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PubMed2003

Cloning, expression, purification and crystallization of the N-domain from the alpha(2) subunit of the membrane-spanning Na,K-ATPase protein.

The nucleotide-binding domain of the Na,K-ATPase ion pump was expressed with a His tag in Escherichia coli and purified. The soluble 24 kDa derivative consists of 214 amino-acid residues and was crystallized in the presence of NiCl(2). The crystals belong to space group F23, with unit-cell parameters a = b = c = 147.5 A, and diffract to 3.1 A. Complete data sets could be collected from native and thimerosal-treated crystals frozen in 50% sucrose. Five mercury positions were found and initial SIR phases calculated.

Amino Acid Sequence↗
PubMed2001

Voltage-dependent Ca2+ release in rat megakaryocytes requires functional IP3 receptors.

Using simultaneous whole-cell patch-clamp and fluorescence measurements of [Ca2+]i in rat megakaryocytes we have investigated the requirement for functional inositol 1,4,5-trisphosphate (IP3) receptors in Ca2+ release induced by membrane depolarization during agonist stimulation. Voltage-dependent Ca2+ release was observed during application of the IP3-generating agonists U46619 (a thromboxane A2 analogue) and ADP. Furthermore, voltage-dependent Ca2+ release was observed in the absence of exogenous agonist following sensitization of IP3 receptors with thimerosal. Depolarization-induced Ca2+ release was not detected during depletion of intracellular Ca2+ stores by thapsigargin. Thus, depletion of stores alone is not sufficient to confer voltage dependence upon the Ca2+ release mechanism. Block of IP3 receptors by carbacyclin-stimulated elevations in cAMP, uncaging of cAMP or exposure to a high concentration of caffeine reversibly abolished Ca2+ increases stimulated by both ADP and depolarization. The cAMP-dependent block was prevented by a peptide inhibitor of protein kinase A, indicating that an alteration of adenylate cyclase activity leading to modulation of protein kinase A activity does not underlie the control of Ca2+ release by voltage. These results are consistent with the requirement for functional IP3 receptors for voltage control of Ca2+ release from intracellular stores during inositol lipid signalling. The data also indicate the involvement of a voltage sensor downstream of surface membrane receptors in the depolarization-evoked Ca2+ response.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMedDate not supplied

Patch testing of Singapore children and adolescents: our experience over 18 years.

This is a retrospective epidemiologic study of allergic contact dermatitis in children and adolescents in Singapore who had undergone patch testing from January 1, 1986 to December 31, 2003. A total of 2,340 patients below the age of 21 years had undergone patch testing. Of these, 1,063 (583 girls, 480 boys) were positive to one or more allergens. The most common allergens were: nickel (40%), thimerosal (15%), colophony (9%), lanolin (8%), cobalt (8%), fragrance mix (5%), and neomycin (4%). Each of these allergens will be discussed individually. This is the first study with over 1000 patients on this subject from Asia.

Adolescent↗
PubMed1975

The frequency of contact sensitivity in North America 1972-74.

Three thousand subjects in North America were patch-tested with 19 allergens. The most frequent sensitizers observed include nickel sulfate, caine mixture, potassium dichromate, balsam of Peru, thimerosal, ethylenediamine hydrochloride, paraphenylenediamine and thiram. The prevalence data compared with an earlier study indicates that the initial allergens reported warrant their continued inclusion in a routine patch test screening series in North America.

Balsams↗
PubMed1976

Effects of vehicles and elicitation concentration in contact dermatitis testing. I. Experimental contact sensitization in humans.

A study was made to evaluate the effects of vehicle and challenge concentration on response of human subjects to potential allergens. In the vehicle studies the modified Draize test was used to test the response of subjects to cinnamic aldehyde and to costus oil, administered at two skin sites, in petrolatum and in 95% ethyl alcohol. In two tests of costus oil, alcohol proved to be more effective in eliciting a response than petrolatum; on the other hand, in one test with cinnamic aldehyde, no difference in results was obtained with these two vehicles. In the concentration studies, subjects known to be sensitive to the test substance were tested by the Al test with costus oil (three concentrations), chloracetamide (four concentrations), or thimerosal (three concentrations); petrolatum was used as the vehicle in each case. Results of the vehicle test showed no compelling reason for the selection of one vehicle rather than another. Results of the concentration tests indicated that concentration does have an effect on the intensity and frequency of reactions to potential allergens.

Acetamides↗
PubMed1978

Contact dermatitis in Salvador, Brazil.

In Salvador, Brazil, 536 patients were patch tested with 24 contact allergens. The most common sensitizers included potassium dichromate, thimerosal, hydroquinone, nitrofurazone ointment and nickel sulfate. The results of this South American study are compared with those published by North American and European dermatologists. Low frequencies of positive reactions were encountered in Salvador to some substances, including mercury bichloride and p-phenylenediamine. The influences of climate and life style in determining patterns of contact sensitivity are discussed.

Adult↗
PubMed1990

Patch and prick test study of 593 healthy subjects.

593 recruits selected by the Military Health Service as being healthy and without a history of present or previous dermatitis, or ocular refraction defects, were patch tested with the GIRDCA (Italian Research Group on Contact and Environmental Dermatitis) standard series. Of these, 336 were also patch tested with substances used in the processing and dyeing of textiles and prick tested with 8 major allergens. 74 (12.5%) reacted to 1 or more substances. The most frequent sensitizers were: thimerosal (28 cases), ammoniated mercury (7 cases), phenol-formaldehyde resin (6 cases), parabens, nickel and Disperse Red 17 (4 cases each). 113 recruits reacted to 1 or more prick test allergens. We have demonstrated the importance of establishing such reference values in healthy groups for the correct evaluation of data collected from selected groups.

Adolescent↗
PubMed1994

Hand eczema in car mechanics.

To estimate the prevalence of hand eczema, a questionnaire was distributed to 901 male car mechanics. Of the 801 persons who responded, 15% reported hand eczema on some occasion in the previous 12 months, and 57% admitted dry skin on the hands. In a 2nd part of the study, those who reported hand eczema were examined and patch tested with a standard series and a special "car mechanics' series". The most common diagnosis was irritant contact dermatitis, 55%, and 2nd was allergic contact dermatitis, 19%. 35/105 (33%) had a total of 51 positive patch test reactions, all to substances in the standard series, except for 2 persons who reacted to oxidized d-limonene. The most frequent reactions were to thimerosal (9%), nickel (8%) and colophony (5%). One plausible explanation for the high prevalence of nickel allergy was the common use of nickel-plated tools. 5 individuals had a history of contact urticaria, but scratch tests were negative. It was concluded that car mechanics are at high risk for contact dermatitis on the hands, irritant as well as allergic.

Adolescent↗
PubMed1995

Oral lichenoid lesions caused by allergy to mercury in amalgam fillings.

Oral lichenoid lesions (OLL) or lichen-planus-like lesions are often idiopathic. Our aim was to determine whether OLL can be caused by allergy to mercury in amalgam fillings, and whether resolution of OLL occurs after replacement of amalgam with other dental fillings. Patients with only OLL (except for 1 case with cutaneous lichen planus) referred for patch testing during 1985-1994 to the Contact and Occupational Dermatitis Clinic of the Skin & Cancer Foundation, Darlinghurst, were reviewed. Patch tests were performed with 1% mercury, 1% ammoniated mercury, 0.1% thimerosal, 0.1% mercuric chloride, 0.05% phenylmercuric nitrate and an amalgam disc, using Finn Chambers occluded for 2 days, 19 patients (17 women and 2 men; age range: 28-72 years) had OLL in close contact with amalgam fillings and showed positive patch test reactions to mercury compounds, 16 out of 19 patients had their amalgam fillings replaced. In 13 patients, the OLL healed. 1 patient had marked improvement. 1 patient had no improvement and developed multiple oral squamous cell carcinoma. In conclusion, OLL can be caused by allergy to mercury in amalgam fillings. Replacement of amalgam with other dental fillings usually results in resolution of OLL and is recommended for cases with positive patch test reactions to mercury compounds.

Adult↗
PubMed2006

Preservatives sensitivity in Israel: a 10-year overview (1995-2004).

This study presents a 10-year survey of 2285 patients tested to common preservatives in northern Israel. The demographic and clinical data were analysed using a revised MOAHLFA index. Patch testing was based on the International Contact Dermatitis Research Group (ICDRG) guidelines. 411 (18%) patients were found to have positive reactions to 1 or more preservatives. The most frequent allergens tested positively were thimerosal, methylchloroisothiazolinone/methylisothiazolinone (MCI/MI), and formaldehyde. The reactions to MCI/MI remained stable around 1.7% throughout the years 1995-1998, with a significant increase to 3.7%-4% (P = 0.029) during 1999-2004. The reactions to formaldehyde remained low around 0.3% until 1999; however, a considerable increase throughout the years 2000-2004 was noted, with rates of 1.5%-1.9% (P = 0.028). The sensitivity rates for parabens, quaternium-15, imidazolidinylurea, and diazolidinylurea were low and unchanged, mostly under 0.5%. As for methyldibromoglutaronitrile (MDBGN), sensitivity rates around 2% were detected for the years 1996 and throughout 2000-2004. Overall, this extensive survey showed a concerning increase in the sensitivity rates for MCI/MI, formaldehyde, and MDBGN.

Adult↗
PubMed1982

The effect of ophthalmic preservatives on the shape of corneal endothelial cells.

The mean area (293.6 +/- 53.6 micrometers 2) and perimeter (65.5 +/- 5.8 micrometers) of individual cells were determined from the photographed endothelium of a total of 54 guinea pig corneas, after staining with silver nitrate and removal of the epithelium and stroma. In addition a quantitative assessment of cell shape S (= P2/A) was calculated. The mean cell density (3406 +/- 619 cells per mm2) was similar to that for young humans, and the S value (14.76 +/- 0.73) was as expected for slightly irregular hexagonal and pentagonal shapes. Incubation of the corneas in glutathione-bicarbonate-Ringer solution for periods up to 4 h resulted in only slight changes in the endothelial cell shape (S = 15.21 +/- 0.89). However, incubation with thimerosal (0.01%) for 2 h (S = 16.78 +/- 1.79) or with disodium ethylenediaminetetraacetate (Na2EDTA) (0.2%) for 1 h (S = 17.20 +/- 2.36) gave a marked increase in the tortuosity of the cell 'outlines'. Longer periods of contact with Na2EDTA resulted in a rounding up and separation of endothelial cells.

Animals↗
PubMed1986

Ultrastructural changes to corneal stromal cells due to ophthalmic preservatives.

Normal and keratectomised rabbit corneas received drops containing preservatives over a two day period. All preservatives investigated caused some change in the morphology of the stromal keratocytes, particularly in the absence of the anterior epithelial barrier. Thimerosal (0.004%) or disodium ethylenediaminetetraacetate (0.1% Na2EDTA) produced only minimum change. Chlorbutanol (0.5%) applied to intact corneas resulted in swelling of the cytoplasm and occasionally breaks in the external cell membrane, whilst in the keratectomised corneas some mitochondria showed swelling and distortion of cristae. In the normal cornea, benzalkonium chloride (0.02% or 0.01% with 0.1% Na2EDTA) resulted in intra and extracellular oedema and contraction of keratocytes. On removal of the superficial layers, there was extensive disruption of mitochondria, other organelles and outer cell membranes resulting in total destruction of some cells. Benzalkonium chloride should not be used on corneas with abnormalities of the epithelium.

Animals↗
PubMed1987

Susceptibility of Torulopsis glabrata in the presence of six antifungal agents determined by comparison of growth at several pHs.

We describe a method using an automated system whereby fungistatic activities can be determined in several conditions. The process was adapted to Torulopsis glabrata, and it showed that benzalkonium chloride, chlorhexidine gluconate, and thimerosal preserve fungistatic activities in acidic medium, whereas acidification reduces the activity of povidone iodine and poloxamer.

Antifungal Agents↗
PubMed2003

SH oxidation coordinates subunits of rat brain ryanodine receptor channels activated by calcium and ATP.

We have reported that ryanodine receptor (RyR) channels display three different responses to cytoplasmic free Ca2+ concentration ([Ca2+]) depending on their redox state (Marengo JJ, Hidalgo C, and Bull R. Biophys J 74: 1263-1277, 1998), with low, moderate, and high maximal fractional open times (Po). Activation by ATP of single RyR channels from rat brain cortex was tested in planar lipid bilayers with 10 or 0.1 microM cytoplasmic [Ca2+]. At 10 microM [Ca2+], low-Po channels presented lower apparent affinity to activation by ATP [[ATP] for half-maximal activation (KaATP) = 422 microM] than moderate-Po channels (KaATP = 82 microM). Oxidation of low-Po channels with thimerosal or 2,2'-dithiodipyridine (DTDP) gave rise to moderate-Po channels and decreased KaATP from 422 to 82 microM. At 0.1 microM cytoplasmic [Ca2+], ATP induced an almost negligible activation of low-Po channels. After oxidation to high-Po behavior, activation by ATP was markedly increased. Noise analysis of single-channel fluctuations of low-Po channels at 10 microM [Ca2+] plus ATP revealed the presence of subconductance states, suggesting a conduction mechanism that involves four independent subchannels. On oxidation the subchannels opened and closed in a concerted mode.

2,2'-Dipyridyl↗
PubMed2003

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes.

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 microM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 microM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in approximately 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 microM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 microM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 microM), which sensitizes the IP3R to IP3. In cells voltage clamped at -40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.

Animals↗
PubMed1997

Mechanism of enhancement of slow delayed rectifier current by extracellular sulfhydryl modification.

To explore the role of sulfhydryl (SH) groups in the function of cardiac slow delayed rectifier channels, we tested the effects of extracellular thimerosal (TMS, a hydrophilic SH modifier) on slow delayed rectifier current (IKs) induced by human IsK (hIsK) in oocytes and on the native IKs in canine ventricular myocytes. TMS (25 or 50 microM) had similar effects on the two currents: current amplitude increased, and there was an acceleration of activation and a slowing of deactivation. These effects showed little or no reversal after washout of TMS. The effects did not depend on intracellular Ca release or protein kinase activities but could be suppressed by dithiothreitol pretreatment. According to the current model of transmembrane topology, there is no cystein in the extracellular domain of hIsK. A likely candidate for TMS modification is the SH group on another subunit in oocyte cell membrane that interacts with IsK to form a functional channel. To explore the domain of hIsK involved in the interaction, extracellular serines of hIsK were mutated to cysteines at three locations: S37C (close to the transmembrane domain), S4C (close to the NH2-terminus), and S28C (in between). S37C and S28C mutations did not affect channel properties or hIsK response to TMS. On the other hand, S4C mutation reduced current expression even when S4C cRNA was injected at a quantity 50-fold higher than that of the other three proteins. Importantly, the response to TMS was markedly reduced in S4C compared with the other three proteins. Therefore, the NH2-terminus of hIsK may be involved in hIsK interaction with the SH-bearing subunit, and this interaction modulates slow delayed rectifier channel function.

Animals↗
PubMed1995

Functional consequences of sulfhydryl modification in the pore-forming subunits of cardiovascular Ca2+ and Na+ channels.

The structure and function of many cysteine-containing proteins critically depend on the oxidation state of the sulfhydryl groups. In such proteins, selective modification of sulfhydryl groups can be used to probe the relation between structure and function. We examined the effects of sulfhydryloxidizing and -reducing agents on the function of the heterologously expressed pore-forming subunits of the cloned rabbit smooth muscle L-type Ca2+ channel and the human cardiac tetrodotoxin-insensitive Na+ channel. The known sequences of the channels suggest the presence of three or four cysteine residues within the putative pores of Ca2+ or Na+ channels, respectively, as well as multiple other cysteines in regions of unknown function. We determined the effects of sulfhydryl modification on Ca2+ and Na+ channel gating and permeation by using the whole-cell and single-channel variants of the patch-clamp technique. Within 10 minutes of exposure to 2,2'-dithiodipyridine (DTDP, a specific lipophilic oxidizer of sulfhydryl groups), Ca2+ current was reduced compared with the control value, with no significant change in the kinetics and no shift in the current-voltage relations. The effect could be readily reversed by 1,4-dithiothreitol (an agent that reduces disulfide bonds). Similar results were obtained by using the hydrophilic sulfhydryl-oxidizing agent thimerosal. The effects were Ca(2+)-channel specific: DTDP induced no changes in expressed human cardiac Na+ current. Single-channel Ba2+ current recordings revealed a reduction in open probability and mean open time by DTDP but no change in single-channel conductance, implying that the reduction of macroscopic Ca2+ current reflects changes in gating and not permeation. In summary, the pore-forming (alpha 1) subunit of the L-type Ca2+ channel contains functionally important free sulfhydryl groups that modulate gating. These free sulfhydryl groups are accessible from the extracellular side by an aqueous pathway.

2,2'-Dipyridyl↗
PubMedDate not supplied

Nitric oxide-independent relaxations to acetylcholine and A23187 involve different routes of heterocellular communication. Role of Gap junctions and phospholipase A2.

NO- and prostanoid-independent relaxations are generally assumed to be mediated by an endothelium-derived hyperpolarizing factor (EDHF) that has been postulated to be an arachidonic acid metabolite. Recent evidence also suggests that direct heterocellular gap junctional communication (GJC) between endothelium and smooth muscle contributes to NO-independent relaxations. In the present study we have investigated the contribution of phospholipase A2 (PLA2)-linked metabolites and GJC to EDHF-type relaxations in rabbit mesenteric artery. In isolated rings preconstricted with 10 micromol/L phenylephrine in the presence of NG-nitro-L-arginine methyl ester (L-NAME) and indomethacin, acetylcholine (ACh) and the Ca2+ ionophore A23187 evoked relaxations that were markedly attenuated by the Ca2+-dependent PLA2 inhibitors 2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (3 micromol/L) and arachidonyl trifluoromethyl ketone (3 micromol/L), but were potentiated by the sulfhydryl agent thimerosal (300 nmol/L). In intact rings, relaxations to ACh were attenuated synergistically by L-NAME and Gap 27 peptide, an inhibitor of GJC, whereas ACh-evoked relaxations of "sandwich" preparations were unaffected by the peptide but were abolished by L-NAME. In both ring and sandwich preparations A23187-induced relaxations were attenuated by inhibition of PLA2 but were insensitive to L-NAME and Gap 27 peptide. We conclude that EDHF-type relaxations of rabbit mesenteric artery to ACh and A23187 depend on a common pathway that involves activation of PLA2. In the case of ACh, relaxation requires transfer of a factor or factors from the endothelium to smooth muscle via gap junctions, whereas A23187 permits release directly into the extracellular space.

8,11,14-Eicosatrienoic Acid↗