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PubMed1982

High-affinity 86Rb-binding and structural changes in the alpha-subunit of Na+,K+-ATPase as detected by tryptic digestion and fluorescence analysis.

High-affinity 86Rb-binding has been related to tryptic cleavage and fluorescence from intrinsic and extrinsic probes in order to examine the relationship of cation binding to structural transitions in the alpha-subunit of pure membrane-bound Na+,K+-ATPase from the outer renal medulla. Native Na+,K+-Atpase binds two Rb+ ions per alpha-subunit (12.3 nmol/mg protein) with high affinity (Kd = 7.5 microM) in 25 mM Tris-HCl, pH 7.5. Enzyme with one molecule of covalently attached fluorescein per alpha-subunit has the same capacity (12.8 nmol/mg protein) but a much lower affinity for Rb+ (Kd = 29.2 microM). The changes in conformational state of the protein are correlated with occupancy of the high-affinity sites for Rb+, also at concentrations of Rb+ below the Kd. Titration at varying ionic strength suggests that the E2-form is the relaxed or native conformation of the alpha-subunit. Changes in tryptic digestion pattern and in fluorescence are parallel events both in the conditions of the binding assay and at physiological ionic strength. Reversible blocking of sulfhydryl groups with Thimerosal (ethylmercurythiosalicylate) abolishes the fluorescence responses to K+ or Rb+ without affecting the capacity or the affinity for binding of 86Rb. The demonstration of high-affinity binding of Rb+ without coupling to a conformational change suggests that the E1-form of the protein exposes sites for tight binding of K+ or Rb+ at the cytoplasmic membrane surface.

Animals↗

PubMed1986

Effects of elevated temperatures on the opacity and toxicity of pertussis vaccines manufactured with different inactivating agents.

Plain pertussis vaccine manufactured with different inactivating agents was evaluated for its stability at 4-8, 25 and 35 degrees C with regard to the opacity and the toxicity [mouse weight gain test (MWGT) and histamine sensitizing (HS) activity]. Two pools each of heat inactivated pertussis vaccine (HIPV), formaldehyde inactivated pertussis vaccine (FIPV), glutaraldehyde inactivated pertussis vaccine (GIPV), thimerosal inactivated pertussis vaccine (TIPV) and acetone (I) treated pertussis vaccine [A(I)TPV] were taken for the study. The test for determination of opacity and MWGT were performed at monthly intervals for three months and the test for HS activity was performed after three months exposure of the samples at various temperatures. There was almost no loss in the opacity of GIPV and TIPV at all the selected storage temperatures and of HIPV and FIPV at 4-8 degrees C, while A(I)TPV lost about 16% opacity in three months at 4-8 degrees C. At 25 degrees C after three months, the percentage loss in opacity of HIPV averaged about 12, of FIPV 17 and of A(I)TPV 33. At 35 degrees C after three months, the average percentage loss in opacity of HIPV was 16, of FIPV was 25 and of A(I)TPV was 33. Regarding the effect of elevated temperatures on the toxicity of the vaccine, it was observed that the samples held at 35 degrees C for three months were the least toxic followed by those held at 25 and 4-8 degrees C as detected by MWGT and the test for HS activity.

Acetone↗

PubMed1993

Piroxicam has at least two epitopes for contact photoallergy.

We have demonstrated previously in guinea pigs that the induction of photocontact sensitivity to piroxicam (PXM) also induces a state of cross-reactive contact hypersensitivity to two compounds having structurally related elements, thimerosal (TMS) and thiosalicylate (TOS). The present study was conducted to determine whether oral administration of TOS would desensitize guinea pigs previously photosensitized with PXM. At the same time, the spectrum of reactivities against these compounds and against tenoxicam (TXM) which resembles only piroxicam was assessed by appropriate sensitizing and eliciting protocols. As expected, animals photosensitized to PXM developed reactivities against all four compounds, PXM and TXM (photosensitivity) and TMS and TOS (contact sensitivity). By contrast, photosensitization with TXM induced cross-reactivity only against PXM. Moreover, the induction of contact sensitivity against TMS or TOS induced photosensitive cross-reactivity to PXM, but not to TXM. Finally, the oral administration of TOS produced a transient desensitization only for TMS and TOS. These results suggest that photosensitization with PXM induces two distinct reactivities. The first reactivity cross-reacts with TMS and TOS and is suppressible with orally administered TOS. The second cross-reacts only with TXM and is not suppressible with oral TOS. We conclude that PXM acquires at least two distinct immunogenic epitopes when exposed to UVA irradiation.

Administration, Oral↗

PubMed2006

Discovery of potent inhibitors of pseudomonal quorum sensing via pharmacophore modeling and in silico screening.

HipHop-Refine was employed to derive a binding hypothesis for pseudomonal quorum sensing (QS) antagonists. The model was employed as 3D search query to screen the National Cancer Institute (NCI) database. One of the hits illustrated nanomolar QS inhibitory activity. The fact that this compound contained tetravalent lead (Pb) prompted us to evaluate the activities of phenyl mercuric nitrate and thimerosal, both fit the binding pharmacophore. The two mercurials illustrated nanomolar to low micromolar IC50 inhibitory values against pseudomonal QS. The three compounds represent a new class of QS inhibitors.

Algorithms↗

PubMed2004

Lactic acid triggers, in vitro, thiomersal to degrade protein in the presence of PLGA microspheres.

Microspheres of polymers like poly(lactic-co-glycolic acid) (PLGA) have been studied as a vehicle for controlled release vaccines. They require materials and processes that might change the protein antigenicity. Lactic acid is produced during microsphere degradation that occurs in tandem with protein liberation. In addition, most of the proteins that have been used in microencapsulation studies contain Thimerosal((R))(TM) and this can introduce another undesirable effect for their stability. We demonstrated in vitro that the thiosalycilic acid (TSA), produced after the reduction of TM by lactic acid, reduces the S-S bridge of the previously incubated diphtheric toxoid (Dtxd). This reduction is immediately followed by blocking the two -SH formed by the same TSA molecules. In the light of these conclusions it is necessary now, to reinterpret the in vitro protein degradation-stabilization data in the presence of PLGA microspheres, mainly for those proteins which contain S-S. We propose that all the PLGA microspheres microencapsulation studies and protein structural considerations should be done in the absence of TM as preservative.

Diphtheria Toxoid↗

PubMed2006

Cell death and cytotoxic effects in YAC-1 lymphoma cells following exposure to various forms of mercury.

The effects of 1 min-4 h exposures to four Hg compounds (mercuric chloride [HgCl2], methyl mercuric chloride [CH3HgCl], p-chloromercuribenzoate [p-CMB] and thimerosal [TMS; ethylmercurithiosalicylate]) on cell death, microtubules, actin, CD3 receptor expression, protein tyrosine phosphorylation (PTyr-P) and intracellular calcium ([Ca2+]i) levels were investigated in YAC-1 lymphoma cells using flow cytometry. YOPRO-1 (YP) and propidium iodide (PI) dye uptake indicated all forms of Hg tested were toxic at concentrations ranging from 25.8-48.4 microM, with two distinct patterns of effects. Early apoptosis was prolonged for CH3HgCl- and TMS-treated cells, with more than 50% remaining YP+/PI- after 4h. Both CH3HgCl and TMS induced complete loss of beta-tubulin fluorescence, indicative of microtubule depolymerization and inhibition of tubulin synthesis and/or beta-tubulin degradation, while F-actin fluorescence diminished to a lesser degree and only after loss beta-tubulin. CH3HgCl and TMS induced an almost immediate two-fold increase in CD3 fluorescence, with levels returning to baseline within minutes. With continued exposure, CD3 fluorescence was reduced to approximately 50% of baseline values. Both compounds also increased PTyr-P two- to three-fold immediately, with levels returning to baseline at 4h. Similarly, two- to three-fold increases in [Ca2+]i were noted after 1 min exposure. [Ca2+]i increased progressively, reaching levels five- to eight-fold greater than control values. In contrast, dye uptake was delayed with HgCl2 and p-CMB, although cell death proceeded rapidly, with almost all non-viable cells being late apoptotic (YP+/PI+) by 4h. p-CMB produced early reductions in F-actin, and after 4h, complete loss of F-actin with only partial reduction of total beta-tubulin was seen with both p-CMB and HgCl2. HgCl2 reduced CD3 expression and PTyr-P slightly within minutes, while p-CMB produced similar effects on CD3 only at 4h, at which time PTyr-P was increased two- to three-fold. Both compounds increased [Ca2+]i within minutes, though levels remained under twice the baseline concentration after 15 min exposure. With continued exposure, [Ca2+]i increased to levels two- to five-fold greater than control values. These findings indicate the two groups of Hg compounds may induce cell death by distinct pathways, reflecting interactions with different cellular targets leading to cell death.

Actins↗

PubMed2003

Kinetic studies of calcium-induced calcium release in cardiac sarcoplasmic reticulum vesicles.

Fast Ca(2+) release kinetics were measured in cardiac sarcoplasmic reticulum vesicles actively loaded with Ca(2+). Release was induced in solutions containing 1.2 mM free ATP and variable free [Ca(2+)] and [Mg(2+)]. Release rate constants (k) were 10-fold higher at pCa 6 than at pCa 5 whereas Ryanodine binding was highest at pCa < or =5. These results suggest that channels respond differently when exposed to sudden [Ca(2+)] changes than when exposed to Ca(2+) for longer periods. Vesicles with severalfold different luminal calcium contents exhibited double exponential release kinetics at pCa 6, suggesting that channels undergo time-dependent activity changes. Addition of Mg(2+) produced a marked inhibition of release kinetics at pCa 6 (K(0.5) = 63 microM) but not at pCa 5. Coexistence of calcium activation and inhibition sites with equally fast binding kinetics is proposed to explain this behavior. Thimerosal activated release kinetics at pCa 5 at all [Mg(2+)] tested and increased at pCa 6 the K(0.5) for Mg(2+) inhibition, from 63 microM to 136 microM. We discuss the possible relevance of these results, which suggest release through RyR2 channels is subject to fast regulation by Ca(2+) and Mg(2+) followed by time-dependent regulation, to the physiological mechanisms of cardiac channel opening and closing.

Animals↗

PubMed2000

Calcium transient activity in cultured murine neural crest cells is regulated at the IP(3) receptor.

In a previous study we have shown that cultured neural crest cells exhibit spontaneous calcium transients and that these events are required for neurogenesis. In this study, we examine the mechanism that generates these calcium transients. Extracellular Ca(2+) modulates calcium transient activity. Lanthanum (La(3+)), a general calcium channel antagonist and zero extracellular Ca(2+), reduces the percentage of cells exhibiting calcium transients (26.2 and 40. 5%, respectively) and decreases calcium spiking frequency (4.5 to 1. 0 and 2.5 to 1.0 spikes/30 min, respectively). Intracellular calcium stores also contribute to the generation of calcium transients. Depleting the calcium stores of the endoplasmic reticulum (ER) reduces the percentage of active cells (15.7%) and calcium spiking frequency (2.8 to 1.5 spikes/30 min). Ryanodine (100 microM), which blocks calcium release regulated by the ryanodine receptor (RyR), had no effect on calcium transient activity. Blocking inositol 1,4, 5-triphosphate receptor (IP(3)R)-dependent calcium release, with elevated extracellular Mg(2+) (20 mM), abolished calcium transient activity. Mg(2+) did not block caffeine-sensitive calcium release (RyR-dependent) or voltage dependent calcium channels. Mg(2+) also suppressed thimerosal-induced calcium oscillations (IP(3)R-dependent). Small increases in the intracellular calcium concentration ([Ca(2+)](i)), increases the percentage of active cells and the calcium spiking frequency, while larger increases in [Ca(2+)](i) block the transients. Reducing intracellular IP(3) levels reduces the percentage of active cells and the calcium spiking frequency. We conclude that the mechanism for generating spontaneous calcium transients in cultured neural crest cells fits the model for IP(3)R-dependent calcium excitability of the ER.

Animals↗

PubMed2002

Inhibition of Ca2+-activated and voltage-dependent K+ currents by 2-mercaptophenyl-1,4-naphthoquinone in pituitary GH3 cells: contribution to its antiproliferative effect.

Quinones have been shown to possess antineoplastic activity; however, their effects on ionic currents remain unclear. The effects of 2-mercaptophenyl-1,4-naphthoquinone (2-MPNQ), menadione (MD) and 1,4-naphthoquinone (1,4 NQ) on cell proliferation and ionic currents in pituitary GH3 lactotrophs were investigated in this study. 2-MPNQ was more potent than menadione or 1,4-naphthoquinone in inhibiting the growth of GH3 cells. 2-MPNQ decreased cell proliferation in a concentration-dependent manner with an IC50 value of 3 microM. In whole-cell recording experiments, 2-MPNQ reversibly caused an inhibition of Ca2+-activated K+ current (I(K(Ca)) in a concentration-dependent manner. The IC50 value for 2-MPNQ-induced inhibition of I(K(Ca)) was 7 microM. In the inside-out configuration of single channel recording, 2-MPNQ (30 microM) applied intracellularly suppressed the activity of large-conductance Ca2+-activated K+ (BK(Ca)) channels but did not modify single channel conductance. Menadione (30 microM) had no effect on the channel activity, whereas 1,4-naphthoquinone (30 microM) suppressed it by about 26%. Both 2-MPNQ and thimerosal suppressed the dithiothreitol-stimulated channel activity. 2-MPNQ also blocked voltage-dependent K+ currents, but it produced a slight reduction of L-type Ca2+ inward current. However, unlike E-4031, 2-MPNQ (30 microM) did not suppress inwardly rectifying K+ current present in GH3 cells. Under the current clamp configuration, the presence of 2-MPNQ (30 microM) depolarized the cells, and increased the frequency and duration of spontaneous action potentials. The 2-MPNQ-mediated inhibition of K+ currents would affect hormone secretion and cell excitability. The blockade of these ionic channels by 2-MPNQ may partly explain its inhibitory effect on the proliferation of GH3 cells.

Action Potentials↗

PubMed1998

Effect of sulfhydryl oxidoreduction on permeability of cardiac tetrodotoxin-insensitive sodium channel.

Effects of sulfhydryl oxidizing and reducing agents on permeability of the tetrodotoxin (TTX)-insensitive Na-channel were investigated in guinea-pig ventricular myocytes using the whole-cell patch-clamp technique. Mercury chloride (HgCl2) at 1-100 microM irreversibly blocked Na+ currents with no significant changes in the gating kinetics. In contrast, the hydrophilic sulfhydryl oxidizing agent, thimerosal at 50-100 microM little affected Na+ permeation through the Na-channel. The Hg2+-induced block of Na+ current could be readily reversed by 1,4-dithiothreitol (DTT), an agent that reduces disulfide bonds. These results indicate that the formation of sulfur-Hg-sulfur bridge is essential for Hg2+ block. Pretreatment with DTT prevented the Hg2+ block of Na+ current, whereas Zn2+ and Cd2+ retained their abilities to block Na+ current after DTT treatment. An application of Zn2+ or Cd2+ resulted in the restoration of Hg2+ sensitivity of the DTT-treated channel. A conformational model for the Na-channel with multiple free sulfhydryl groups and native disulfide bonds could account for our experimental data regarding the effects of sulfhydryl modifying agents on the channel permeability. We conclude that the cardiac TTX-insensitive Na-channel contains functionally important free sulfhydryl groups and disulfide bonds which are accessible from the extracellular side by an aqueous pathway. These sulfhydryls would be capable of modulating the Na-channel permeability by affecting the conformation of channel pore region.

Algorithms↗

PubMed1998

Prostaglandin production by guinea-pig placenta and other uterine tissues during mid-pregnancy.

Prostaglandin (PG) output from cultured placenta, sub-placenta, endometrium and fetal membranes of guinea-pigs was measured on days 22, 29 and 36 of pregnancy to establish the source of increased PGF2alpha production during mid-pregnancy. PGF2alpha and 6-keto-PGF1alpha were produced in larger quantities than PGE2 by the placenta, sub-placenta and endometrium; 6-keto-PGF1alpha was in the major prostaglandin produced by the fetal membranes. The initial outputs of PGF2alpha, PGE2 and 6-keto-PGF1alpha from the sub-placenta, fetal membranes and endometrium either decreased or remained fairly constant between days 22 and 36. In contrast, the initial outputs of PGF2alpha, PGE2 and 6-keto-PGF1alpha from the placenta increased 14.7-, 2.5- and 2.0-fold, respectively, between days 22 and 36, indicating that the placenta is the tissue responsible for the increase in PGF2alpha output from the mid-pregnant guinea-pig uterus. Aristolochic acid (a phospholipase A2 inhibitor) inhibited prostaglandin output from the endometrium, but had a more variable effect in prostaglandin output from the other tissues. Thimerosal (an arachidonic acid uptake inhibitor) inhibited PGF2alpha and PGE2 outputs from the endometrium, but generally potentiated 6-keto-PGF1alpha output and prostaglandin output from the other tissues. Arachidonic acid release for prostaglandin synthesis in the endometrium, but not the placenta, sub-placental or fetal membranes, is apparently dependent upon a constant level of phospholipase A2 activity.

Animals↗

PubMed1996

Spatial and temporal aspects of Ca2+ oscillations in Xenopus laevis melanotrope cells.

Spatio-temporal aspects of Ca2+ signaling in melanotrope cells of Xenopus laevis have been studied with confocal laser-scanning microscopy. In the whole-frame scanning mode, two major intracellular Ca2+ compartments, the cytoplasm and the nucleus, were visualized. The basal [Ca2+] in the nucleus appeared to be lower than that in the cytoplasm and Ca2+ oscillations seemed to arise synchronously in both compartments. The N-type channel blocker omega-conotoxin eliminated oscillations in both regions, indicating a strong coupling between the two compartments with respect to Ca2+ dynamics. Line-scanning mode, which gives higher time resolution, revealed that the rise phase of a Ca2+ oscillation is not a continuous process but consists of 3 or 4 discrete steps. Each step can be seen as a Ca(2+)-wave starting at the cell membrane and going through the cytoplasm at a speed of 33.3 +/- 4.3 microns/s. Before the Ca(2+)-wave enters the nucleus, a delay of 120.0 +/- 24.1 ms occurred. In the nucleus, the speed of a wave was 80.0 +/- 3.0 microns/s. Treatment with the Ca(2+)-ATPase inhibitor thapsigargin (1 MicroM) almost completely eliminated the apparent difference in the basal [Ca2+] in the cytoplasm and the nucleus, reduced the delay of a Ca(2+)-wave before entering the nucleus to 79.8 +/- 8.7 ms, and diminished the nuclear wave speed to 35.0 +/- 4.9 microns/s. These results indicate that a cytoplasmic thapsigargin-sensitive ATPase near the nuclear envelope is involved in buffering Ca2+ before the Ca2+ wave enters the nucleus. After sensitizing IP3 receptors by thimerosal (10 microM) the speed of the cytoplasmic Ca(2+)-wave was increased to 70.3 +/- 3.6 microns/s, suggesting that IP3 receptors may be involved in the propagation of the cytoplasmic Ca2+ wave. Our results indicate that in melanotropes the generation and propagation of Ca2+ oscillations is a complex event involving influx of Ca2+ through N-type Ca2+ channels, propagation of the cytoplasmic Ca2+ wave through mobilization of intracellular stores and a regulated Ca2+ entry into the nucleus. We propose that Ca(2+)-binding proteins may act as a Ca2+ store for propagation of the wave in the nucleus.

Animals↗

PubMed1998

Determination of the inositol (1,4,5) trisphosphate requirement for histamine- and substance P-induced Ca2+ mobilisation in human U373 MG astrocytoma cells.

In human U373 MG astrocytoma cells, histamine and substance P stimulated similar peak increases in intracellular free calcium concentrations ([Ca2+]i), as measured by single cell imaging of Fura-2 fluorescence. Best-fit EC50 values for the peak Ca2+ response were 1.86 microM for histamine and 0.93 nM for substance P. The histamine Ca2+ response was manifest as either a series of repetitive spikes, or, at higher concentrations, a peak followed by a lower plateau level of Ca2+. In contrast, the substance P response became more transient at higher agonist concentrations. Substance P (10 nM) stimulated a biphasic increase in levels of inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) with a peak of 97 +/- 5 pmoles/mg protein at 10 s. In contrast, the Ins(1,4,5)P3 response to 100 microM histamine was only marginally above basal levels of around 12 pmoles/mg protein. However, concentrations of histamine and substance P giving similar Ins(1,4,5)P3 responses produce similar peak increases in [Ca2+]i. HPLC analysis indicated that histamine stimulated the production of [3H]-Ins(1,4,5)P3 and its metabolites, although the magnitude of response was smaller than that observed with substance P. The initial Ca2+ responses to histamine and substance P did not require the presence of extracellular Ca2+. The Ca2+ response to histamine was unaffected by treatment with ryanodine, and was shifted to areas of lower agonist concentration by thimerosal. These results demonstrate that extremely small increases in Ins(1,4,5)P3 can stimulate large increases in [Ca2+]i in U373 MG cells, and suggest a marked redundancy for Ins(1,4,5)P3 production in the Ca2+ signalling pathway.

Astrocytoma↗

PubMed2001

Predicted mercury concentrations in hair from infant immunizations: cause for concern.

Mercury (Hg) is considered one of the worlds most toxic metals. Current thinking suggests that exposure to mercury occurs primarily from seafood contamination and rare catastrophic events. Recently, another common source of exposure has been identified. Thimerosal (TMS), a preservative found in many infant vaccines, contains 49.6% ethyl mercury (EtHg) by weight and typically contributes 25 microg of EtHg per dose of infant vaccine. As part of an ongoing review, the Food and Drug Administration (FDA) announced in 1999 that infants who received multiple TMS-preserved vaccines may have been exposed to cumulative Hg in excess of Federal safety guidelines. According to the centers for disease control (CDC) recommended immunization schedule, infants may have been exposed to 12.5 microg Hg at birth, 62.5 microg EtHg at 2 months, 50 microg EtHg at 4 months, 62.5 microg EtHg at 6 months, and 50 microg EtHg at approximately 18 months, for a total of 237.5 microg EtHg during the first 18 months of life, if all TMS-containing vaccines were administered. Neurobehavioral alterations, especially to the more susceptible fetus and infant, are known to occur after relatively low dose exposures to organic mercury compounds. In effort, to further elucidate the levels of ethyl mercury resulting from exposure to vaccinal TMS, we estimated hair Hg concentrations expected to result from the recommended CDC schedule utilizing a one compartment pharmacokinetic model. This model was developed to predict hair concentrations from acute exposure to methymercury (MeHg) in fish. Modeled hair Hg concentrations in infants exposed to vaccinal TMS are in excess of the Environmental Protection Agency (EPA) safety guidelines of 1 ppm for up to 365 days, with several peak concentrations within this period. More sensitive individuals and those with additional sources of exposure would have higher Hg concentrations. Given that exposure to low levels of mercury during critical stages of development has been associated with neurological disorders in children, including ADD, learning difficulties, and speech delays, the predicted hair Hg concentration resulting from childhood immunizations is cause for concern. Based on these findings, the impact which vaccinal mercury has had on the health of American children warrants further investigation.

Forecasting↗

PubMed1997

Leukotriene B4 stimulates the release of arachidonate in human neutrophils via the action of cytosolic phospholipase A2.

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation and is involved in the receptor-mediated activation of a number of leukocyte responses including degranulation, superoxide formation, and chemotaxis. In the present research, stimulation of unprimed polymorphonuclear leukocytes (neutrophils) with LTB4 results in the transient release of arachidonate as measured by mass. This release of arachidonate was maximal at an LTB4 concentration of 50-75 nM and peaked at 45 s after stimulation with LTB4. The transient nature of this release can be attributed, in part, to a fast (< 60 s) metabolism of the added LTB4. Moreover, the inhibition of the reacylation of the released arachidonate with thimerosal results in greater than 4-times as much arachidonate released. Thus, a rapid reacylation of the released arachidonate also contributes to the transient nature of its measured release. Multiple additions of LTB4, which would be expected to more closely resemble the situation in vivo where the cell may come into contact with an environment where LTB4 is in near constant supply, yielded a more sustained release of arachidonate. No release of [3H]arachidonate was observed when using [3H]arachidonate-labeled cells. This indicates that the release of arachidonate as measured by mass is most probably the result of hydrolysis of arachidonate-containing phosphatidylethanolamine within the cell since the radiolabeled arachidonate is almost exclusively incorporated into phosphatidylcholine and phosphatidylinositol pools under the non-equilibrium radiolabeling conditions used. Consistent with the role of cytosolic phospholipase A2 (cPLA2) in the release of arachidonate, potent inhibition of the LTB4-stimulated release was observed with methylarachidonylfluorophosphonate, an inhibitor of cPLA2 (IC50 of 1 microM). The bromoenol lactone of the calcium-independent phosphospholipase A2. failed to affect LTB4-stimulated release of arachidonate in these cells.

Arachidonic Acid↗

PubMed1998

Liquid or solid ultrasonically tissue-mimicking materials with very low scatter.

A new tissue-mimicking material for ultrasound, using evaporated milk as the primary absorption component, is described. It has very low backscatter but still exhibits the 1540 m s-1 propagation speed and proportionality of attenuation coefficient and frequency over the diagnostic frequency range. The material can be produced in solid or liquid form with attenuation coefficient slopes spanning the range 0.1-0.7 dB cm-1 MHz-1. The liquid form is useful in phantoms where detailed beam patterns are to be determined, either involving translation of measurement devices in the liquid or phantoms with fibers present for causing the only detectable echoes. In the latter case, the liquid quality allows removal of liquid with one attenuation coefficient slope and replacement with another. The solid form may be more useful than the liquid for two reasons. First, many simulated lesions (including ones that produce essentially no internal echoes) can lie in the scan slice with positions extending over the entire image area without enhancement or shadowing effects being of concern. Second, the lack of significant backscatter from the material in the absence of added scatterers allows the backscatter coefficient to be varied over a considerable range. A critical result is that intrinsic material contrast between targets and surroundings can be accurately predicted in terms of the concentrations of added scatterers and, assuming all scatterers are of the same type, the contrast will be completely independent of frequency. Use of the fungicide thimerosal eliminates deterioration, and ultrasonic properties have been shown to be stable over 2.5 years.

Animals↗

PubMed2002

Parthenogenesis of rabbit oocytes activated by different stimuli.

Oocyte activation is one of the essential elements determining the success of nuclear transfer and the subsequent development of cloned embryos both in vitro and in vivo. Experiments were conducted to optimize the protocol for oocyte activation in a regular nuclear transfer study. In vivo derived oocytes were collected at 14-15 h from New Zealand white rabbits after ovulation treatment and were activated +18 h post-ovulation treatment. Single activation agents including calcium ionophore (A23187, 5 microM, 5 min), ethanol (Eth, 7%, 7 min), and thimerosal (200 microM, 10 min) were tested. Cleavage rates were highest in the ethanol-treated group (37%) compared to other treatments (19-25%). Very low blastocyst rates (2-3%) resulted which were not significantly different among treatments (P>0.05). Combined single agent treatment (calcium stimulators) with protein kinase inhibitor, 6-DMAP were used to achieve a full oocyte activation. Both pronuclear and blastocyst formation rates were significantly higher (P<0.05) in the Eth+6-DMAP treatment group (38 and 27%) than in the other groups (16-21 and 7-9%, respectively, P<0.05). Low (0.2mM) and high (2.5mM) concentrations of 6-DMAP treatments with different treatment lengths (1.5 and 3.5h) in the combined groups were also compared. Blastocyst formation and cleavage rates were greater in the high concentration with less treatment time groups (36% versus 4-20%, P<0.05). In conclusion, single activation agents, either Ca2+ stimulators or protein kinase inhibitors, could not fully activate mature rabbit oocytes. The best activation procedure obtained in this study was the Eth+6-DMAP combined treatment, which may be incorporated into regular nuclear transfer or cloning protocols.

Adenine↗

PubMed2000

Microscopic properties of elementary Ca2+ release sites in non-excitable cells.

BACKGROUND: Elementary Ca2+ signals, such as 'Ca2+ puffs', that arise from the activation of clusters of inositol 1 ,4,5,-trisphosphate (InsP3) receptors are the building blocks for local and global Ca2+ signalling. We previously found that one, or a few, Ca2+ puff sites within agonist-stimulated cells act as 'pacemakers' to initiate global Ca2+ waves. The factors that distinguish these pacemaker Ca2+ puff sites from the other Ca2+ release sites that simply participate in Ca2+ wave propagation are unknown. RESULTS: The spatiotemporal properties of Ca2+ puffs were investigated using confocal microscopy of fluo3-loaded HeLa cells. The same pacemaker Ca2+ puff sites were activated during stimulation of cells with different agonists. The majority of agonist-stimulated pacemaker Ca2+ puffs originated in a perinuclear location. The positions of such Ca2+ puff sites were stable for up to 2 hours, and were not affected by disruption of the actin cytoskeleton. A similar perinuclear distribution of Ca2+ puff sites was also observed when InsP3 receptors were directly stimulated with thimerosal or membrane-permeant InsP3 esters. Immunostaining indicated that the perinuclear position of pacemaker Ca2+ puffs was not due to the localised expression of InsP3 receptors. CONCLUSIONS: The pacemaker Ca2+ puff sites that initiate Ca2+ responses are temporally and spatially stable within cells. These Ca2+ release sites are distinguished from their neighbours by an intrinsically higher InsP3 sensitivity.

Acetylcholine↗