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PubMed1998

Acanthamoeba adherence to contact lenses, removal by rinsing procedures, and survival to some ophthalmic products.

Unworn soft and rigid gas-permeable contact lenses were inoculated with an Acanthamoeba keratitis strain to study the protozoon's ability to adhere. Furthermore, the efficacy of the rinsing in saline on acanthamoeba removal was evaluated, as well as the amebicidal activity of five commercial cleaning/disinfecting products: hydrogen peroxide, chlorhexidine, polyaminopropyl biguanide-poloxamine, thimerosal-polyquaternium, and thimerosal-chlorhexidine. Microscopic count of cells showed that Acanthamoeba trophozoites and cysts adhered to all types of contact lenses. A significantly greater adherence of trophozoites than cysts was recorded. The rinsing in saline using a flow-method was significantly more effective than the immersion-method, particularly in removing trophozoites from rigid gas-permeable lenses. The cleaning/disinfecting solutions tested were ineffective in removing or in affecting the viability of all Acanthamoeba trophozoites or cysts in the 17 hours allotted for the experiment. The need for a better care in mechanical and physical hygiene procedures is stressed.

Acanthamoeba↗
PubMed1975

The effect of formaldehyde on the toxicity and potency of pertussis vaccine.

Storage of concentrated suspensions of Bordetella pertussis organisms at 5 C for long periods of time in the presence of 0.02% thimerosal as well as heating at 56 C for 30 minutes cannot be relied upon to detoxify the vaccines. Our data show that the toxic thimerosal-killed vaccines can be rapidly detoxified by adding formalin to a final concentration of 0.025-0.05% and incubating at temperatures of 5-20 C for from one to three months. These formalin-detoxified vaccines are protective and meet present potency requirements for pertussis vaccine. Addition of larger amounts of formalin at higher temperatures may detoxify the vaccine but may also reduce the protective effect.

Animals↗
PubMed1992

Morphometric and physical investigations of segmental cortical bone autografts and allografts in canine ulnar defects.

Cortical bone grafts were implanted for six months in mature dogs using an osteoperiosteal 3-cm defect in the ulna to evaluate their respective morphometric and physical values compared with autografts. The bone-grafting material included fresh auto- and allografts, frozen and thimerosal preserved allografts, and partially demineralized bone allografts. The grafts were evaluated by roentgenograms, microradiograms, photon absorptiometry, porosity, fluorescence labeling measurements, and torsional loading at failure. Autografts achieved a better union score than the allografts, but intracortical bone porosity, percentage of cumulative new bone, and mineral apposition rate were not variables with statistical significance. Lamellar bone was found earlier and in greater quantity in autografts. Within the graft, new bone was deposited at a slower rate than in the recipient bone. Autografts showed less peripheral resorption and a greater torsional resistance than allografts. Photon absorptiometry demonstrated that nondemineralized allografts underwent a substantial loss of peripheral bone. This marked reduction in the outer diameter of the graft had more influence on torsional resistance than did the intracortical porosity of the graft. Demineralized allografts were osteoinductive in only 28% of the cases and appeared to respond in an all-or-nothing pattern. Frozen and thimerosal preserved allografts were the most acceptable substitutes to autografts.

Absorptiometry, Photon↗
PubMed1992

Spontaneous cytosolic calcium oscillations driven by inositol trisphosphate occur during in vitro maturation of mouse oocytes.

Immature mouse oocytes undergo spontaneous meiotic maturation when released from antral follicles into culture media. The first sign of meiotic resumption is germinal vesicle breakdown (GVB). Cytosolic free Ca2+ was measured in mouse oocytes during spontaneous maturation by monitoring fluorescence of indo-1 or fluo-3. The majority of oocytes showed a series of Ca2+ oscillations that continued for 1-3 h. Repetitive Ca2+ increases occurred every 1-3 min and lasted for 10-60 s. The Ca2+ oscillations appeared to be caused by an increase in inositol 1,4,5-trisphosphate (InsP3) because once they ceased, similar oscillations were triggered by injection of exogenous InsP3. Also, injection of the InsP3 receptor antagonist heparin (final concentration, 100 micrograms/ml) blocked the spontaneous Ca2+ oscillations. In contrast, Ca2+ oscillations induced by thimerosal were not inhibited by heparin. Treating oocytes with media containing 20 microM BAPTA/AM abolished Ca2+ oscillations in oocytes but did not affect the rate of GVB. The data show that cytosolic Ca2+ oscillations apparently caused by polyphosphoinositide turnover occur during mammalian oocyte maturation. However, the spontaneous oscillations do not appear to trigger GVB. Also, the data indicate that there are two separate Ca2+ release mechanisms in mouse oocytes, one sensitive to InsP3, the other to thimerosal.

Animals↗
PubMed1989

[Patch test of mercury compounds and disinfectant solutions and the clinical picture of mercury contact dermatitis].

The results of patch tests with mercury compounds and disinfectant solutions as well as the clinical picture of mercury contact dermatitis are presented and discussed. During the period from Nov. 1983 to Dec. 1986, 686 patients were tested with mercury compounds. During the 13 year period from 1974-1986, 118 patients were tested with disinfectant solutions and 59 patients with mercury contact dermatitis were seen. Among the disinfectant solutions tested, the highest incidence of positive reaction, 11.5%, was obtained with thimersal. Cross sensitization has been shown between mercurochrome and ammoniated mercury, but no statistical correlation existed that demonstrated cross sensitization between mercury and thimerosal until our study. Therefore, the allergen in mercurochrome contact dermatitis is mercury. No statistical correlation could be established between the occurrence of atopic dermatitis and the patch test reactions to mercury compounds. The contactants were mercurochrome, thimerosal, and broken thermometers in 49 out of 59 cases of mercury contact dermatitis. Consequently, the number of occurrence of mercury dermatitis can be reduced by the elimination of the use of these two disinfectant solutions and the substitution of electric thermometers for mercury thermometers.

Adolescent↗
PubMed1983

[Improvement of the quality control of thioglycol medium].

The effect of thimerosal used at different concentrations on the growth of S. haemolyticus, strain Dick I, and A. faecalis, strain 415, has been studied. A. faecalis, strain 415, in contrast to S. haemolyticus, strain Dick I, has been shown to be highly sensitive to thimerosal. The growth and neutralizing properties of a number of lots of thyoglycol medium have been studied with the use of the above-mentioned strains. The advantages of A. faecalis, strain 415, over S. haemolyticus, strain Dick I, for the evaluation of these properties of thioglycol medium have been established. A. faecalis, strain 415, can be recommended for use as a test strain for evaluating the quality of thioglycol medium instead of S. haemolyticus, strain Dick I.

Alcaligenes↗
PubMed1994

A third generation immunoassay for tumor necrosis factor alpha.

Tumor necrosis factor alpha (TNF alpha) is a polypeptide cytokine produced primarily by monocytes and macrophages. It is involved in a wide variety of immune reactions. Measurement of TNF alpha originally depended upon bioassays that are of varying reliability and reproducibility. Early immunoassays for TNF alpha required handling of radioisotopes and costly disposal of radioactive waste. Subsequent use of enzymes as reporter molecules in enzyme immunoassay (EIA) has eliminated the burden of radioisotope handling and its associated costs. However, EIA has presented new challenges. Use of thimerosal as a preservative in EIAs may require high disposal costs due to its mercury content. In addition, many EIAs lack the sensitivity achievable in radioimmunoassay (RIA). We have developed a simple microplate enzyme-linked immunosorbent assay (ELISA) for the detection of TNF alpha in serum, plasma and culture supernatants. Our high affinity capture antibody has enabled us to achieve a sensitivity of 1.5 pg/mL. The assay is calibrated to the World Health Organization (W.H.O.) first international standard for TNF alpha (87/650) and exhibits excellent precision and reproducibility. Tetramethylbenzidine is used to generate the colored end product of the reaction, and thimerosal has been removed from all components.

Enzyme-Linked Immunosorbent Assay↗
PubMed1994

Control of calcium spiking frequency in pituitary gonadotrophs by a single-pool cytoplasmic oscillator.

The mechanisms by which the generation and frequency of cytoplasmic Ca2+ oscillations are controlled were investigated in pituitary gonadotrophs. In these cells, two Ca(2+)-mobilizing receptors, the gonadotropin-releasing hormone and endothelin receptors, induce frequency-modulated Ca2+ spiking at the rate of up to 30 min-1. The cytoplasmic oscillator is also activated by discharge of luminal Ca2+ (initiated by ionomycin, thapsigargin, or thimerosal) but not by increased voltage-sensitive Ca2+ influx or treatment with caffeine. The basic difference between these two types of Ca2+ oscillations is related to their requirement for inositol-1,4,5-triphosphate (InsP3). Thapsigargin-, thimerosal-, and ionomycin-induced spiking occurs without the rise in InsP3 production that is essential for the generation of receptor-controlled oscillatory responses. The differential requirement for InsP3 in the two types of Ca2+ spiking is indicated by two lines of evidence. First, agonist-induced Ca2+ spiking of frequency similar to that of non-receptor-mediated oscillations was accompanied by a significant increase in InsP3, whereas none of the non-receptor-mediated oscillations was associated with measurable changes in inositol phosphate production. Second, agonist-induced InsP3 formation and Ca2+ spiking were abolished by treatment with the phospholipase C inhibitors U73122 and neomycin sulfate, whereas non-receptor-mediated Ca2+ spiking was not affected by these agents. When the oscillator was activated by agents that do not increase InsP3 formation, it operated only at the basal rate of approximately 5 min-1 and spiking frequency did not rise with increasing drug concentrations, in contrast to the situation in agonist-stimulated gonadotrophs. However, both types of oscillations were affected by depletion of luminal Ca2+ and by changes in the intracellular Ca2+ concentration ([Ca2+]i) but were not inhibited by ryanodine. These findings are consistent with the operation of a single-pool Ca2+ oscillator that is responsible for generation of both types of Ca2+ oscillations. The oscillator is controlled by the coagonist actions of InsP3 and Ca2+ on the InsP3 receptor channels and by the activation of Ca(2+)-ATPase by rising [Ca2+]i. It can be induced to operate at low frequency without an increase in InsP3 production by agents that reduce intraluminal [Ca2+]i, and it exhibits a dose-dependent increase in spiking frequency during agonist stimulation.

Animals↗
PubMed1983

Cianidanol ( [+]-cyanidanol-3) prevents the development of abdominal adhesions in rats.

Abdominal adhesions were experimentally induced in rats by gentle scraping. Severe adhesions developed in 38.7% of the control animals. The flavonoid cianidanol ( [+]-cyanidanol-3), an in vitro inhibitor of procollagen production, was administered intragastrically or intraperitoneally in doses of 9 to 72 mg per rat. Administered intraperitoneally at a dose of 36 or 72 mg per rat, cianidanol substantially inhibited adhesion formulation, when given immediately, three days, or five days after surgery. Oral administration of the drug was less effective in preventing the formation of adhesions. Thimerosal, another connective-tissue inhibitor, was found to be toxic at doses of 2.5 or 5 mg per rat, and its effect on the prevention of adhesion formation was poor. The ability of cianidanol to inhibit the development of abdominal adhesions in rats suggests that a possible approach to treatment is to inhibit the production of procollagen and thus prevent the formation of the collagenous fibers that are the cause of adhesions.

Abdomen↗
PubMed1996

Pharmacological characterization of inositol-1,4,5,-trisphosphate binding to membranes from retina and retinal cultures.

Light and excitatory amino acids (EAA) stimulate the phosphoinositide cycle in the vertebrate retina. The regulation of Ca2+ release from intracellular stores by inositol-1,4, 5-trisphosphate (IP3) involves an interaction of this compound with specific receptors. By means of [3H]IP3-specific binding, we studied the kinetic and pharmacological properties of IP3 receptors in the chick retina as well as in primary cultures of neurons and glia from this tissue. The equilibrium time for the binding reaction was 15 min and was optimal at alkaline pH (8.3). IP3 receptor displayed high affinity (K(B) approximately 40 nM) and selectivity for D-IP3, compared to D-IP4 > L-IP3 > D-IP2 > D-IP1. These characteristics were the same in subcellular fractions from outer (P1) and thinner (P2) plexiform layers, binding sites being more abundant in P2 (2.65 pmol/mg protein). IP3 receptors were present in both neuronal and glial cultures, but were concentrated in neuronal cultures. Binding was not affected by ryanodine, or caffeine, related to calcium-induced calcium release (CICR) channels, nor by the endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin, while heparin affectively inhibited IP3 binding. GSSG and thimerosal increased the affinity of [3H]IP3 binding from IC50 approximately 80 nM to IC50 approximately 40 nM; this effect was reversed by DTT. Binding in zero Ca2+ was decreased by low concentrations of Ca2+ (350 nM). These results suggest that actions of IP3 in the retina are regulated by physiological changes in intracellular pH and Ca2+ concentrations, as well as by the oxidation state of the receptor. Additionally, the presence of IP3 receptors in Müller glia opens the possibility of IP3 participation in nonsynaptic signalling through Ca2+ waves in glial cells.

Analysis of Variance↗
PubMed1980

Validation of washing procedures for maintaining a microbiologically clean gel filtration column.

Chromatographic gel filtration matrixes used in various separation techniques are subject to microbial contamination. The need for a microbe-free column is critical when preparing materials that require a low or zero microbial count. This report proposes two alternative washing systems: 0.02 N HCl containing 0.81% NaCl, and 0.1 M tromethamine--hydrochloride buffer (pH 7.0) containing 0.81% NaCl and 0.02% thimerosal. These washing systems were validated using a 100 x 2.6-cm column packed with a modified dextran gel slurry previously inoculated with known counts of USP test organisms. After each wash, the column separation characteristics were verified further with appropriate test proteins.

Bacteria↗
PubMed2005

Application of a capillary electrophoresis method for simultaneous determination of preservatives in pharmaceutical formulations.

Preservatives are used to protect pharmaceutical formulations from microbial attack during the period of administration to the patient. Because of their biological activity, preservatives have to be identified and assayed according to the same rules as apply to active components. A number of methods for separation of preservatives are reported, to account for the heterogeneity of their chemical structures. A capillary electrophoretic method was devised for simple and simultaneous qualification and quantification of the preservatives most often included in pharmaceuticals, such as benzyl alcohol, parabens, phenol, m-cresol, chlorobutanol, thimerosal. After systematic method development, the electrophoretic conditions were defined as: 50 mM borate buffer pH 9.0 containing 20 mM SDS. Separations were performed at a temperature of 20 degrees C and with detection at 214 nm. Preservatives under examination can be analyzed within a 10 min run. The method was successfully validated and applied to the determination of preservatives in a number of pharmaceuticals. Results from the CE method were compared with those from reference methods.

Benzyl Alcohol↗
PubMed2006

Development and apoptosis of pre-implantation porcine nuclear transfer embryos activated with different combination of chemicals.

Artificial activation of oocytes is a pre-requisite for successful cloning by nuclear transfer (NT). This study investigated effect of different combination of activation chemicals such as electric pulse (E), thimerosal (Thi) + dithiothreitol (DTT), 6-dimethylaminopurine (6-DMAP), or cycloheximide (CH) on the developmental ability and the frequency of apoptosis of porcine NT embryos during the culture in vitro. NT embryos activated with chemicals showed significantly higher developmental rate to blastocyst stage compared to embryos activated with E alone (21.5%-26.6% vs. 15.7%, respectively). Of chemicals, Thi + DTT supported higher development to blastocyst stage as compared to 6-DMAP or CH (26.6% vs. 21.5%-23.4%, respectively). Apoptosis of NT embryos were analyzed by using a terminal deoxynucleatidyl transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) assay. The onset of apoptosis of embryos activated E alone was on Day 4, whereas embryos activated with chemicals showed apoptosis on Day 3 post-activation NT embryos exposed to chemicals for activation had higher frequency of apoptosis compared to that of embryos exposed to E alone from Day 3 to Day 7 during the culture. In conclusion, this study shows that chemical activation after fusion could increase not only the developmental ability of porcine NT embryos but also the mean cell number with an increased ratio of inner cell mass (ICM) to trophectoderm (TE) cells. However, the chemical activation also could increase the frequency of apoptosis and induced apoptosis earlier in porcine NT embryos.

Adenine↗
PubMed1993

Formation and release of dinitrosyl iron complexes by endothelial cells.

The release of dinitrosyl non-heme iron complexes from cytotoxic macrophages accounts for NO-mediated iron loss. We have now investigated whether or not a similar mechanism operates in endothelial cells. Following stimulation with bradykinin or calcium ionophore A23187 NO and intracellular dinitrosyl iron complexes were detected by ESR spectroscopic analysis of frozen cells. In addition, endothelial cells released dinitrosyl iron complexes which bound to extracellular albumin. In transferrin and iron-free medium stimulation of endothelial cells by bradykinin or thimerosal resulted in a loss of non-heme iron. These effects were prevented by inhibition of NO synthase. Thus NO generated by the constitutive NO synthase appears to be incorporated into dinitrosyl iron complexes, which potentially account for endothelium-dependent relaxation.

Animals↗
PubMed1996

Bioassay of endothelium-derived hyperpolarizing factor.

Endothelium-derived hyperpolarizing factor (EDHF) mediates vasodilatation in certain blood vessels, together with prostacyclin and NO. However, its chemical nature is not known. A perfusion-superfusion cascade was developed to confirm the diffusible nature of EDHF. Canine carotid arteries with endothelium were used as donors of vasoactive substances, whereas rings of coronary artery without endothelium were used as detectors. Inhibitors of NO synthesis and cyclooxygenase were present throughout, to avoid interference from NO and prostanoids. Measurements of membrane potential and isometric tension, in coronary arteries without endothelium (used as detectors), demonstrated the release of EDHF from the carotid arteries, following treatment with 8-methoxypsoralen, bradykinin and thimerosal. The K+-channel blocker tetraethylammonium inhibited the action od EDHF in the detectors. Thus, these results demonstrate that endothelial cells release a diffusible activator of K+-channels in vascular smooth muscle.

Animals↗
PubMed1994

Competence to undergo normal, fertilization-induced cortical activation develops after metaphase I of meiosis in mouse oocytes.

The purpose of this study was to determine at what stage of meiotic maturation mouse oocytes develop the ability to undergo sperm-induced activation, assayed by cortical granule (CG) release. Germinal vesicle breakdown (GVBD), prometaphase I (proMI), metaphase I (MI), and metaphase II (MII, the normal stage at fertilization) stages were evaluated by quantitative image analysis. At 2 hr, fertilized MII eggs underwent a mean CG loss of 69%; loss was global--in the entire cortex occupied by CGs. In contrast, fertilized GVBD and proMI oocytes had no significant CG loss in the cortex after 2 hr. After 4 hr, 63, 67, and 88% of fertilized GVBD, proMI, and MI oocytes, respectively, had localized CG release only in the vicinity of the fertilizing sperm. At 4 hr, GVBD oocytes had small sperm-associated, CG-depleted domains, approximately 100 microns 2 (< 1% of the oocyte cortex), whereas those for proMI oocytes were approximately 2500 microns 2. The CG density was reduced by 90% in these domains, whereas the remaining cortex showed no evidence of CG loss. Both the extent and time course of CG loss were altered in oocytes fertilized before MII and were not significantly affected with 25 microM thimerosal, which sensitizes egg calcium stores. Unlike the ability to undergo CG release, the ability to initiate sperm chromatin decondensation was not stage specific and the extent of decondensation was cell cycle related. Competence to undergo CG loss develops in two phases: the ability to undergo localized CG release increases between the GV and MI stages, whereas the mechanism of propagating a normal wave of global CG loss from the site of sperm entry develops between MI and MII.

Animals↗
PubMed1998

Depletion of intracellular Ca2+ stores, mediated by Mg2+-stimulated InsP3 liberation or thapsigargin, induces a capacitative Ca2+ influx in prawn oocytes.

By voltage clamp technique and intracellular calcium measurements, we recorded in prawn oocytes simultaneous [Ca2+]i and ionic current changes stimulated by external Mg2+. The [Ca2+]i response consists of an oscillation period followed by a second state of sustained [Ca2+]i level. The oscillation period successively comprises a first [Ca2+]i peak, a series of [Ca2+]i transients, and a [Ca2+]i oscillatory plateau respectively concurrent with an initial transient outward K+Ca current, an inward Na+Ca current, and a final K+Ca outward current. By using inhibitor (heparin) or sensitizers (thimerosal or caffeine) of calcium release ER channels, and caged InsP3, we established that InsP3 is the sole second messenger releasing Ca2+ from intracellular stores. By sequential substitutions and reapplications of external Ca2+, and using econazole (50 microM), a Ca2+ influx inhibitor, we documented Ca2+ influx during the [Ca2+]i oscillatory plateau. The intracellular Ca2+ store was depleted with thapsigargin (75-350 nM) in Ca2+-free ASW. Reapplication of external Ca2+ evoked a rise in [Ca2+]i, indicating a store-dependent capacitative Ca2+ influx, correlated with a K+Ca outward current increase. No measurable Ca2+ release-activated Ca2+ current (Icrac) could be detected, but was indirectly demonstrated using the sensitivity of the K+Ca channels to [Ca2+]i. We propose that the involvement of external Ca2+, in the physiological [Ca2+]i response of prawn oocytes to external Mg2+, consists of a store-dependent capacitative Ca2+ influx.

Animals↗
PubMed2000

Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic activation.

Fertilization in mammalian eggs is characterized by the presence of intracellular calcium ([Ca(2+)]i) oscillations. In mouse eggs, these oscillations cease after a variable period of time and this is accompanied by a decrease in inositol 1,4,5-trisphosphate receptor (IP3R) responsiveness and down-regulation of the IP3R type 1 (IP3R-1). To investigate the signaling pathway responsible for inducing IP3R-1 down-regulation during fertilization, mouse eggs were exposed to or injected with several Ca(2+)-releasing agonists and the amounts of IP3R-1 immunoreactivity evaluated by Western blotting. Exposure to ethanol or ionomycin, which induce a single [Ca(2+)]i rise, failed to signal down-regulation of IP3R-1. However, [Ca(2+)]i oscillations induced by injection of boar sperm fractions (SF), which presumably stimulate production of IP3, or adenophostin A, an IP3R agonist, both induced down-regulation of IP3R-1 of a magnitude similar to or greater than that observed after fertilization. Exposure to thimerosal, an oxidizing agent that modifies the IP3R without stimulating production of IP3, also initiated down-regulation of IP3R-1, although oscillations initiated by SrCl(2) failed to evoke down-regulation of IP3R-1. The degradation of IP3R-1 in mouse eggs appears to be mediated by the proteasome pathway because it was inhibited by preincubation with lactacystin, a very specific proteasome inhibitor. We therefore suggest that persistent stimulation of the phosphoinositide pathway in mouse eggs by the sperm during fertilization or by injection of SF leads to down-regulation of the IP3R-1.

Adenosine↗