[Quantitative determination of sulfhydryl groups with "Mercurochrome". II. Detection of fast reacting protein thiols in native cells].
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Mercurochrom (2,7-dibromo-4-(hydroxymercuri)-fluoresceine-disodium salt) reacts histochemically not only with protein-SH-groups, but is also bound unspecifically to cellular proteins. The amount of the unspecifically staining approximately equals the specific SH-staining. Two methods are described to remove the unspecifically bound Mercurochrom, without influencing the specific reaction with the protein thiols. The first applies 0,1 m thioglycolate pH 4.0 (MT4-method), the other a special tris-cyanide-buffer pH 7.4 (MCN)-method). Aliquots from preparations of rat hepatocytes, Yoshida ascites tumor cells, Ehrlich ascites tumor cells, isolated nuclei of Ehrlich-cells and chicken thymocytes were investigated as well for the protein thiol content of the cells macroscopically with DTNB as microspectrophotometrically for the extinctions of the cells after staining with MT4- or MCN-method. A strong correlation was found between the macroscopically determined total-protein-SH-contents and the microphotometrically determined mean-total-extinctions of the cells. Additionally the molar absorptivities determined macroscopically by Schauenstein and Scheuringer (1980) coincide excellently with the values found microspectrometrically on MT4- and MCN-stained cells.
Two rapid flourescence procedures are described for detecting sulphydryl, disulphide and isothiocyanate groups of scented and pungent principles present in the vacuolar sap of onion, garlic and cabbage. To localize compounds containing sulphydryl groups, fresh or fixed frozen sections of the plants were treated with mercurochrome. After the fluorochromization, strongly-positive sulphydryl sites emitted an intense orange-red fluorescence, while weakly-positive sites emitted a distinctive red-brown fluorescence. Disulphide groups were detected by first reducing with thioglycolic acid to thiol groups before treating with mercurochrome. To effect isothiocyanate localization, frozen sections were exposed to ammonia: isothiocyanates were converted to thioureas and the engendered amino groups were revealed with fluorescamine.
Mercurochrom [2,7-dibromo-4-(hydroxymercuri)-fluorescein disodium salt] used for staining of protein thiols in addition binds to other groups of proteins. Experimental evidence is provided that mercurochrom bound to non-thiol groups forms a 1:1 adduct with protein (mixed) disulfides. The disulfide contents of three different types of cells determined biochemically correlated with the corresponding mean integrated optical densities determined microphotometrically after mercurochrom staining of groups other than thiols. Intracellular disulfide exchange has been studied, leading to a transformation of protein mixed disulfides to protein disulfides and an equimolar loss of protein thiols. Protein mixed disulfides were generated from protein thiols using both methyl methanethiosulfonate (MMTS) and 2,2'-dihydroxy-6,6'-dinaphthyldisulfide (DDD). Loss of thiols as well as the equimolar increase of protein mixed disulfides were followed using both mercurochrom staining for thiols and for disulfides. Generation of protein mixed disulfides due to the DDD reaction was also followed by azocoupling with Fast blue B. On the basis of the observed stoichiometry between the loss of protein thiols and the quantity, increase or conversion of protein disulfides determined microphotometrically using both mercurochrom staining and DDD Fast blue B staining, we conclude that: (1) 1 mol of mercurochrom is bound per mol of protein (mixed) disulfide; and (2) the molar absorptivity of mercurochrom bound to disulfides is epsilon 520 = 34940. This study demonstrates that mercurochrom can be used for the quantitative determination of the oxidative status of protein thiols in cells.
The authors present a case of accidental intrathecal mercury application. A 69-year-old white woman was admitted to our department with suspected meningitis following surgery for spinal stenosis at another hospital. Postoperatively, she had developed a cerebro-spinal fluid (CSF) fistula with a subcutaneous cavity. Local wound irritation had been suspected and, unfortunately, mercury-containing disinfectant was injected into the cavity. Within 24 h the patient demonstrated acute neurological deterioration due to meningitis and encephalitis and was admitted to our clinic with suspected meningitis due to postoperative CSF fistula. Lumbar puncture revealed desinfectant-stained, non-bloody CSF, while lumbar MRI demonstrated the large lumbar subcutaneous cavity. Additionally, CSF fistula was visualized on MRI. Laboratory examination revealed extremely high mercury levels in CSF, blood and urine. Treatment consisted in insertion of a lumbar drainage to wash out the mercury. The patient underwent medical detoxication using chelating agents (DMPS: RS-2,3-dimercapto-1-propansulfonacid, DMSA: meso-2,3-dimercaptosuccinatacid). Surgery was performed in order to close the cavity and the fistula. Postoperatively, the patient was admitted to the intensive care unit and remained intubated for 3 days. Within 4 weeks after surgery, she demonstrated good recovery. Eighteen months after intoxication, polyneuropathy and slight neuropsychological deficiencies were detectable.
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Measurement of the important performance parameters shows that Kodak Kodirex film is more suitable than conventional ones for dark-field transmission electron microscopy of molecules at very high mignification. Results are cited for 120 kV; but the relation ship is valid up to 3 MV.
Thirty-seven patients with open wounds from surgical intervention of pilonidal cyst (14; 37.8%) or pilonidal fistula (23; 62.2%) were enrolled on a voluntary basis in a comparative blinded clinical trial and randomly assigned to a topical treatment with a Rhizophora mangle aqueous bark extract once a day or twice a day or mercurocrome twice a day. The efficacy of the treatments was evaluated weekly from day 10 to 12 until 6 weeks after surgery by measuring the area of the wounds by image digital planimetry and the tolerability by recording adverse effects. The initial size of the wounds was taken in consideration as covariable in the Generalized Lineal Model used. A thin dark red colored film covering the wound was observed in all the cases treated with the extract. The wound areas of the groups treated with the extract once or twice per day showed a greater reduction (P < 0.05) compared to the group treated with mercurocrome. No differences between the two regimes of application of the extract of R. mangle were observed. No subject showed any sign of adverse effects and no secondary infections were observed.
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Mercurochrome strongly inhibits aspartate transaminase and 2,3-dicarboxyethylated aspartate transaminase. The native enzyme exhibits a biphasic time-course of inactivation by mercurochrome with second-order rate constants 1.62 x 10(4) M-1 - min-1 and 2.15 x 10(3) M-1 - min-1, whereas the modified enzyme is inactivated more slowly (second-order rate constant 6.1 x 10(2) M-1 - min-1) under the same conditions. The inhibitor inactivates native and modified enzyme in the absence as well as in the presence of substrates. Mercurochrome-transaminase interaction is accompanied by a red shift in the absorption maximum of the fluorochrome of about 10 nm. Difference spectra of the mercurochrome-enzyme system versus mercurochrome, compared with analogous spectra of mercurochrome-ethanol, revealed that the spectral shifts recorded during mercurochrome-transaminase interaction are similar to those that occur when mercurochrome is dissolved in non-polar solvents. Studies of mercurochrome complexes with native or modified transaminase, isolated by chromatography on Sephadex G-25, revealed that native transaminase is able to conjugate with four mercurochrome molecules per molecule, but the modified enzyme is able to conjugate with only two mercurochrome molecules per molecule.
Seventy-two mycologically proven cases of otomycosis (38 males, 34 females) aged 8-80 years were investigated. Aspergillus niger was the commonest aetiological agent (43.1%) followed by Candida spp. (22.2%), A. flavus (19.4%) and A. fumigatus (15.3%). Three topical drugs, namely mercurochrome, clotrimazole and locacorten-vioform, were evaluated for otomycosis therapy in separate groups of 24 patients each. Mercurochrome was found to be the most efficacious in terms of healing, relief from symptoms of the disease and production of negative fungal cultures. Mercurochrome is recommended as a safe and economical drug for the topical treatment of otomycosis in developing countries like Nigeria.
Aspergillus niger, an opportunistic filamentous fungus, was identified as the cause of chronic bilateral otomycosis in a 46-year-old female patient who was unresponsive to different drugs. The patient showed signs of erythema, otalgia, itching, otorrhoea and presence of greyish black coloured mass in both the ear canals. The direct microscopical examination of the ear debris in potassium hydroxide preparations, Giemsa, phase contrast and Gram revealed many thin, branched septate hyphae, condia and conidiophores morphologically indistinguishable from Aspergillus spp. The histopathological section of the ear wax mass by haematoxylin and eosin and periodic acid-Schiff techniques also showed similar fungal elements. The patient responded to 1% solution of mercurochrome. The use of mercurochrome in developing countries like India may be recommended to treat the fungal otitis in patients. We also emphasize that 'Narayan' stain should be routinely employed by microbiology and public health laboratories to study the morphology of pathogenic fungi.
OBJECTIVE: To determine whether the local application of mercurochrome over genital skin before surgery, in addition to soap/water scrubs, can help to decrease the incidence of infection patients undergoing hypospadias repair, and thereby the incidence of fistula formation. PATIENTS AND METHODS: The study comprised 44 consecutive boys undergoing primary or repeat surgery for hypospadias between October 1999 and April 2000. They were arbitrarily divided into two groups; group 1 received a conventional local scrub with soap/water 48 h before surgery and group 2, a conventional local scrub with soap/water was followed by a local application of 2% mercurochrome for 48 h before surgery. Both groups were comparable in age, location of the meatus and stage of repair. All patients were exposed to similar conditions during and after surgery. Urine, skin and wound swabs taken before during and after surgery were assessed microbiologically, using standard precautions. The incidence of infection and complications was then compared. RESULTS: Mercurochrome significantly decreased local infection, especially before surgery, but its effect in decreasing fistula formation, although appreciable, was not statistically significant. CONCLUSION: The local application of mercurochrome after a soap/water scrub for at least 48 h before surgery is a simple, economic and effective means to decrease postoperative wound infection. Future studies are needed, keeping other determinants of fistula formation constant, to evaluate its role in decreasing fistula formation in patients undergoing hypospadias repair.