The mechanism mediating regenerative intercellular Ca2+ waves in the blowfly salivary gland.
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Intercellular Ca2+ signaling in intact salivary glands of the blowfly Calliphora erythrocephala was studied by fluorimetric digital imaging combined with microinjection of putative messenger molecules. Iontophoretic injection of D-myo-inositol 1,4, 5-trisphosphate (InsP3) into salivary gland cells evoked regenerative intercellular Ca2+ waves that spread through the impaled cell and several rows of surrounding cells. Ca2+ increases induced by microinjection of Ca2+ ions were confined to the injected cells and their nearest neighbors. Depletion of intracellular Ca2+ stores by thapsigargin pre-treatment did not alter the time course of the Ca2+ increase caused by Ca2+ injection. However, activation of Ca2+ release became clearly evident when Ca2+ was injected in the presence of serotonin (5-HT). Under these conditions, injection of Ca2+ triggered intercellular Ca2+ waves that consecutively passed through >10 cells. The phospholipase C inhibitor U73122 blocked 5-HT-induced Ca2+ increases but did not affect InsP3-dependent Ca2+ spiking and intercellular Ca2+ wave propagation. The results demonstrate that propagation of agonist-evoked Ca2+ waves in the blowfly salivary gland requires supra-basal [InsP3] but does not depend on feedback activation of phospholipase C. We conclude that the intra- and intercellular transmission of these Ca2+ waves is mediated by diffusion of Ca2+ and Ca2+-induced Ca2+ release via the InsP3 receptor channel. (+info)
Increased doxorubicin uptake and toxicity in multicellular tumour spheroids treated with DC electrical fields.
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Electrochemotherapy (ECT) is a new approach to the treatment of tumours. In the present study, multicellular prostate tumour spheroids were treated with non-lethal direct current (DC) electrical fields, and uptake and toxicity of doxorubicin were investigated. An electrical field with a field strength of 500 Vm(-1) applied for a duration of 90 s resulted in neither reversible nor irreversible membrane breakdown as revealed by fluid phase uptake studies of the membrane impermeant tracer Lucifer yellow. However, treated spheroids showed an increased uptake of doxorubicin and, consequently, an increased toxicity following electrical field exposure. The electrical field raised intracellular reactive oxygen species (ROS) as revealed using 2',7'-dichlorofluorescein diacetate (H2DCFDA) as an indicator. ROS induced membrane lipid peroxidation since the lipid peroxidation end products malondialdehyde (MDA) and 4-hydroxy-2-(E)-nonenal (4-HNE) were detected after electrical field treatment. Moreover, lipid peroxidation decreased the lipid diffusion coefficient D from 4.2 x 10(-10) cm2 s(-1) to 2.7 x 10(-10) cm2 s(-1) in the control and treated sample, respectively, as revealed by fluorescence recovery after photobleaching (FRAP) experiments. The field effects could be mimicked by incubating spheroids with 100 nM hydrogen peroxide and were inhibited by the radical scavengers dehydroascorbate (DHA) and alpha-tocopherol (vitamin E), indicating that the increased uptake of doxorubicin after electrical field treatment is owing to lipid peroxidation and decreased membrane lipid mobility. Treatment of tumours with low intensity electrical fields may be useful to improve the cytotoxic capacity of anthracyclines. (+info)
Differential regulation of corticotropin-releasing hormone and vasopressin gene transcription in the hypothalamus by norepinephrine.
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All stress-related inputs are conveyed to the hypothalamus via several brain areas and integrated in the parvocellular division of the paraventricular nucleus (PVN) where corticotropin-releasing hormone (CRH) is synthesized. Arginine vasopressin (AVP) is present in both magnocellular and parvocellular divisions of the PVN, and the latter population of AVP is colocalized with CRH. CRH and AVP are co-secreted in the face of certain stressful stimuli, and synthesis of both peptides is suppressed by glucocorticoid. CRH and AVP stimulate corticotropin (ACTH) secretion synergistically, but the physiological relevance of the dual corticotroph regulation is not understood. Norepinephrine (NE) is a well known neurotransmitter that regulates CRH neurons in the PVN. We explored the mode of action of NE on CRH and AVP gene transcription in the PVN to examine the effect of the neurotransmitter on multiple genes that are responsible for a common physiological function. After NE injection into the PVN of conscious rats, CRH heteronuclear (hn) RNA increased rapidly and markedly in the parvocellular division of the PVN. AVP hnRNA did not change significantly in either the parvocellular or magnocellular division of the PVN after NE injection. The present results show that the transcription of CRH and AVP genes is differentially regulated by NE, indicating the complexity of neurotransmitter regulation of multiple releasing hormone genes in a discrete hypothalamic neuronal population. (+info)
Quinolone accumulation by Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli.
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The accumulation of nalidixic acid and 14 fluoroquinolones over a range of external drug concentrations (10-100 mg/L; c. 25-231 microM) into intact cells of Escherichia coli KL-16, Staphylococcus aureus NCTC 8532, Pseudomonas aeruginosa NCTC 10662 and spheroplasts of E. coli was investigated. The effect of 100 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) upon the concentration of quinolone accumulated by intact cells and spheroplasts of E. coli was also determined. Except for pefloxacin, there was an increase in the concentration of the six quinolones examined accumulated by E. coli, despite a reduction in fluorescence at alkaline pH. For ciprofloxacin the partition coefficient (P(app)) was constant despite an increase in the pH; however, the P(app) for nalidixic acid decreased significantly with an increase in pH. The concentration of nalidixic acid, ciprofloxacin and enrofloxacin accumulated by E. coli and S. aureus increased with an increase in temperature up to 40 degrees C and 50 degrees C, respectively. Above these temperatures the cell viability decreased. With an increase in drug concentration there was, for intact E. coli and 12/15 agents, and for S. aureus and 10/15 agents, a linear increase in the concentration of drug accumulated. However, for P. aeruginosa and 13/15 agents there was apparent saturation of an accumulation pathway. Assuming 100% accumulation into intact cells of E. coli, for 10/14 fluoroquinolones < or = 40% was accumulated by spheroplasts. CCCP increased the concentration of quinolone accumulated but the increase varied with the agent and the bacterial species. The variation in the effect of CCCP upon accumulation of the different quinolones into E. coli could result from chemical interactions or from different affinities of the proposed efflux transporter for each quinolone. Overall, these data suggest that accumulation of most quinolones into E. coli and S. aureus proceeds by simple diffusion, but that P. aeruginosa behaves differently. (+info)
Permeability of single nuclear pores.
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In this first application of optical single transporter recording (OSTR), a recently established technique for optically monitoring the activity of single transporters in membrane patches (Tschodrich-Rotter and Peters. 1998. J. Microsc. 192:114-125), the passive permeability of the nuclear pore complex (NPC) was measured for a homologous series of hydrophilic probe molecules. Nuclei were isolated from Xenopus oocytes and firmly attached to filters containing small cylindrical pores. Transport through membrane patches spanning filter pores was measured by scanning microphotolysis. Thus the permeability coefficients of single NPCs were determined for fluorescently labeled dextrans of approximately 4, 10, and 20 kDa. Dextrans of >/=40 kDa could not permeate the NPC. The data were consistent with a model in which the NPC contains a single diffusion channel. By application of established theories for the restricted diffusion through small pores, the diffusion channel was approximated as a cylinder with a radius of 4.4-6.1 nm (mean 5. 35 nm). Because the transport rate constant of the single NPC was known, the equivalent length of the channel could be also determined and was found to be 40-50 nm (mean 44.5 nm). The symmetry of the NPC implies that a singular component such as the diffusion channel is located at the center of the NPC. Therefore a common transport pathway apparently mediates both passive and signal-dependent transport. To test this hypothesis, measurements of signal-dependent transport and of the mutual effects signal-dependent and passive transport may exert on each other are in progress. (+info)
Conformational changes of the in situ nuclear pore complex.
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By bridging the double membrane separating the cell nucleus and cytoplasm, nuclear pore complexes (NPCs) are crucial pathways for the exchange of ions, proteins, and RNA between these two cellular compartments. A structure in the central lumen of the NPC, called the nuclear transport protein, central granule, or nuclear plug, appeared to gate diffusion of intermediate-sized molecules (10-40 kDa) across the nuclear membranes. Visualization of the NPC required drying and fixation of the specimen for electron and atomic force microscopy (AFM), a requirement that has raised doubts about the physiological relevance of the observation. Here we present AFM images of the outer nuclear membranes and NPCs of Xenopus laevis oocytes under more physiological conditions. Measured under a variety of Ca2+ depletion conditions, the central granule appeared to occupy and occlude the lumen of the pore in >80% of NPCs compared to <10% in controls. In a few instances images were obtained of the same NPCs as the solution was changed from control saline to store depletion conditions, and finally to store repletion conditions. We conclude that the central lumen of the nuclear pore complex undergoes a conformational change in response to depletion of nuclear cisternal Ca2+ levels. (+info)
Anisotropic motion of cholesterol in oriented DPPC bilayers studied by quasielastic neutron scattering: the liquid-ordered phase.
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Quasielastic neutron scattering (QENS) at two energy resolutions (1 and 14 microeV) was employed to study high-frequency cholesterol motion in the liquid ordered phase (lo-phase) of oriented multilayers of dipalmitoylphosphatidylcholine at three temperatures: T = 20 degrees C, T = 36 degrees C, and T = 50 degrees C. We studied two orientations of the bilayer stack with respect to the incident neutron beam. This and the two energy resolutions for each orientation allowed us to determine the cholesterol dynamics parallel to the normal of the membrane stack and in the plane of the membrane separately at two different time scales in the GHz range. We find a surprisingly high, model-independent motional anisotropy of cholesterol within the bilayer. The data analysis using explicit models of molecular motion suggests a superposition of two motions of cholesterol: an out-of-plane diffusion of the molecule parallel to the bilayer normal combined with a locally confined motion within the bilayer plane. The rather high amplitude of the out-of-plane diffusion observed at higher temperatures (T >/= 36 degrees C) strongly suggests that cholesterol can move between the opposite leaflets of the bilayer while it remains predominantly confined within its host monolayer at lower temperatures (T = 20 degrees C). The locally confined in-plane cholesterol motion is dominated by discrete, large-angle rotational jumps of the steroid body rather than a quasicontinous rotational diffusion by small angle jumps. We observe a significant increase of the rotational jump rate between T = 20 degrees C and T = 36 degrees C, whereas a further temperature increase to T = 50 degrees C leaves this rate essentially unchanged. (+info)
Evolution of the internal dynamics of two globular proteins from dry powder to solution.
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Myoglobin and lysozyme picosecond internal dynamics in solution is compared to that in hydrated powders by quasielastic incoherent neutron scattering. This technique is sensitive to the motions of the nonexchangeable hydrogen atoms in a sample. Because these are homogeneously distributed throughout the protein structure, the average dynamics of the protein is described. We first propose an original data treatment to deal with the protein global motions in the case of solution samples. The validity of this treatment is checked by comparison with classical measurements of the diffusion constants. The evolution with the scattering vector of the width and relative contribution of the quasielastic component was then used to derive information on the amount of local diffusive motions and their characteristic average relaxation time. From dry powder to coverage by one water layer, the surface side chains progressively acquire the possibility to diffuse locally. On subsequent hydration, the main effect of water is to improve the rate of these diffusive motions. Motions with higher average amplitude occur in solution, about three times more than for a hydrated powder at complete coverage, with a shorter average relaxation time, approximately 4.5 ps compared to 9.4 ps for one water monolayer. (+info)