarities on electrical properties and expression of ion channel proteins show that B. marinus oocytes can be used effectively to express these proteins, making these cells a viable heterologous system for the expression of ion channel proteins and their electrophysiological characterization. 相似文献
2.
The morphology of Vibrio marinus MP-1 was studied by phase and electron microscopy. The ultrastructure of the vibrio form of V. marinus was found to be typically gram-negative with a trilaminar plasma membrane and cell wall. The coccoid or round bodies noted in otherwise pure cultures of V. marinus were frequently found in early and late stationary phase of growth. The round bodies in ultrathin section were found to contain at least one, and often three or four, cell units. Three types of round bodies were observed in ultrathin section, each differing in size and behavior: "spherules," "spheres" or the "round body," and "giant cells" or "macrospheres." The round bodies appeared to be associated with, or to result from, the constrictive cell division of V. marinus. 相似文献
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An electron microscope study of Vibrio marinus strains MP-1, an obligate psychrophile, and PS-207, a moderate psychrophile, revealed numerous intracellular membranous structures. The structures were found to occur more frequently in V. marinus strain MP-1 than in strain PS-207. The frequency of occurrence and complexity of structure were related to age of the culture. In early logarithmic phase, cells revealed invaginations of the plasma membrane. More complex membrane forms, found in late logarithmic and stationary phase, were either myelin-like sheaths, for which the term "myelemma" is proposed, or membranes randomly arranged throughout the cells. The complex membrane forms were not observed to be directly connected with the plasma membrane. However, they were often found in approximation to the plasma membrane or associated with vacuoles and circular membrane profiles. Individual membranes were of a tripartite structure and of dimensions similar to the cell wall and plasma membrane. 相似文献
4.
Nunes OC Manaia CM Da Costa MS Santos H 《Applied and environmental microbiology》1995,61(6):2351-2357
(sup13)C nuclear magnetic resonance spectroscopy and (sup1)H nuclear magnetic resonance spectroscopy were used to identify and quantify the organic solutes of several strains of halophilic or halotolerant thermophilic bacteria. Two strains of Rhodothermus marinus and four strains of "Thermus thermophilus" grown in complex medium containing NaCl were examined. 2-O-Mannosylglycerate was a major compatible solute in all strains: the Thermus strains accumulated the (beta)-anomer only, whereas both anomers were found in R. marinus. 2-O-(beta)-mannosylglycerate and 2-O-(alpha)-mannosylglycerate were the major compatible solutes in R. marinus. The former was the predominant solute in cells grown in 2.0 and 4.0% NaCl-containing medium, while the latter was the predominant compatible solute at higher salinities. Glutamate, trehalose, and glucose were also present as minor components. The intracellular K(sup+) concentration, as determined by (sup39)K nuclear magnetic resonance spectroscopy, in R. marinus increased with salinity and was sufficient to balance the negative charges of the mannosylglycerate. In addition to 2-O-(beta)-mannosylglycerate, trehalose was a major compatible solute of "T. thermophilus." 2-O-(beta)-Mannosylglycerate was the main solute in medium containing 1.0 or 2.0% NaCl, while trehalose predominated in cells grown in medium supplemented with 3.0 or 4.0% NaCl. Glycine betaine, in lower concentrations, was also detected in two "T. thermophilus" strains. This is the first report of mannosylglycerate as a compatible solute in bacteria. 相似文献
5.
Plant regeneration from protoplasts isolated from haploid cell suspensions of commercial supersweet maize (SS 7700) was achieved and the plants were survival after transfer into soil in pots. Protoplast plating efficiency obtained from feeder layer system was 130 folds higher as compared with conventional liquid culture method, the composition of protoplast culture medium, the pore size of supportive membrane filter and the relationship between protoplasts and feeder cells were critical for callus formation. An enriched medium containing vitamins, organic acids, amino-acids and other organic substances such as coconut water could extremely improve callus formation. Filters with pore size within the range of 0.22–8.0 μm in diameter was useful. Filters with smaller pore size of 0.04 μm or larger 11 μm appeared to decrease the frequency of protocolony formation. The feeder cells which belong to the same species (Zea mays) as protoplasts greatly increased protoplast plating efficiencies as compared to those of feeder cells belonging to other species such as Avena nuda and Nicotiana tabacum. Among 11 protoplast-regenerated plants examined, 10 plants were haploid and one plant was diploid. 相似文献
6.
Summary Plants were regenerated from maize (Zea mays L.) protoplasts isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures. 相似文献
7.
BRL条件培养基在ES细胞培养中的应用方法探讨 总被引:1,自引:0,他引:1
目的:探讨布法罗大鼠肝细胞条件培养基(Buffalo rat liver cell conditioned medium,BRL)在ES细胞培养中的应用方法。方法:ES细胞复苏后分别培养在BRL条件培养基、小鼠胚胎成纤维细胞饲养层(mouse enbryonic fibroblast,MEF)及合并应用BRL条件培养基和MEF饲养层的环境中,通过细胞计数、拟胚体计数和ES细胞集落边缘细胞分化状态比较ES细胞在三种培养基中生长和分化差异。结果:与BRL组比较,MEF组和BRL+MEF组细胞生长较快(P<0.01),ES细胞集落边缘分化细胞较少;MEF组和BRL+MEF组无明显差异。结论:在复苏后早期阶段ES细胞培养中,不宜单独应用BRL条件培养基,须用MEF饲养层或合并应用BRL条件培养基和MEF饲养层。 相似文献
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Anderson IJ Sun H Lapidus A Copeland A Glavina Del Rio T Tice H Dalin E Lucas S Barry K Land M Richardson P Huber H Kyrpides NC 《Standards in genomic sciences》2009,1(2):183-188
Staphylothermus marinus Fiala and Stetter 1986 belongs to the order Desulfurococcales within the archaeal phylum Crenarchaeota. S. marinus is a hyperthermophilic, sulfur-dependent, anaerobic heterotroph. Strain F1 was isolated from geothermally heated sediments at Vulcano, Italy, but S. marinus has also been isolated from a hydrothermal vent on the East Pacific Rise. We report the complete genome of S. marinus strain F1, the type strain of the species. This is the fifth reported complete genome sequence from the order Desulfurococcales. 相似文献
10.
目的 分离和培养 6 15小鼠的ES细胞集落 ,为建系打下基础。方法 以PMEF为饲养层分离 6 15小鼠的ES细胞集落 ,进行无饲养层培养 ,并对其进行初步鉴定。结果 ES细胞集落的出现率和传代成功率为 2 2 6 %和 0 94 % ,其ALP染色阳性 ,具有稳定的二倍体核型 ,可自发分化为多种类型的细胞。结论 成功分离和培养了6 15小鼠的ES细胞集落 相似文献
11.
Duerr JM Tucker K 《Journal of experimental zoology. Part A, Ecological genetics and physiology》2007,307(8):425-438
Cardiac mitochondria were isolated from Bufo marinus and Rana catesbeiana, two species of amphibian whose cardiovascular systems are adapted to either predominantly aerobic or glycolytic modes of locomotion. Mitochondrial oxidative capacity was compared using VO2 max and respiratory control ratios in the presence of a variety of substrates including pyruvate, lactate, oxaloacetate, beta-hydroxybutyrate, and octanoyl-carnitine. B. marinus cardiac mitochondria exhibited VO2 max values twice that of R. catesbeiana cardiac mitochondria when oxidizing carbohydrate substrates. Pyruvate transport was measured via a radiolabeled-tracer assay in isolated B. marinus and R. catesbeiana cardiac mitochondria. Time-course experiments described both alpha-cyano-4-hydroxycinnamate-sensitive (MCT-like) and phenylsuccinate-sensitive pyruvate uptake mechanisms in both species. Pyruvate uptake by the MCT-like transporter was enhanced in the presence of a pH gradient, whereas the phenylsuccinate-sensitive transporter was inhibited. Notably, anuran cardiac mitochondria exhibited activities of lactate dehydrogenase and pyruvate carboxylase. The presence of both transporters on the inner mitochondrial membrane affords the net uptake of monocarboxylates including pyruvate, beta-hydroxybutyrate, and lactate; the latter potentially indicating the presence of a lactate/pyruvate shuttle allowing oxidation of extramitochondrial NADH. Intramitochondrial lactate dehydrogenase and pyruvate carboxylase enables lactate to be oxidized to pyruvate or converted to anaplerotic oxaloacetate. Kinetics of the MCT-like transporter differed significantly between the two species, suggesting differences in aerobic scope may be in part attributable to differences in mitochondrial carbohydrate utilization. 相似文献
12.
Perkinsus marinus is a highly contagious pathogen of the eastern oyster Crassostrea virginica. Until recently, transmission studies have employed wild-type parasites isolated directly from infected oysters. Newly developed methods to propagate P. marinus in vitro have led to using cultured parasites for infection studies, but results suggest that cultured parasites are less virulent than wild-type parasites In this paper, we report results of experiments designed to quantify differences between wild-type and cultured P. marinus virulence and to test the following hypotheses: (1) in vitro-cultured parasites are less virulent than wild-type parasites; (2) virulence decreases gradually during in vitro culture; (3) virulence of in vitro cultures can be restored by in vivo passage; (4) virulence changes with culture phase. Our results demonstrate that parasites freshly isolated from infected hosts are much more virulent than those propagated in culture, indicating a potential deficiency in the culture medium used. Virulence was lost immediately in culture and, for that reason, the practice of repassing cultured cells through the host to restore virulence does not work for P. marinus. Virulence was also associated with culture phase: log-phase parasites were significantly more virulent than those obtained from lag- or stationary-phase cultures. 相似文献
13.
In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice,the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF).Compared with the feeder layer of MEF cells,medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyotypic normality of ES cells only in short term cell propagation.Besides,ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras.Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more converient and efficient than the conventional microdrop method. 相似文献
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人孤雌胚胎干细胞(human parthenogenetic embryonic stem cells,hPESCs)体外培养常需饲养层的支持以保持干细胞特性.通过原代培养获得人包皮成纤维细胞(human foreskin fibroblasts,hFFs)并将其制备成饲养层,使hPESCs在hFFs上进行体外培养及传代.倒置显微镜下观察hPESCs的生长状态,采用碱性磷酸酶(alkalinephosphatase,AKP)检测、核型分析和体内分化实验研究hPESCs的生物学特性及分化潜能,以探索hFFs能否长期支持hPESCs的生长并维持其未分化状态.经原代培养成功获得了hFFs,通过形态学观察和免疫细胞化学染色鉴定符合成纤维细胞的生物学特性;在hFFs上生长的hPESCs克隆形态规则,不易分化;已成功在体外培养20余代,hPESCs仍能够保持基本生物学特性和正常核型,在裸鼠体内可形成含有3个胚层组织成分的畸胎瘤.作为人源性饲养层,hFFs可长期支持hPESCs的生长并维持其未分化状态. 相似文献
16.
Establishment of the Embryo-derived Stem (ES) Cell Lines from Mouse Blastocysts: Effects of the Feeder Cell Layer. 总被引:5,自引:0,他引:5
The ES ceii lines are embryo-derived stem cell lines directly isolated from the inner cell mass of mouse blastocysts using feeder cell layer. We have established a number of ES cell lines from 129 or C57BL/6 strain mice by using the feeder layer of the STO cells (from ATCC) or the primary embryonic fibroblasts, which was obtained by trypsinizing the 16-day-old BALB/c mouse fetus. The ES cell lines established on the STO feeder layer showed differentiation into various tissues in solid tumors when injected into syngenic mice. Karyotype was, however, nearly tetraploid. The ES cell lines established on the primary fibroblasts exhibited differentiation into larger variety of tissues in solid tumors. Karyotype was almost diploid and majority of the cells kept normal set of chromosomes in G-banding. We conclude that the primary fibroblasts are better feeder layer than the STO cells for establishment and maintenance of the ES cell lines. 相似文献
17.
用小鼠腹腔细胞等作为滋养细胞培养人杂交瘤,国内外均有报道。我们曾建立了人包皮传代细胞株。本文利用我们所建立的人包皮细胞做滋养细胞,并与小鼠腹腔细胞及不加滋养细胞的空白作对照,观察人包皮细胞对杂交瘤克隆生长的影响。 将人包皮细胞和小鼠腹腔细胞分别接种同一96孔板,次日将两株不同的人杂交瘤细胞1B4及1C8稀释至每毫升含10个或50个细胞,每孔接种0.1ml,使每孔分别含1个及5个细胞,经不同时间观察每孔杂交瘤克隆的生长情况,记录有杂交瘤生长的孔数。表1结果表明, 相似文献
18.
This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.Download video file.(75M, mov) 相似文献
19.
M González M Qui?onez J A Argibay 《Comparative biochemistry and physiology. A, Comparative physiology》1985,81(3):619-625
Inward rectification of the resting potassium conductance was studied in skeletal muscle fibres of the toad Bufo marinus. This conductance was shown to be blocked by Ba and Cs and located both in the surface membrane and the membranes of the tubular system. Some differences were found between the properties of this conductance channel in Bufo marinus and those reported for various species of Rana. The possible adaptative value of these differences is pointed out. 相似文献
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