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91.
Takashi Aoki Ikuo Hirono Teresa de Castro Tadatoshi Kitao 《Zeitschrift fur angewandte Ichthyologie》1989,5(2):67-73
A 562 base pair fragment of DNA from a serotype A strain of Vibrio anguillarum was cloned into pUC9 and used as a hybridization probe for the rapid identification of Vibrio anguillarum by colony hybridization. The probe was tested on nine different fish pathogens, 15 Vibrio isolates, 2 organisms closely related to Vibrio, and 9 serotypes of V. anguillarum. The probe hybridized only with the DNA of V. anguillarum serotypes A and H. The sequence of the 562 nucleotides have been determined. This probe allows rapid, reliable, and specific detection of V. anguillarum in freshwater ayu, Plecoglossus altivelis. 相似文献
92.
Giovanna Cacciapuoti Maria Angela Moretti Sabrina Forte Assunta Brio Laura Camardella Vincenzo Zappia Marina Porcelli 《European journal of biochemistry》2004,271(23-24):4834-4844
The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins. 相似文献
93.
Marina S. Trofimova Inna M. Zhestkova Valentina P. Kholodova Igor M. Andreev Evgeny M. Sorokin Anna G. Kruglova Vladimir V. Kuznetsov 《Physiologia plantarum》2003,118(2):232-239
The osmotic water permeability ( P os ) of cell membranes isolated from leaves of 40-, 50- and 60-day-old Mesembryanthemum crystallinum plants was estimated by measuring light-scattering kinetics using stopped-flow spectrophotometry. The measurements were performed on the plasma membrane (PM), purified tonoplast (TP), and TP-enriched vesicles. The PM and TP-enriched vesicles were obtained by partitioning the microsomal fraction in an aqueous polymer two-phase system, whereas the purified TP vesicles were prepared by microsomal vesicle flotation on a sucrose cushion. The P os of isolated membranes declined with plant age. The kinetic experiments showed that there was no difference between the P os of the PM and TP isolated from plants of all ages. A 24-h exposure of plants to 400 m M NaCl caused a decline in the P os as well. These findings suggest that, during M. crystallinum transition to CAM, which was induced by plant ageing or salinity, plant osmoregulatory responses included changes in the P os of the leaf-cell membranes. These variations in the P os are discussed in the context of adaptive mechanisms responsible for the maintenance of the water balance in the common ice plant. 相似文献
94.
Antonio del Castillo-Olivares Alicia Esteban del Valle Javier Márquez Ignacio NÚñez de Castro Miguel ángel Medina 《Journal of bioenergetics and biomembranes》1995,27(6):605-611
Ehrlich cell plasma membrane ferricyanide reductase activity increased in the presence of mastoparan, a generic activator of G proteins, using either whole cells or isolated plasma membrane fractions. Agents that increase intracellularcAMP also increased the rate of ferricyanide reduction by Ehrlich cells. For the first time, evidence is shown on a modulation of plasma membrane redox system bycGMP. In fact, permeant analogs ofcGMP, dibutyrylcGMP, and 8-bromo-cGMP increased the rate of ferricyanide reduction by the Ehrlich cell plasma membrane redox system. Furthermore, specific inhibition ofcGMP-phosphodiesterases by dipyridamole was also accompanied by an enhancement in the rate of ferricyanide reduction. On the other hand, treatments expected to increase cytoplasmic Ca2+ concentrations were accompanied by a remarkable stimulation of the reductase activity. Taking all these data together, it seems that the Ehrlich cell plasma membrane redox system is under a multiple and complex regulation by different signal transduction pathways involving G proteins, cyclic nucleotides, and Ca2+ ions. 相似文献
95.
Abstract: We studied the action of H2 O2 on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H2 O2 (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca2+ -dependent exocytosis of glutamate, induced by KCl or by the K+ -channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H2 O2 , although a transient decrease was observed after the treatment. H2 O2 did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H2 O2 did not modify significantly the KCl-induced increase of [Ca2+ ]i . H2 O2 inhibited exocytosis also when the latter was induced by increasing [Ca2+ ]i with the Ca2+ ionophore ionomycin. The effects of H2 O2 were unchanged in the presence of superoxide dismutase and the presence of the Fe3+ chelator deferoxamine. These results appear to indicate that H2 O2 , apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process. 相似文献
96.
Chronic photoinhibition in seedlings of tropical trees 总被引:1,自引:0,他引:1
Seedlings of five canopy species of tropical trees from Costa Rica and Puerto Rico were grown in full shade (midday range of photosynthetic photon flux density [PPFD], 100–140 μmol m?2 s?1), partial shade (midday PPFD, 400–600 μmol m?2 s?1) and full sun (midday PPFD, 1 500–1 800 μmol m?2 s?1) for 3 months. The species were Ochroma lagopus (Bombacaceae), a pioneer species; Inga edulis (Fabaceae), found in secondary forest; and Dipteryx panamensis (Fabaceae), Hampea appendiculata (Malvaceae), and Manilkara bidentata (Sapotaceae), three species characteristic of primary forest. After the plants were placed in the dark overnight, chlorophyll fluorescence characteristics were measured for recently expanded and mature leaves. The ratio of variable fluorescence to maximum fluorescence (Fv/Fm) was used to estimate the degree of chronic photoinhibition. Only individuals of one species, Dipteryx panamensis, showed significant depression of Fv/Fm after long-term exposure to full sun. The depression was highly correlated with quantum yield of O2 evolution which also declined after exposure to full sun. The decline may have been related to foliar N concentration. Although all plants were supplied with ample nutrients, foliar N did not increase significantly for Dipteryx seedlings in full sun, whereas it did for Ochroma and Inga. Leaf age affected Fv/Fm only in the cases of Manilkara, where it was slightly lower in recently expanded leaves, and of Dipteryx where it interacted with the effects of light regime. We conclude that chronic photoinhibition is not common in seedlings of canopy trees of tropical rain forests except when availability of mineral nutrients may be limiting. 相似文献
97.
98.
Ana Alonso-Varona Yolanda Calle Teodoro Palomares Begoa Castro Emilio Barber-Guillem 《Biology of the cell / under the auspices of the European Cell Biology Organization》1995,83(1):87-92
Summary— We designed a protocol for cell labeling with the lectin wheat germ agglutinin (WGA) linked to the fluorochrome tetramethyl-rhodamine isothiocyanate (TRITC) for effective detection of the B16F10 melanoma and Lewis lung carcinoma (LLc) cells on pulmonary histological sections from C57BL6; mice. We have also determined a suitable concentration of WGA-TRITC (10 μg/ml), which leads to a very intense and homogeneous labeling of the cells, as it avoids cell clumping due to the presence of the lectin WGA. In order to determine to what extent the method affects these tumor cells, we have studied some important aspects related to their metastatic behavior, taking into account three parameters: a) viability and rate of proliferation of the cells cultured in vitro; b) percentage of animals C57BL6 mice) bearing metastasis 15 days after intravenous inoculation with 105 B16F10 or LLc cells; and c) pattern of distribution of tumor foci in lung. There were no significant differences in these three parameters between the WGA-TRITC labeled-cells compared to the cultures of non-labeled cells in either of the cell lines (B16F10, LLc). Thus, we conclude that B16F10 and LLc tumor cells can be labeled following the protocol set-up in our study, as it allows these cells to be neatly identified on tissue sections and it causes no important physiological changes in the cells, with regard to metastatic behavior. These points make this technique very suitable for the detection of B16F10 and LLc cells on histological sections in studying their behavior during the first stages of the metastatic process. 相似文献
99.
De Castro LD 《Bioethics》1995,9(3-4):259-268
Relatively subtle forms of exploitation of human subjects may arise from the inefficiency or incompetence of a researcher, from the existence of a power imbalance between principal and subject, or from the uneven distribution of research risks among various segments of the population. A powerful and knowledgeable person (or institution) may perpetrate the exploitation of an unempowered and ignorant individual even without intending to. There is an ethical burden on the former to protect the interests of the vulnerable. Excessive or insufficient compensation may be exploitative. However, genuine economic imperatives motivating needy volunteers have to be considered. These forms of exploitation should be appreciated in the context of social and cultural factors suggesting that the relationship between researcher and subject cannot properly be appraised as a contractual undertaking. While compliance with pertinent codes and regulations minimises the exploitative potential, they cannot be enforced in a way that does not recognize a society's peculiar characteristics. The experience with some Filipino cultural traits illustrates this point. 相似文献
100.
Jesús Mateo Enrique Castro Jean Zwiller Dominique Aunis M. T. Miras-Portugal 《Journal of neurochemistry》1995,64(1):77-84
Abstract: We investigated the effect of the adenosine receptor agonist 5'-( N -ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30–40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 ± 3.3%), slowly developing inhibition of [Ca2+ ]i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca2+ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca2+ ]i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca2+ /protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca2+ influx pathway (e.g., L-type Ca2+ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells. 相似文献