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PubMedDate not supplied

The effects of different decalcification protocols on TUNEL and general cartilage staining.

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.

Acetic Acid↗
PubMed2004

Microanalysis of non-heme iron in animal tissues.

The method recommended by the Iron Panel of the International Committee for the Standardization in Haematology for measurement of serum iron was adapted for measurement of non-heme iron in animal tissues. The method developed was designed specifically to facilitate measurement of non-heme iron using as little as 10 mg of tissue, in a final reaction volume of 60 microl. In this assay, tissue homogenates are treated with hydrochloric acid and trichloroacetic acid and heated at 95 degrees C. Non-heme iron is released and protein is precipitated. Following centrifugation, iron in the supernatant is reacted with ferrozine in the presence of the reducing agent thioglycolic acid, and the complex is quantified by spectrophotometry. The method was validated by analysis of two Standard Reference Materials (bovine liver), comparing results of this assay against certified values and concentrations determined by flame atomic absorption spectrometry following acid digestion. Results using this method for analysis of non-heme iron in guinea pig tissues (liver, kidney and heart) compared favorably with those obtained using micro-scale adaptations of three published reference methods. The new method was more sensitive, required less time, and was less cumbersome than the three published methods to which it was compared.

Animals↗
PubMed2006

Key issues in the modes of action and effects of trichloroethylene metabolites for liver and kidney tumorigenesis.

Trichloroethylene (TCE) exposure has been associated with increased risk of liver and kidney cancer in both laboratory animal and epidemiologic studies. The U.S. Environmental Protection Agency 2001 draft TCE risk assessment concluded that it is difficult to determine which TCE metabolites may be responsible for these effects, the key events involved in their modes of action (MOAs) , and the relevance of these MOAs to humans. In this article, which is part of a mini-monograph on key issues in the health risk assessment of TCE, we present a review of recently published scientific literature examining the effects of TCE metabolites in the context of the preceding questions. Studies of the TCE metabolites dichloroacetic acid (DCA) , trichloroacetic acid (TCA) , and chloral hydrate suggest that both DCA and TCA are involved in TCE-induced liver tumorigenesis and that many DCA effects are consistent with conditions that increase the risk of liver cancer in humans. Studies of S-(1,2-dichlorovinyl) -l-cysteine have revealed a number of different possible cell signaling effects that may be related to kidney tumorigenesis at lower concentrations than those leading to cytotoxicity. Recent studies of trichloroethanol exploring an alternative hypothesis for kidney tumorigenesis have failed to establish the formation of formate as a key event for TCE-induced kidney tumors. Overall, although MOAs and key events for TCE-induced liver and kidney tumors have yet to be definitively established, these results support the likelihood that toxicity is due to multiple metabolites through several MOAs, none of which appear to be irrelevant to humans.

Animals↗
PubMed1991

The influence of assay conditions on measurement of excitatory dibasic sulphinic and sulphonic alpha-amino acids in nervous tissue.

Major improvements to the HPLC separation of fluorescent derivatives of excitatory sulphur-containing amino acids have been made. Quisqualate was used as the internal standard since no endogenous derivatives coeluted with it. The artefactual generation of sulphinic and sulphonic amino acids from the oxidation of cysteine (56 microM) and homocysteine (1.2 microM) has been investigated using deionised water, an acidic phosphate/methanol mixture, perchloric acid and trichloroacetic acid (TCA) as extraction media. Of the four extraction media examined, TCA in combination with ether extraction was shown to be the most potent oxidative treatment and resulted in 23% oxidation of original cysteine or homocysteine to sulphinic and sulphonic acids. This oxidation was partially resistant to the presence of physiological concentrations of glutathione (1.5 mM) such that in the case of cysteine, 6% oxidation was observed. A 10% (v/v) mixture of methanol in 75 mM phosphate solution (pH 4.6) was found to be the most artefact-free extraction method and in spinal cord tissue processed with this medium, cysteine sulphinic acid was the only excitatory sulphur-containing amino acid consistently detectable (0.24 +/- 0.01 pmol/mg wet weight, n = 6).

Amino Acids, Sulfur↗
PubMed2006

Comparative study of effects of polyols, salts, and alcohols on trichloroacetic acid-induced state of cytochrome C.

A systematic investigation of the effect of polyethylene glycols, salts, and alcohols on the trichloroacetic acid (TCA)-induced state of ferricytochrome c was made using various spectroscopic techniques. Native cytochrome c (Cyt c) has a fluorescence maximum at 335 nm, whereas the TCA-induced state of Cyt c has a red shift of 7 nm with enhanced fluorescence intensity. The near- and far-UV CD spectra showed a significant loss of tertiary and secondary structure, although the protein is relatively less unfolded as compared with a conformation at pH 2.0. Addition of 70% (v/v) polyols to TCA (3.3 mM)-induced state of Cyt c resulted in increased 1-anilino-8-naphthalene sulfonate binding and increased mean residue ellipticity at 222 nm, indicating increase in compactness with enhanced exposure of hydrophobic surface area. Also, the stabilizing effect of salts and alcohols on the TCA-induced state was studied and compared with their effect on trifluoroacetic acid-unfolded state of Cyt c. Among all the polyols, salts, and alcohols studied, PEG-400, K3[Fe(CN)6], and butanol were the most efficient in inducing secondary structure in TCA-induced state as examined by the above-mentioned spectroscopic techniques. For salts, the efficiency in inducing the secondary structure followed the order K3[Fe(CN)6] > KClO4 > K2SO4 > KCl. For alcohols, this order was found to be as follows: butanol > propanol > ethanol > methanol.

Alcohols↗
PubMed1982

Fixed protein A-containing staphylococci as solid-phase immunoadsorbents.

Staphylococcus aureus Cowan I containing protein A were rapidly fixed in hot 5% (w/v) trichloroacetic acid (TCA) for 6 min in order to remove the negatively charged cell wall polymer teichoic acid. This resulted in a stable IgG adsorbent able to bind 1.4 mg of human IgG/ml 10% (v/v) suspension for at least 5 months. This IgG binding ability is 25% less than for formalin-fixed bacteria, which were stable for at least 1 year. Preincubation of the bacterial adsorbents in 0.5% (v/v) Tween 20 after fixation prolonged stability and rendered prewashes prior to use unnecessary. IgG could be quantitatively eluted from TCA-fixed bacteria at pH 3.0, but not from formalin-fixed bacteria unless 80 mM MgCl2 was included in the acid buffer. This is explained by ionic interaction between IgG and teichoic acid at the bacterial surface at low pH. Non-specific binding of different proteins to the bacterial adsorbents was also studied and buffers for reducing this effect are suggested.

Animals↗
PubMed1994

Purification and characterization of colicin V from Escherichia coli culture supernatants.

The peptide antibiotic, colicin V (ColV), has been purified and characterized from Escherichia coli culture supernatants by precipitation with trichloroacetic acid (TCA) and high-performance liquid chromatography (HPLC). Polyacrylamide gel electrophoresis (PAGE) and Western analysis identifies ColV as a polypeptide with an apparent molecular mass of 5.8 kDa. The protein identified remains biologically active after purification and SDS-PAGE. A mutant form of ColV, ColV-1, removes the carboxy-terminal 21 amino acids and replaces them with eight heterologous residues. The ColV-1 mutant is also secreted into the extracellular medium, demonstrating that the carboxy-terminal 21 amino acids are not required for secretion by the dedicated ColV export system, CvaAB/TolC. N-Terminal amino acid sequencing shows that the primary translation product of cvaC, the ColV structural gene, is processed to remove the N-terminal 15 amino acids. The cleavage site is preceded by the sequence Ser-Gly-Gly, making it a potential substrate for leader peptidase. The ColV leader sequence has many characteristics in common with the amino-terminal leader sequences of the lactococcins, lactacins, and pediocins from Gram-positive bacteria. Mass spectroscopy of purified ColV shows that it has a mass of 8741.0 amu, consistent with the mass of the unmodified 88 amino acid polypeptide. The purification scheme provides a rapid and simple way to obtain ColV for further biochemical analysis.

Amino Acid Sequence↗
PubMed1972

Polypeptide secretion from the isolated parietovisceral ganglion of Aplysia californica.

In vitro studies of the secretory behavior of the parietovisceral ganglion in Aplysia californica were performed. The aim of these studies was to investigate the release of polypeptides in response to depolarizing stimuli, and, in particular, to determine if a specific polypeptide known to induce egg laying in the intact animal is secreted into the bathing medium. During continuous perfusion of a ganglion preincubated in leucine-(3)H the application of either high-potassium medium or a burst of electrical stimuli (via the pleurovisceral connective nerve) evoked a marked increase in the amount of trichloroacetic acid (TCA)-precipitable radioactivity recovered in the perfusate. Enhanced release could be detected within 80 sec of the initial exposure to high potassium; however, incubation of a ganglion in calcium-free media before the application of high-potassium medium abolished the increase of precipitable radioactivity. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of perfusate samples revealed a significant change in the polypeptide species washed from the ganglion during high-potassium depolarization. Bioassays confirmed that egg laying is induced when high-potassium medium used to bathe a ganglion is injected into a recipient animal. These and other results permit the conclusion that the bulk of the polypeptide material secreted from the ganglion in response to depolarization is a specific neurohormone produced by two identified cell clusters, the so-called bag cells.

Acrylamides↗
PubMed1980

Localization of bacteriophage receptor, clumping factor, and protein A on the cell surface of Staphylococcus aureus.

The surface of several laboratory strains of Staphylococcus aureus were observed with a scanning electron microscope, and the presence of two morphologically characteristic structures--a ridge separating cell surface into old and new surfaces and a concentric circular structure--are described. These two structures seemed to be present universally on the surfaces of cells of the genus Staphylococcus. The removal of the circular structures by a mild treatment of the cell with trichloroacetic acid suggested that this structure seemed to represent circularly arranged teichoic acid. With experiments using morphologically recognizable markers among three of the cell wall components, clumping factor, phage receptor, and protein A, the clumping factor was proven to be specifically localized on the old surface; and more phage receptors were detected on the old surface than on the new surface, but protein A was present all over the cell surface. This indicated that the clumping factor and most of the phage receptors appeared on the cell wall surface in a late stage of the cell growth cycle, but protein A was present in an early stage of the growth. The idea of aging of the cell wall is discussed.

Agglutinins↗
PubMed1989

Energy-dependent in vitro translocation of a model protein into Escherichia coli inverted membrane vesicles can take place efficiently in the complete absence of the cytosol fraction.

The translocation of the precursor of a secretory protein into Escherichia coli inverted membrane vesicles was demonstrated in the absence of the cytosol fraction. A small, hydrophilic chimeric protein, OmpF-Lpp, possessing an uncleavable signal peptide was used as the model protein. As much as 80% translocation of the precursor protein into membrane vesicles was observed within 6 min in the absence of the cytosol fraction, when the precursor protein purified by means of immunoaffinity chromatography was used. The translocation was dependent on both ATP and respiratory substrates such as succinate. ATP could be replaced by a higher concentration of CTP or UTP, whereas GTP was inactive. Trichloroacetic acid treatment of the precursor protein, which is reported to result in removal of the trigger factor that is attached to a precursor protein (Crooke, E., and Wickner, W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5216-5220), did not lower the translocation efficiency significantly. Finally, the precursor protein, which was highly purified by means of successive immunoaffinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, could still be efficiently translocated into the membrane vesicles. The precursor proteins purified in the presence and absence of bovine serum albumin were both active. Neither washing of the membrane vesicles for prevention of possible contamination by cytosolic factors nor the addition of the cytosol fraction to the reaction mixture affected the translocation efficiency. These results indicate that the in vitro translocation of the OmpF-Lpp precursor protein can take place in the complete absence of cytosolic soluble proteins.

Adenosine Triphosphate↗
PubMed1965

DISTRIBUTION OF GRISEOFULVIN TAKEN UP BY MICROSPORUM GYPSEUM: COMPLEXES OF THE ANTIBIOTIC WITH CELL CONSTITUENTS.

El-Nakeeb, Moustafa A. (Rutgers, The State University, New Brunswick, N.J.), and J. O. Lampen. Distribution of griseofulvin taken up by Microsporum gypseum: complexes of the antibiotic with cell constituents. J. Bacteriol. 89:1075-1081. 1965.-The griseofulvin-(4-methoxy-H(3)) accumulated by Microsporum gypseum was bound to fungal constituents. Mild procedures (hot-water or acetone extraction) removed between 40 and 60% of the total concentration of antibiotic. The remainder was recovered only after treatment with hot trichloroacetic acid followed by hot NaOH. The ratio of griseofulvin-H(3) in the water-soluble pool to that in the nucleic acid (soluble in hot trichloroacetic acid) and protein (soluble in NaOH) fractions was about 2:1:1, respectively. Only small amounts of radioactivity could be detected subsequent to hot-water extraction in the cold trichloroacetic acid extracts and in the cell-wall residues. Essentially all the activity in the soluble pool and in the nucleic acid extracts was chemically unchanged griseofulvin, much of which was present in various complexes, especially after long periods of incubation. One cannot be certain of the original form of all the radioactivity which was extracted in the protein fraction, because some degradation of the antibiotic occurred during the hot NaOH treatment. Nevertheless, a considerable amount of griseofulvin-H(3) was still present, and there had been no significant incorporation of radioactivity into the amino acid residues. It is probable, therefore, that the radioactivity in both protein and nucleic acid fractions represents complexes of the antibiotic with macromolecular components which were formed in vivo by the sensitive organisms.

Amino Acids↗
PubMedDate not supplied

Skin rejuvenation regimens: a profilometry and histopathologic study.

OBJECTIVE: To quantitatively examine the effects of skin rejuvenation regimens in treating photodamaged skin. METHODS: Fourteen patients with photodamaged skin were considered for analysis. Skin rejuvenation regimens were as follows: (1) 10 weeks of treatment with topical 0.05% tretinoin emollient cream, (2) 10 weeks of treatment with 0.05% tretinoin emollient cream and topical ascorbic acid lotion, (3) 6 superficial trichloroacetic acid peels, and (4) a combination of the topical treatments and superficial peels. Comparisons of the treatments were analyzed using profilometry and histologic findings. RESULTS: Profilometry analysis provided quantification of the changes from each treatment group and among the treatment programs. Each group showed improvements from baseline. Trichloroacetic acid peels combined with application of the topical products improved skin topography to a greater extent than the less aggressive regimens. Histologic changes correlated well with the skin replica findings. CONCLUSIONS: A 10-week skin rejuvenation regimen objectively improved photodamaged facial skin. Significant changes are noted when combining topical treatments with superficial peels. Hence, use of a combination of trichloroacetic acid peels, 0.05% tretinoin emollient cream, and ascorbic acid lotions is well tolerated and superior to either component alone as part of a comprehensive skin care and sun protection program.

Administration, Topical↗
PubMed1975

Studies on non-haemoglobin erythrocyte iron; the influence of haemolysis on plasma iron determinations.

Approximately 2% of iron contained in mouse erythrocytes is transferred into supernatant when haemolysed erythrocytes are precipitated with trichloroacetic acid. The component unprecipitable with trichloroacetic acid is probably bound predominantly to reticulocytes and it is larger the younger is the reticulocyte. Under the conditions of postirradiation suppression of erythropoiesis this component grows strongly and in the phase of overrecovery of erythropoiesis decreases. The numerical values determined in the paper can be used for a correction of the disturbing influence of haemolysis on concentration and radioactivity of plasma iron in the resting state of erythropoiesis, if a preliminary precipitation of a sample with trichloroacetic acid was carried out.

Animals↗
PubMed1989

[Solid-phase immunoenzyme analysis (ELISA)--a test for diagnosing Q fever (comparison with the complement fixation and immunofluorescence methods)].

Purified phase I Coxiella burnetii corpuscles treated chemically in different ways (by potassium periodate, mild acid hydrolysis or trichloroacetic acid) were compared by ELISA for their ability to detect phase II (directed to antigen 2) and phase I (directed to antigen 1) antibodies in sera from human Q fever convalescents. As to the absorbance values, the most sensitive was the antigen obtained by mild acid hydrolysis (0.1 mol/l HCl for 30 min. at 100 degrees C), followed by phase I corpuscular antigen treated trichloroacetic acid. Phase I antigen treated by K10(4) gave lower absorbance values. The performances of the commonly used complement fixation test and the more recently developed indirect immunofluorescence test were compared on 45 human sera. Immunofluorescence revealed specific antibodies in 62.2% of the sera. In the complement fixation test only 2.2% of sera were positive. ELISA proved superior (76.8% positive sera) to the immunofluorescence and complement fixation tests in detection of Q fever.

Complement Fixation Tests↗
PubMed1986

Identification of S-1,2,2-trichlorovinyl-N-acetylcysteine as a urinary metabolite of tetrachloroethylene: bioactivation through glutathione conjugation as a possible explanation of its nephrocarcinogenicity.

The elimination and metabolism of [14-C]-tetrachloroethylene (Tetra) was studied in female rats and mice after the oral administration of 800 mg/kg [14-C]-Tetra. Elimination of unchanged Tetra was the main pathway of elimination in both species and amounted to 91.2% of the dose in rats and 85.1% in mice. [14-C]-Carbon dioxide (CO2) was found to be a trace metabolite of [14-C]-Tetra. Only a small part of the applied dose was transformed to urinary (rats = 2.3%, mice = 7.1%) and fecal (rats = 2.0%, mice = 0.5%) metabolites. The urinary metabolites were separated and quantified by high performance liquid chromatography (HPLC) and identified by gas liquid chromatography/mass spectrometry (GC/MS). The following metabolites could be identified: oxalic acid (8.0% of urinary radioactivity in rats, 2.9% in mice), dichloroacetic acid (5.1%, 4.4%), trichloroacetic acid (54.0%, 57.8%), N-trichloroacetyl-aminoethanol (5.4%, 5.7%), trichloroethanol, free and conjugated (8.7%, 8.0%), S-1,2,2-trichlorovinyl-N-acetylcysteine (N-acetyl TCVC) (1.6%, 0.5%), and another conjugate of trichloroacetic acid (1.8%, 1.3%). The structures of the identified metabolites indicate two different pathways operative in Tetra biotransformation: cytochrome P-450-mediated epoxidation forming reactive metabolites in the liver and conjugation of Tetra with glutathione (GSH) catalyzed by glutathione transferase(s). The formation of reactive intermediates by renal processing of the glutathione conjugates may provide a molecular mechanism for the nephrotoxicity and nephrocarcinogenicity of Tetra in male rats.

Acetylcysteine↗
PubMed2002

Guinea pig maximization test for trichloroethylene and its metabolites.

OBJECTIVES: To study the contact allergenic activities of trichloroethylene (TCE) and its three metabolites trichloroacetic acid, trichloroethanol and chloral hydrate. METHODS: A modified guinea pig maximization test (GPMT) was adopted. The skin sensitization (edema and erythema) was observed in trichloroethylene, trichloroacetic acid, trichloroethanol, chloral hydrate and 2,4-dinitrochlorobenzene. RESULTS: The allergenic rate of TCE, trichloroacetic acid and 2,4-dinitrochlorobenzene was 71.4%, 58.3% and 100.0% respectively, and that of trichloroethanol and chloral hydrate was 0%. The mean response score of TCE, trichloroacetic acid and 2,4-dinitrochlorobenzene was 2.3, 1.1, 6.0 respectively. The histopathological analysis also showed an induction of allergenic transformation in guinea pig skin by both TCE and trichloroacetic acid. CONCLUSION: TCE appears to be a strong allergen while trichloroacetic acid a moderate one. On the other hand, both trichloroethanol and chloral hydrate are weak sensitization potentials. Immunologic reaction induced by TCE might be postulated as the pathological process of this illness. Consequently, it is suggested that in the mechanism of Occupational Dermatitis Medicamentose-Like (ODML) induced by TCE, the chemical itself might be the main cause of allergy. As one of its metabolic products, trichloroacetic acid might be a subordinate factor.

Allergens↗
PubMed2003

Airborne haloacetic acids.

Haloacetic acid (HAA) concentrations were measured in air samples from a semi-rural and a highly urbanized site in southern Ontario throughout 2000 to investigate their sources and gas-particle partitioning behavior. Denuders were efficient for collection of gaseous HAAs, and the particle phase was collected on a downstream quartz filter with negligible breakthrough. Total HAA concentrations (i.e., gas + particles) ranged between <0.025 and 19 ng m(-3) for individual HAAs at both sites. The dominant airborne HAA was monochloroacetic acid (MCA), followed in decreasing order by dichloroacetic acid (DCA), trifluoroacetic acid (TFA), and trichloroacetic acid (TCA). Difluoroacetic acid (DFA), monofluoroacetic acid (MFA), and chlorodifluoroacetic acid (CDFA) were also frequently detected at lower concentrations. Between sites, TFA, DFA, MFA, and TCA concentrations were significantly higher in Toronto, while CDFA concentrations were higher in Guelph. HAAs were primarily in the gas phase all year; however, during colder months, particle-phase HAA concentrations increased relative to the gas phase. Trichloroacetic acid had the highest particle fraction (phi) for all detected HAAs, with a mean phi of 0.51 and 0.56 for Guelph and Toronto, respectively, and both vapor pressure and acid strength appeared to influence gas-particle partitioning. Temporal trends at both sites were partially explained by temperature, short-wave radiation, and particle mass (PM10), leading to indications of the respective sources. A simple deposition model indicated that dry deposition of TFA and TCA should not be neglected in temperate mid-latitude environments and that precipitation concentrations can be successfully predicted by the Henry's law constant.

Acetates↗