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PubMed1987

Effect of heat or chemical treatment on leptospiral antigens.

The use of bacterins is one of the primary methods in the control of leptospirosis in domestic animals, especially cattle, swine, and dogs. Bacterins have been made using chemically treated or heat-inactivated organisms. Many of the initial studies indicated that the bacterins gave complete protection, whereas later reports found that some inactivation methods resulted in bacterins that protect against death but not against kidney infection and renal shedding. This raised the possibility that some of the antigens had been altered. To investigate this possibility, we heat or chemically treated Leptospira interrogans serovar icterohaemorrhagiae CF-1, L. interrogans serovar canicola Moulton, L. interrogans serovar grippotyphosa SC4397, L. interrogans serovar hardjo Hardjoprajitno, and L. interrogans serovar pomona MLS. Temperatures used to determine alterations in antigens were 50, 56, 80, 100, and 121 degrees C. The chemicals used were formaldehyde, phenol, and thimerosal. Analysis was done using Laemmli gels and Western blots. We found that heating at 50 or 56 degrees C had the smallest effect on antigens, whereas heating at 80 or 100 degrees C caused the appearance of high-molecular-weight bands on Western blots. Heating at 121 degrees C caused almost all the bands to disappear, in both the stained gel and the Western blot. Both phenol and Formalin treatment altered proteins, whereas thimerosal treatment appeared to have little effect compared with heating at 50 degrees C.

Antigens, Bacterial↗
PubMed1989

Time-dependent effects of extraluminally-applied oxyhemoglobin and endothelial removal on vasodilator responses in isolated, perfused canine basilar arteries.

The effects of oxyhemoglobin (oxyHb) and removal of the endothelium have been measured in isolated, perfused canine basilar arteries treated with intraluminal vasodilator drugs. In preparations with an intact endothelium, acetylcholine (ACh) produces a biphasic response, where a small vasodilation precedes a vasoconstriction. Calcium ionophore A23187 and thimerosal induce a vasodilation at low doses and a biphasic response at high doses. Sodium nitroprusside (SNP) evokes only a vasodilation. Extraluminally applied oxyHb itself produces a transient vasoconstriction and then a gradual increase in perfusion pressure. After this treatment, the vasoconstriction to ACh was enhanced as was the vasodilation to SNP. Significant attenuation of the vasodilator action of A23187 and thimerosal was also observed. Subsequent removal of endothelium produced no further change in response. This implies that the action of extraluminal oxyHb is to eliminate any endothelium-dependent responses. If cerebral vasospasm after subarachnoid hemorrhage is initiated by hemoglobin released from the clot surrounding the artery, at least one component of its action appears to involve interference with endothelial function.

Acetylcholine↗
PubMed1982

Cytotoxicity of mercurial preservatives in cell culture.

The cytotoxicity of two kinds of mercurial preservatives, thimerosal and phenylmercuric acetate, was studied using Chang's human conjunctival epithelia in cell culture. The cultured cells were exposed for 5 s, 2 min and 24 h to each of the two preservatives at various concentrations, obtained by serial dilution. The cytopathic effect and cell desquamation from the wall of culture flask were observed with an inverted microscope and LD50 was calculated by Van der Waerden's method. The LD50 values of thimerosal were 291.6, 47.4 and 2.2 micrograms/ml at exposure times of 5 s, 2 min and 24 h, respectively. Those of phenylmercuric acetate were 1,120.2, 227.5 and 2.6 microgram/ml at exposure times of 5 s, 2 min and 24 h, respectively. Cytotoxicity can be expressed quantitatively and precisely by LD50. LD50 is lower than the concentrations that are necessary to induce observable morphological changes. The results may help to choose the least toxic concentration of mercurial preservatives for addition to ophthalmic solutions.

Cells, Cultured↗
PubMed2001

Thiol-reactive agents biphasically regulate inositol 1,4,5-trisphosphate binding and Ca(2+) release activities in bovine adrenal cortex microsomes.

Within all endocrine cells, the inositol 1,4,5-trisphosphate (InsP(3)) receptor plays an important role in regulation of the intracellular Ca(2+) concentration. In the present study we showed that a single short-term treatment with either N-ethylmaleimide (known to decrease InsP(3) receptor activity) or thimerosal (known to increase InsP(3) receptor activity) caused time-dependent biphasic effects on the InsP(3) binding activity of bovine adrenal cortex microsomes. The early potentiating effect of thiol-reactive agents translated into a 2-fold increase in binding affinity and Ca(2+) release efficiency. The late dampening effect of thiol-reactive agents translated into a continuous reduction of the maximal binding capacity of the microsomes with a concomitant decrease in Ca(2+) release efficiency. Under these conditions, Western blot analyses demonstrated that the level of InsP(3) receptor protein was not modified. Sequential treatments with thimerosal and the reducing agent dithiothreitol showed that the InsP(3) receptor can readily oscillate between high and low affinity states that are related to its alkylation state. Our results suggest a common mode of action of thiol-reactive agents on the InsP(3) receptor. These results also support the contention that cellular mechanisms of thiol group modification could play important roles in regulation of the intracellular Ca(2+) concentration.

Adrenal Cortex↗
PubMed1991

Quantitative evaluation of the corneal epithelial barrier: effect of artificial tears and preservatives.

The effect of artificial tear solutions on the corneal epithelial barrier was evaluated by measuring corneal uptake of 5, 6 carboxyfluorescein (CF) after exposure of rabbit corneas to various formulations in a conjunctival cup. Four tear solutions containing 0.01% benzalkonium chloride (BAC), a formulation containing 0.001% Polyquad, a contact lens re-wetting solution containing 0.004% thimerosal, and each of the above preservatives in balanced salt solution (BSS) were evaluated. Four non-preserved solutions were also tested. After treatment with the solutions corneas were exposed to the CF, removed from the eyes and dialyzed in balanced salt solution. The CF concentration in the dialysate was measured by fluorometry. Solutions containing 0.01% BAC caused a 9.24 to 99.28 fold increase in CF uptake as compared to control. Solutions preserved with Polyquad or thimerosal caused only a 0 to 4 fold increase in CF uptake while non-preserved solutions caused no change compared to control. Corneas prepared for transmission electron microscopy using fixative containing ruthenium red exhibited damage which correlated well with CF uptake; the ruthenium red penetrated the epithelium to the basal cell layer after corneal exposure to solutions containing BAC while only superficial cell layers were stained after exposure to the other test solutions. The method used in this study allows statistical comparison of artificial tear formulations. The data show that patients with severe dry-eye who use artificial tears frequently should avoid tear solutions containing BAC and that non-preserved solutions are preferable for treatment of these patients.

Animals↗
PubMed2002

Redox regulation of calcium release in skeletal and cardiac muscle.

In skeletal and cardiac muscle cells, specific isoforms of the Ryanodine receptor channels mediate Ca2+ release from the sarcoplasmic reticulum. These channels are highly susceptible to redox modifications, which regulate channel activity. In this work, we studied the effects of Ca2+ (endogenous agonist) and Mg2+ (endogenous inhibitor) on the kinetics of Ca2+ release from sarcoplasmic reticulum vesicles isolated from skeletal or cardiac mammalian muscle. Native skeletal vesicles exhibited maximal stimulation of release kinetics by 10-20 microM [Ca2+], whereas in native cardiac vesicles, maximal stimulation of release required only 1 microM [Ca2+]. In 10 microM [Ca2+], free [Mg2+] < 0.1 mM produced marked inhibition of release from skeletal vesicles but free [Mg2+] < or = 0.8 mM did not affect release from cardiac vesicles. Incubation of skeletal or cardiac vesicles with the oxidant thimerosal increased their susceptibility to stimulation by Ca2+ and decreased the inhibitory effect of Mg2+ in skeletal vesicles. Sulfhydryl-reducing agents fully reversed the effects of thimerosal. The endogenous redox species, glutathione disulfide and S-nitrosoglutathione, also stimulated release from skeletal sarcoplasmic reticulum vesicles. In 10 microM [Ca2+], 35S-nitrosoglutathione labeled a protein fraction enriched in release channels through S-glutathiolation. Free [Mg2+] 1 mM or decreasing free [Ca2+] to the nM range prevented this reaction. Possible physiological and pathological consequences of redox modification of release channels on Ca2+ signaling in heart and muscle cells are discussed.

Animals↗
PubMed1997

Microscopic diagnosis of blood parasites following a cytoconcentration technique.

An isotonic fixative (formalin and thimerosal) solution, with a saponin additive to lyse erythrocytes and platelets, has been developed. The formalin and thimerosal ensure good preservation of blood parasites. This fixative has led to the development of a new concentration technique using cytocentrifugation (cytospin) in the search for Plasmodium spp., Leishmania spp., and microfilariae, as well as leukocytes in which parasites or pigment may be present. The concentration of the parasites present in the sediment from 100 microl of blood spread on a 6-mm diameter circle results in good morphology that is well stained using the usual Giemsa or Wright techniques. This new technique has the advantage of a relatively low cost and offers the possibility of isolating and identifying in the same sediment the main blood-stage parasites, with the exception of young trophozoites, of Plasmodium falciparum.

AIDS-Related Opportunistic Infections↗
PubMed1987

Abluminal release and asymmetrical response of the rabbit arterial wall to endothelium-derived relaxing factor.

A marked functional polarity of endothelial cells as well as an asymmetry of the vascular wall in response to vasoactive compounds is well established. Therefore, we investigated the polarity of endothelium-derived relaxant factor (EDRF) release from native endothelial cells, its diffusion characteristics, and its dilator effects on inner and outer muscle layers of the vascular wall in isolated rabbit arteries. Following exposure of rabbit aortae (with intact endothelium) to the EDRF stimulators acetylcholine, A23187, or thimerosal, a humoral dilator compound could be assayed in the adventitial superfusate of the vessels. The vasodilator effects were blocked by the EDRF inhibitors hemoglobin, dithiothreitol, and gossypol. Penetration of the dilator through the arterial wall following stimulation by acetylcholine, A23187, or thimerosal was observed only when dilator EDRF activity in the luminal perfusate was maximal. Luminally administered EDRF, released from cultured endothelial cells, did not cross the aortic wall in detectable amounts. EDRF (from cultured cells) elicited significantly smaller dilations (9 +/- 4%) when applied to the adventitial side of endothelium-denuded rabbit aortae or femoral arteries as compared with luminal application (92 +/- 7%). In contrast, sodium nitroprusside was equieffective by both routes of administration. We conclude that EDRF in native endothelial cells is released in both luminal and abluminal directions and can penetrate the entire vascular wall. However, the lengthy diffusion time and the decreasing sensitivity of outer smooth muscle layers suggest that mechanisms other than EDRF diffusion contribute significantly to the propagation of endothelium-mediated relaxation through the arterial wall.

Acetylcholine↗
PubMed1981

Conformational changes of renal sodium plus potassium ion-transport adenosine triphosphatase labeled with fluorescein.

We studied conformational changes of purified renal sodium plus potassium ion-transport adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3) labeled with fluorescein isothiocyanate. Fluorescein covalently binds to the alpha-subunit of the enzyme and inhibits the ATPase but not the p-nitrophenylphosphatase activity. Four unphosphorylated and three phosphorylated conformations were distinguished by the level of fluorescence and by the rate of its change (relative fluorescence is shown in percentages). Fluorescence of the ligand-free form (E1, 100%) was increased by Na+ (E1.Na form, 103%) and quenched by K+ (E2.K, 78%) at a site of high affinity (K0.5 for K+ = 0.07 mM). Mg2+ did not alter fluorescence of E1 or E1.Na but raised that of E2.K (E2.K.Mg form, 85-90%). Addition of excess Na+ to the E2.K.Mg form restored high fluorescence but the rate of transition from E2.K.Mg to E1.Na became progressively slower with increasing Mg2+ concentration. Two phosphorylated conformations, (E2-P).Mg (82%) and (E2-P).Mg.K (82%) were differentiated by a faster turnover of the latter form. A third conformation, (E2-P).Mg.ouabain, had the lowest fluorescence (56%) and its formation allowed the binding of ouabain to the phosphoenzyme. Reversible blocking of sulfhydryl groups with thimerosal inhibited the formation of E2.K and (E2-P).Mg.ouabain but not that of the other conformations of the fluorescein-enzyme. The thimerosal-treated fluorescein-enzyme retained K+-p-nitrophenylphosphatase activity, inhibition of this activity by ouabain and ouabain binding. The unphosphorylated enzyme had low (K0.5 = 1.2 mM) and the phosphoenzyme had high affinity (K0.5 = 0.03 - 0.09 mM) for Mg2+ in the absence of nucleotides. Since low and high affinity for Mg2+ alternates as the enzyme turns over, Mg2+ may be bound and released sequentially during the catalytic cycle.

Animals↗
PubMed1984

Contact lens wear problems: implications of penicillin allergy, diabetic relatives, and use of birth control pills.

A study was carried out on a random sample of contact lens patients seen in private practice. Some of these patients experienced contact lens wear problems including keratitis, hyperemia, and discomfort. The results indicate that: (1) Fifty percent of those who experienced problems with the wear of hard contact lenses had diabetic relatives. (2) It is likely that the wear of either hard or soft contact lenses is adversely affected by the use of birth control pills. (3) Forty-five percent of those patients who were allergic to penicillin experienced problems with soft contact lens care regimens containing thimerosal. Some implications of an immunological cross-reactivity between thimerosal-haptenated proteins and penicillin-haptenated proteins are discussed.

Contact Lenses↗
PubMed1983

[Evaluation of the toxic action of prophylactic and therapeutic preparations on cell cultures. III. The detection of toxic properties in medical biological preparations by the degree of cell damage in the L132 continuous cell line].

The methods of the quality control of medical biological preparations, including tests on animals, do not ensure the complete absence of toxicity in a final product. The use of the method of "subcultures with the introduced preparation" makes it possible to determine the toxicity of both specific and nonspecific components of vaccines and sera from the number of dead and damaged cells. The toxic action of preparations kills and damages the cells at the site of injection, thus inducing the formation of autoantigens whose effect on the body cannot be predicted. Thus thimerosal, commonly used as preservative, has been found not only to render its primary toxic effect, but also capable of changing the properties of cells. This fact suggests that the use of thimerosal for the preservation of medical biological preparations, especially those intended for children, is inadmissible.

Allergens↗
PubMed1981

Aerobic responses of the cornea to ophthalmic preservatives, measured in vivo.

A micropolarographic system was used to measure the effects of several concentrations of benzalkonium chloride and thimerosal on oxygen uptake by the cornea. Although the threshold of 0.01% found for benzalkonium chloride generally agreed with other criteria (e.g., clinical and histologic) in the current literature, the threshold for thimerosal was somewhat higher, 1.0%, indicating a substantially greater aerobic activity tolerance of the cornea to initial direct exposures of that agent then to benzalkonium chloride.

Animals↗
PubMed1980

Pseudobacteremia caused by Clostridium sordellii.

Thirteen of 280 (4.6%) blood cultures collected over a 12-day period were positive for Clostridium sordellii, a spore-forming anaerobe, rarely considered a human pathogen. Nosocomial bacteremia and intrinsic contamination of material used to culture blood were excluded as the source of the organism. Contaminated tincture of thimerosal used to swab the rubber stoppers of blood culture bottles prior to venting (aerobic) or during blind subculturing after 24 hours of incubation (anaerobic) in the clinical microbiology laboratory was determined to be the cause of the pseudobacteremia. After appropriate safe-guards were implemented, we have continued to use tincture of thimerosal for these procedures with no further growth of C sordellii from blood cultures. The importance of less-conspicuous steps in the routine processing of culture material have been reemphasized.

Blood Specimen Collection↗
PubMed1995

Sperm-induced calcium oscillations of human oocytes show distinct features in oocyte center and periphery.

Temporal and spatial characteristics of explosive periodic increases (spikes) of intracellular free Ca2+ concentration ([Ca2+]i) induced by sperm in human oocytes (Ca2+ oscillations) were analyzed by confocal laser scanning microscopy and compared to Ca2+ oscillations induced in oocytes by the thiol reagent thimerosal. During the steady-state period of sperm-induced Ca2+ oscillations, each individual [Ca2+]i spike invariably began from a focus in oocyte periphery and spread throughout the entire peripheral region before propagating to the central ooplasm. This peripheral Ca2+ wave was immediately followed by an explosive [Ca2+]i increase in the central ooplasm. However, this central [Ca2+]i rise only peaked when [Ca2+]i in the peripheral ooplasm was already on the decline. Moreover, the peak [Ca2+]i values were always considerably higher in the oocyte center than in the periphery. In contrast, thimerosal-induced Ca2+ oscillations did not show this particular form of propagation. These data show that sperm-induced Ca2+ oscillations have a unique pattern of spatial dynamics and suggest that the bulk of Ca2+ mobilized during each spike is released from stores that have a relatively high threshold for Ca(2+)-induced Ca2+ release (CICR). These stores are poorly developed, if not absent, in the oocyte cortex, and CICR from them is triggered by previous CICR from another type of store with a lower threshold that are preferentially located in the oocyte cortex and act as a detonator.

Calcium↗
PubMed2004

Association of female aging with decreased parthenogenetic activation, raised MPF, and MAPKs activities and reduced levels of glutathione S-transferases activity and thiols in mouse oocytes.

This study aims to determine in the mouse whether oocytes from reproductively old females exhibit a different susceptibility to be parthenogenetically activated when compared to oocytes from young females. At the age of 10-12 (young-female group) or 60-62 (old-female group) weeks, hybrid female mice were superovulated using pregnant mare's serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) 48 hr later. After removing the cumulus cells, oocytes were exposed to any of two different activating protocols: (a) 6-min exposure to 8% ethanol; and (b) treatment with 200 microM thimerosal for 15 min followed by 8 mM dithiothreitol (DTT) for 30 min. Oocytes from old female mice displayed (1) lower total percentage of parthenogenetic activation and extrusion of the second polar body after treatment with either thimerosal + DTT or ethanol; (2) higher M-phase-promoting factor (MPF) and mitogen-activated protein kinases (MAPKs) activities; and (3) lower intracytoplasmic levels of glutathione S-transferases (GSTs) activity and thiols than oocytes from young females. These data show that female aging is associated with higher resistance of oocytes to be parthenogenetically activated, higher MPF and MAPKs activities and lower intracytoplasmic levels of GSTs activity and thiols.

Aging↗
PubMed1995

Calcium-dependent signal transduction by the hypertrehalosemic hormone in the cockroach fat body.

The hypertrehalosemic hormones (HrTH) are insect peptides that stimulate the fat body to synthesize trehalose. The present research investigated the relative importance of extra- and intracellular Ca2+ for HrTH signal transduction in the fat body of the cockroach, Blaberus discoidalis. Although trehalose synthesis could be stimulated significantly by B. discoidalis HrTH (Bld-HrTH) in the absence of Ca2+, the presence of Ca2+ was necessary to attain a maximum hormonal response. Entry of extracellular Ca2+ mediated by ionophores (A23187, ionomycin) produced no significant hypertrehalosemic effects, whereas release of intracellular Ca2+ by thimerosal or thapsigargin significantly stimulated trehalose synthesis. The hypertrehalosemic effect by thimerosal occurred independently of extracellular Ca2+. Dantrolene inhibits intracellular Ca2+ release, and its presence partially suppressed Bld-HrTH-dependent hypertrehalosemia. In conclusion, the presence of either extracellular Ca2+ or intracellular Ca2+ enhanced the Bld-HrTH response, but agents that affected intracellular Ca2+ release produced the more conclusive effects.

Animals↗
PubMed1998

[Differentiated contact allergy lists serve in quality improvement].

Through cooperation between the German Contact Dermatitis Research Group (DKG) and the Information Network of Departments of Dermatology (IVDK), results of standardized patch tests performed by the participating centres are centrally recorded in Germany. For this study, data from 24 departments of dermatology (19678 patients) were evaluated retrospectively and separately for 1995 and 1996. With decreasing frequency, nickel, fragrance mix, balsam of peru, and thimerosal yielded positive reactions most often; surprisingly high numbers of positive reactions were also seen with amerchol L-101 and palladium. Differentiated lists of allergens were compiled for 1995, referring to subgroups of patients (defined by gender, age, localization of eczema, geographical region, occupation, sensitization) and particular problems. In men, percentages of positive reactions were markedly lower for nickel, fragrance mix and balsam of peru than in women. Younger patients reacted more often to thimerosal and older ones to topical medical preparations. Medical allergens were also often positive in patients with leg eczema, whereas occupational allergens were found more frequently in patients with hand eczema. A comparison of positive reactions obtained in distinct geographical regions was problematic because of differences between test populations. The spectrum of allergens found in office workers was similar to that of the whole test population. Patients with positive reactions to nickel and fragrance mix had more positive reactions to unrelated allergens than the total test population. Sex- and age-adjusted frequencies of sensitization revealed a decrease in reactions to nickel and an increase in reactions to mercury allergens from 1995 to 1996. The clinical relevance of mercury reactions was often not apparent. Differentiated lists of allergens can be used to improve the quality of diagnostic and prophylactic procedures in allergic contact dermatitis.

Adolescent↗