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PubMed1969

Role of exogenous adenosine triphosphate in catabolic and synthetic activities of Chlamydia psittaci.

The synthetic activities of isolated cells of the meningopneumonitis strain (MN) of Chlamydia psittaci were investigated and further observations were made on their catabolic reactions. These observations included the demonstration of CO(2) production from aspartate in the presence of pyruvate and the formation of pyruvate from glucose-6-phosphate. Both reactions were enhanced by added adenosine triphosphate (ATP). Of a large number of compounds tested, only glucose-6-phosphate, pyruvate, aspartate, and isoleucine were shown to furnish carbons that were incorporated into molecules precipitated by trichloroacetic acid. The reactions with pyruvate, aspartate, and isoleucine were dependent entirely, or almost entirely, on added ATP, and the reaction with glucose-6-phosphate was enhanced by ATP. Except for CO(2), which greatly stimulated the reactions, the addition of a number of other compounds or a combination of compounds, such as cofactors, amino acids, and purine and pyrimidine bases, did not greatly affect incorporation. About 95% of the activity of the trichloroacetic acid precipitates was recovered in the chloroform-methanol soluble fraction.

Acetates↗

PubMed1987

Altered insulin distribution and metabolism in type I diabetics assessed by [123I]insulin scanning.

Scintigraphic scanning with [123I]insulin provides a direct and quantitative assessment of insulin uptake and disappearance at specific organ sites. Using this technique, the biodistribution and metabolism of insulin were studied in type 1 diabetic patients and normal subjects. The major organ of [123I]insulin uptake in both diabetic and normal subjects was the liver. After iv injection in normal subjects, the uptake of [123I]insulin by the liver was rapid, with peak activity at 7 min. Activity declined rapidly thereafter, consistent with rapid insulin degradation and clearance. Rapid uptake of [123I]insulin also occurred in the kidneys, although the uptake of insulin by the kidneys was about 80% of that by liver. In type 1 diabetic patients, uptake of [123I]insulin in these organ sites was lower than that in normal subjects; peak insulin uptakes in liver and kidneys were 21% and 40% lower than those in normal subjects, respectively. The kinetics of insulin clearance from the liver was comparable in diabetic and normal subjects, whereas clearance from the kidneys was decreased in diabetics. The plasma clearance of [123I]insulin was decreased in diabetic patients, as was insulin degradation, assessed by trichloroacetic acid precipitability. Thirty minutes after injection, 70.9 +/- 3.8% (+/- SEM) of [123I]insulin in the plasma of diabetics was trichloroacetic acid precipitable vs. only 53.9 +/- 4.0% in normal subjects. A positive correlation was present between the organ uptake of [123I]insulin in the liver or kidneys and insulin degradation (r = 0.74; P less than 0.001). Both decreased insulin uptake in the liver and kidneys and decreased insulin degradation were inversely correlated to the binding capacity of antiinsulin antibodies in plasma of diabetics (r = -0.87 to -0.92; P less than 0.001). In summary, type 1 diabetic patients have altered patterns of insulin biodistribution and metabolism, with decreased organ uptake and slow degradation of [123I]insulin, which correlated with the insulin antibody-binding capacity of their serum. Thus, antiinsulin antibodies, even at subclinical concentrations, may modulate the metabolic effects of insulin in the diabetic by prolonging the biological half-life of the hormone as well as by altering its distribution and uptake at specific organ sites.

Adult↗

PubMed1980

Measurement of thermal denaturation of beta-lactoglobulin at pH 2.5.

Solutions of beta-lactoglobulin (approximately 1%) at pH 2.5, 4.5, and 6.5 were heated at 90 C for 30 min, cooled rapidly to room temperature (23 to 25 C), and examined for nature and extent of denaturation by various methods. Heating at pH 4.5 and at 6.5 caused almost complete denaturation as judged by loss of solubility at pH of heating, at pH 4.5, or in 2% trichloroacetic acid. However, after heating at pH 2.5 the protein remained in solution but partially lost solubility in 2% trichloroacetic acid and at pH 4.5. The protein fraction denatured by heating at pH 2.5, rendered insoluable at pH 4.5, was unfolded partially and irreversibly, since when resolubilized at pH 2.5 it showed increased optical levorotation and demonstrated no endothermic reaction by differential scanning calorimetry. This material was eluted at lower eluant volumes from a Sephadex G-100 column. However, it was unchanged in molecular size as judged by electrophoretic analysis and measurements of sedimentation velocity. The pH 4.5-soluble fraction from the sample heated at pH 2.5 was similar to native beta-lactoglobulin by the above methods.

Electrophoresis↗

PubMedDate not supplied

Insulin receptor binding and degradation in IM-9 cultured human lymphocytes - importance of extracellular degradation.

Insulin degradation in IM-9 cultured human lymphocytes occurs extracellularly. In the presence of 0.1% bovine serum albumin, insulin degradation products are formed that are soluble in 5% trichloroacetic acid. In the presence of 5% bovine serum albumin, the major products of insulin degradation are insoluble in 5% trichloroacetic acid and have little or no biological activity. Inhibition of binding with Concanavalin A or antireceptor antibody does not affect the rate of insulin degradation.

Antibodies↗

PubMed1983

Increased fraction of acid-soluble proteins in 0.35 M NaCl extracts of nuclei from rat liver tumors.

1. The fraction of proteins extracted from nuclei with 0.35 M NaCl and soluble in 2% trichloroacetic acid was examined in five Morris hepatomas and rat liver. 2. This fraction was a much greater percentage of the total 0.35 M NaCl soluble proteins in the tumors than in normal or regenerating liver. 3. In part, this difference was due to proteins with molecular weights greater than high mobility group proteins. 4. The conditions for precipitation of high mobility group proteins 1 and 2 with trichloroacetic acid were found to differ in hepatoma and liver fractions.

Animals↗

PubMed1968

The membrane teichoic acid of Staphylococcus lactis I3.

1. Teichoic acid was isolated by extraction with trichloroacetic acid of the membrane fraction of disrupted cells of Staphylococcus lactis I3. 2. The purified material contains glycerol, phosphate and alanine, but little or no sugar or amino sugar. 3. A study of the products of hydrolysis with acid and alkali established that the membrane teichoic acid is a (1-->3)-linked poly(glycerol phosphate) that differs in structure from the glycerol teichoic acid in the wall of this organism. 4. The alanine ester residues show the characteristic high lability to alkali and are thus distinguishable from the more stable alanine ester residues of the wall teichoic acid. 5. The significance of these structural features and the possible function of teichoic acids are discussed.

Acids↗

PubMed1982

Sensitive formaldehyde determination with Nash's reagent and a 'tryptophan reaction'.

The widely applied Nash-method and its modifications have unsatisfactory specificity and restricted sensitivity for the determination of small amounts of formaldehyde. A method using small volumes of reaction mixtures with low background absorbance is described; this method avoids dilution during the protein denaturation step by the use of trichloroacetic acid. Alternatively a 'tryptophan-sulfuric acid-iron' reaction takes advantage of high specificity for formaldehyde detection. A more economical and sensitive variant is described including trichloroacetic acid precipitation of protein. The sensitivity and specificity of the colour development reaction of the 'tryptophan-sulfuric acid-iron' reaction appears to be superior to the Nash-modification. However the detection of formaldehyde by the NASH method can also be amplified by subsequent extraction and concentration of the reaction product diacetyl-dihydrolutidine into n-amyl alcohol so that a formaldehyde amount of few nanomoles can be determined. The application of these methods is recommended in cases of formaldehyde formation in small amounts such as in cell cultures or when kinetic data at low substrate concentrations and slow turnover rates have to be measured.

Animals↗

PubMed2002

Study of competitive binding of enantiomers to protein by affinity capillary electrochromatography.

Affinity capillary electrochromatography (CEC) with zonal elution method was used to probe the competitive interactions of enantiomers with protein. In this approach, a known concentration of a competing agent is continuously applied to a CEC column with bovine serum albumin (BSA) physically adsorbed on SAX packing while injections of a small amount of analyte are made. The binding sites of solutes on the BSA molecule were determined by the changes in the retention factors of the solutes resulted from the addition of competitive agent. By using D- or L-tryptophan as competitive agents and D-, L-tryptophan and benzoin enantiomers as injected analytes showed that BSA molecule has a primary site to strongly bind L-tryptophan, but D-tryptophan dose not bind at this site; D- and L-tryptophan share a weak binding site on the BSA molecule. Benzoin enantiomers do not share any binding sites with either D- or L-tryptophan. Non-chiral compounds of trichloroacetic acid and n-hexanoic acid were applied as the competitive agents to study the binding of warfarin enantiomers to BSA, it was observed that trichloroacetic acid and n-hexanoic acid had a same binding site for warfarin enantiomers binding to BSA molecule.

Binding, Competitive↗

PubMed1962

Oxidative assimilation and distribution of glucose in Bacillus cereus.

Clifton, C. E. (Standford University School of Medicine, Stanford, Calif.). Oxidative assimilation and distribution of glucose in Bacillus cereus. J. Bacteriol. 83:66-69. 1962-Exogenous C(14)-labeled glucose is taken up rapidly by washed cells of Bacillus cereus and apparently enters a metabolic pool soluble in cold 5% trichloroacetic acid. From this pool the label passes rapidly into other cellular fractions, primarily materials soluble and insoluble in hot 5% trichloroacetic acid, which may constitute endogenous reserves of the cells. There is a marked lag in the liberation of C(14)-labeled CO(2); little or no lag is observed in the slightly enhanced rate of oxygen consumption noted in the presence of exogenous glucose. Assimilation, in part at least, appears to be at the expense of cellular reserves which appear to be replenished or replaced by the assimilated substrate.

Bacillus↗

PubMed1988

Spermine binding to subsynaptosomal fractions of rat brain cortex.

Binding sites for [14C]spermine have been identified in rat brain cortex subcellular fractions. The binding, characterized by using synaptosomal membranes, is specific for spermine. It was not detected below 20 degrees C and increased about three/four-fold with a temperature rise of 10 degrees C. Binding occurred only in the presence of -SH reducing agents. It was completely suppressed by metal chelating agents, and was stimulated about four-fold by 1-5 x 10(-5) M Fe2+. Smaller increases were observed in the presence of Mn2+, Ni2+, Ca2+, Mg2+, and Zn2+; in contrast, millimolar concentrations of most divalent cations inhibited the binding differently (Mn2+ = Ni2+ = Zn2+ = Co2+ much greater than Mg2+ greater than Ca2+). Bound radioactive spermine was not displaced by the addition of high concentrations of unlabelled polyamine or chelating agents, nor by precipitation and washing of the membranes with 10 percent trichloroacetic acid, or by boiling of the precipitate in the presence of 1.0 percent SDS and 10 percent beta-mercaptoethanol. The trichloroacetic acid precipitate showed two radioactive bands, corresponding to low Mr (less than 8,000) components, after SDS-polyacrylamide gel electrophoresis and fluorography. The Fe2+-stimulated [14C]spermine binding was neither influenced by a previous heating of the membranes at 100 degrees C for 30 min nor by trypsin or pronase digestion, whereas the heat-treatment increased the binding occurring in the absence of Fe2+ by about two fold. A non-enzymatic formation of a spermine-metal complex tightly bound to some membrane peptide(s) is suggested.

Animals↗

PubMed1979

Soluble cyclic AMP-dependent protein kinases from chick kidney. Effects of dilution and non-protein inhibitors.

Cyclic AMP-dependent protein kinases prepared from crude cytosols of chick kidney, rat kidney and rat liver were found on dilution to exhibit complex kinetics. Dilution of the cytosols appears to increase the state of activation of the enzymes. This effect was due to the presence of inhibitory agents in the cytosol which had a greater inhibitory effect on the cyclic AMP-dependent than on the cyclic AMP-independent enzyme. Two types of inhibitory activity were found by column chromatography, one resistant to trichloroacetic acid precipitation and boiling but affected by trypsin digestion and the other resistant to boiling and trypsin digestion but precipitated by trichloroacetic acid. Inhibitory activity corresponding to the former characteristics has been described previously but the presence of additional soluble inhibitory agents in the cytosol has not been documented. The complete characterisation of this previously undescribed inhibitory activity requires further investigation. The relevance of such cytosolic inhibitory activity to the interpretation of states of activation of protein kinase enzymes is discussed.

Adenosine↗

PubMed1991

Exposure of 1,1,1-trichloroethane and dose-related excretion of metabolites in urine of printing workers.

The quantitative relationship between the time-weighted average exposure by inhalation to 1,1,1-trichloroethane and excretion of metabolites in urine at the end of a work shift was investigated in 48 male printing workers exposed to the solvent at levels below 65 ppm. Male control subjects, 10 in all, were also investigated. Statistical analysis showed that there is a linear relationship between the two exposure indicators, suggesting that occupational exposure to 1,1,1-trichloroethane can be bio-monitored by means of urinalysis for either total trichloro-compounds or trichloroacetic acid. The correlation coefficient was higher for total trichloro-compounds (r = 0.86-0.89 depending on correction for urine density) than trichloroacetic acid (r = 0.50-0.71), which suggests that the former is a better indicator of exposure to 1,1,1-trichloroethane than the latter. The calculations based on the present study suggest that, at the end of the shift, only less than 2% of 1,1,1-trichloroethane absorbed will be excreted as metabolites in urine.

Air Pollutants, Occupational↗

PubMed1970

Composition and properties of a group A streptococcal teichoic acid.

Teichoic acid-like material extracted by cold trichloroacetic acid from lyophilized whole cells of streptococci from groups A,D,E,O, and T was shown to give a positive precipitin reaction with group antisera. Similar material from cells of groups B,C,F,G,H,K,L,M,N,P,Q,R, and S did not give a positive reaction with group antisera. The group A material also reacted with anti-E serum; however, the opposite did not occur. A similar result was also obtained on the group T material and anti-O serum. The group A teichoic acid was purified by Sephadex column chromatography, and was shown to be free of cell wall peptidoglycan and polysaccharide, and ribitol teichoic acid. It was composed of glycerol, phosphate, alanine, and glucosamine. Alkaline hydrolysis showed the presence of ester-linked alanine and glucosaminylglycerol. Phosphorus was released from ester linkage by alkaline phosphatase. N-acetylglucosamine produced a 72% inhibition of the precipitin test at a level of 10 mumoles, and d-alanine methyl ester was significantly stronger than the l-alanine ester. A single precipitin band was seen with group A serum. The data indicate that teichoic acid of group A streptococci is a polymer composed of glycerol phosphate and containing N-acetylglucosamine and alanine. Antisera to these streptococci contain antibodies specific for the alanine and the glucosamine linkages. The use of serum containing antibodies to alanine-polyglycerophosphate shows that the occurrence of this type of teichoic acid is widespread among the streptococci.

Acetone↗

PubMed1968

The glycerol teichoic acid from walls of Staphylococcus epidermidis I2.

1. Walls of Staphylococcus epidermidis I2 contain 30% (w/w) of a glycerol teichoic acid containing phosphate, d-alanine and d-glucose in the molecular proportions 1:0.25:0.50. 2. The teichoic acid was isolated by extraction with trichloroacetic acid and with dilute aqueous NN-dimethylhydrazine at pH7, and was shown to be a (1-->3)-linked poly(glycerol phosphate) containing beta-d-glucopyranosyl and d-alanyl ester substituents. 3. 2-O-beta-d-Glucopyranosylglycerol was isolated and characterized as its crystalline hexa-O-acetate. 4. Unlike that of certain other bacteria, the peptidoglycan component of the wall is not solubilized by NN-dimethylhydrazine. 5. The membrane teichoic acid is also a (1-->3)-linked poly(glycerol phosphate) but contains a smaller proportion of glucosyl substituents.

Alanine↗

PubMed1992

An assay to quantitate the binding of Rhodococcus equi to macrophages.

A Rhodococcus equi radiobinding assay has been developed using organisms labeled with 3H-uracil. These labeled organisms resemble their unlabeled counterparts with respect to colony morphology, viability, and buoyant density. Bacteria routinely incorporate between 5 x 10(-3) and 5 x 10(-2) counts per minute per colony forming unit (cfu) which in this assay allows the detection of fewer than 0.2 cfu per macrophage. Once incorporated, greater than 90% of the label remains bacterial associated for at least 4 h postlabeling. The majority of the label is trichloroacetic acid precipitable, partitions into the aqueous phase following phenol/chloroform extraction and is ethanol precipitable. RNAse treatment of the ethanol precipitate abolishes label trichloroacetic acid precipitation. This radiolabeling technique has been used to quantitate the attachment of R. equi to both murine peritoneal and equine alveolar macrophages adherent to 13 mm glass coverslips. R. equi binding is dose dependent, saturable, and specific to macrophages. Further, binding is enhanced in the presence of fresh serum. Inhibition of radiolabeled bacterial binding can be obtained by competition with cold R. equi. This radiolabeled binding assay represents a crucial step in identifying the receptors on macrophages involved in the recognition of R. equi and may help to provide information on how macrophages recognize intracellular bacteria in general.

Animals↗

PubMed1977

Metabolism of trichloroethylene and tetrachloroethylene in human subjects.

A linear correlation exists between the trichloroethylene concentration in the work environments and the level of total trichloro compounds in the urine of the workers, as expressed by the equation: Gamma = 7.25=chi + 5.5, where Gamma is trichloroethylene in air (ppm) is Chi is total trichloro compounds in urine (mg/l). Trichloroethanol level is also linearly related to trichloroethylene concentration, while trichloroacetic acid level deviates from the linear relation when trichloroethylene level exceeds 50 ppm. In the case of tetrachloroethylene exposure, both trichloroethanol and trichloroacetic acid levels, and consequently the total trichloro compound level, reach a plateau at tetrachloroethylene level well below 100 ppm. The mean urinary biological half-life is 41 hr for trichloroethylene and 144 hr for tetrachloroethylene. The two values are the largest of the values so far obtained with organic solvents. The respiratory half-life is shorter than the urinary half-life, both in richloroethylene and in tetrachloroethylene. Applications of the urinalyses in clinical cases are described. In one case of trichlorethylene dependency, a longer urinary half-life of 73 hr was observed. An automated system is presented for the determination of total trichloro compounds in human urine. The system can analyze the samples at the rate of 20 samples per hour with an accuracy comparable to that of the time-consuming manual analysis.

Air Pollutants, Occupational↗

PubMed1980

A trypsin and chymotrypsin inhibitor from the metacestodes of Taenia pisiformis.

The metacestodes of Taenia pisiformis have been shown to contain a protease inhibitor capable of inactivating the esterolysis of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoyl-L-tyrosine ethyl ester (BTEE) by trypsin and chymotrypsin, respectively, of bovine, dog and rabbit origin, but not affecting the hydrolytic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. This inhibitor has been demonstrated in whole worm extracts and in the incubation medium of in vitro-maintained, intact living metacestodes. The protease inhibitor which was purified by trichloroacetic acid precipitation, Sephadex G-100 chromatography and affinity chromatography on CNBr-activated Sepharose 4B-bovine, chymotrypsin conjugate was soluble in 5% trichloroacetic acid, withstood heat up to 80 degrees C, tolerated the pH range 1.5 to 9.0, was unaffected by 8 M urea or 0.2 M 2-mercaptoethanol and had a molecular weight of about 7000 to 7200, as calculated from its gel chromatographic behaviour. Complex formation between the inhibitor and the enzymes required 3--4 min for completion. The enzyme-inhibitor complex was not dissociated by 4 M KCl. Activity determinations on bovine TPCK-trypsin and bovine chymotrypsin with BAEE and BTEE assays revealed that the inhibitory actions toward both enzymes are functions of the same or closely adjacent sites of the inhibitor molecule. The supposed function of the inhibitor is discussed.

Animals↗

PubMed2001

A multi media load model for the Baltic Sea.

The background of this work is the international decision process with regard to the selection of chemicals to be assessed with priority. In order to stress the precautionary principle, mass flows were analysed rather than concentrations, threshold values, etc., as preferred by the chemical legislation (which still excludes the marine area). Lindane, hexachlorobenzene (HCB), trichloroacetic acid and its sodium salt, medium-chained chlorinated paraffins and tributyltin (TBT) were selected due to their great relevance for the marine area. Trichloroacetic acid is an interesting model compound on account of its accidental formation by degradation of volatile chlorinated hydrocarbons and during chlorination processes. In addition, a hypothetical compound was modelled, representing a highly water-soluble substance with low vapour pressure. The balancing area is the Baltic Sea and its catchment area. In order to model the substance flows, the 'Input/Output-load model' has been developed. The model quantifies the shift and the distribution of a yearly load of the substance investigated from the terrestrial-limnic into the marine compartment (Baltic Sea). Water-soluble substances, which are usually considered to be of no concern, may enter the sea in great amounts and, if not degradable, remain there. It turned out to be necessary to take into account remobilisation, unintended formation and point as well as line-sources.

Baltic States↗