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At least 343 records · Page 19Linked to original sources
PubMed2003

Assessment of element-specific homogeneity in reference materials using microanalytical techniques.

The IAEA established in 1994 a co-ordinated research programme (CRP) on "Reference Materials for Microanalytical Nuclear Techniques" as part of its efforts to promote and strengthen the use of nuclear analytical technologies in member states with the specific aim of improving the quality of analysis of nuclear, environmental, and biological materials. The objectives of this initiative were: to identify suitable biological reference materials which could serve the needs for quality control in microanalytical techniques; to evaluate existing CRMs for use in microanalytical investigations; to evaluate appropriate sample pretreatment procedures for materials being used for analysis with microanalytical techniques; to identify analytical techniques which can be used for characterisation of homogeneity determination, and to apply such techniques to the characterization of candidate reference materials for use with microanalytical techniques. The CRP lasted for 4 years and seven laboratories and the Agency's Laboratories in Seibersdorf participated. A number of materials including the candidate reference materials IAEA 338 (lichen) and IAEA 413 (single cell algae, elevated level) were evaluated for the distribution of elements such as Cl, K, Ca, Cr, Mn, Fe, Zn, As, Br, Rb, Cd, Hg, and Pb. The results obtained during this CRP suggest that: each element exhibits its characteristic distribution in a matrix described by the "Ingamels' sampling constant" or the "relative homogeneity factor" of Kurfuerst; both concepts are valid over a large range of sample mass used for analysis (from 0.1 microg to around 100 mg); and materials being characterised quantitatively for element homogeneity could be used for the experimental determination of total uncertainty of other analytical techniques. As far as we are aware this is the first time the concept of quantitative characterisation of homogeneity has been applied to potential reference materials and the first demonstration of the feasibility and usefulness of the concept with particular emphasis on enhancing quality control opportunities for microanalytical techniques.

Journal Article↗
PubMed1976

Amylases of the genital tract. I. Isoamylases of genital tract tissue homogenates and peritoneal fluid.

Homogenates of tissue from the female genital tract contain isoamylases which are, to a certain extent, electrophoretically distinguishable from the pancreatic and salivary isoamylases. In healthy nonpregnant women, high levels of activity of genital isoamylases were found in tissue homogenates of cervical and Fallopian tube mucosa, whereas activity was weak or absent in homogenates of endometrium. The isoamylases of the cervical mucosa had an electrophoretic migration rate toward the anode identical to that of the salivary main fraction, whereas the isoamylases of the Fallopian tube migrated faster. Specific genital isoamylase activities were also demonstrable in peritoneal fluid collected from the cul-de-sac. During the menstrual cycle, these activities showed a midcycle peak. In pregnant women, the levels of activity of the genital isoamylases in peritoneal fluid were lower than in nonpregnant women. In homogenates of the male accessory genital glands, the isoamylases specific for the genital tract were present in minute amounts. The isoamylases specific for the genital tract were not detectable in serum in either sex.

Adult↗
PubMed1985

A procedure for the purification of ferritin from human liver by heating a methanol-treated homogenate.

A simple, rapid technique for purification of ferritin from human liver tissue is described. Methanol, at a final concentration of 40% (v/v) in liver homogenate, precipitates the majority of proteins but does not affect ferritin. Subsequent heating of this homogenate at 75 degrees C for 10 min results in a purified ferritin preparation as judged by immunoelectrophoresis and polyacrylamide gel electrophoresis. The resultant purified ferritin contained the same amount of iron as the original endogenous ferritin. There were no significant differences (paired t tests) in the amount of protein in the purified ferritin preparation when measured by rocket immunoelectrophoresis and by the Lowry procedure, suggesting that the antigenecity of ferritin was unaffected by the methanol and heat treatment. Both endogenous liver ferritin and radiolabeled human liver ferritin added to liver homogenates were recovered after methanol and heat treatment with similar yields (77 +/- 7% and 70 +/- 2%, respectively) when compared with the standard treatment of heating a homogenate at 75 degrees C. The overall ferritin yield with this rapid procedure was 40%.

Chromatography, High Pressure Liquid↗
PubMed1985

Isolation of enzymes from polyacrylamide disk gels by a centrifugal homogenization method.

A sliced segment of polyacrylamide gel was quickly homogenized without any loss of gel pieces. The gel segment was placed on a disposable pipet tip, which was packed with a small amount of lumped copper wires and held in a microfuge tube. The gel was homogenized by centrifugation for 15 s at 15,000 g at 0 to 4 degrees C. Almost 70% of endodextranase activity could be recovered from homogenized gel within 30 min at 4 degrees C, whereas only 20% of activity was eluted from gel slices. If necessary, copper wire could be replaced by fine stainless-steel wire or by the nylon string used in fishing lines. Proteins could also be recovered from the homogenized gel by charging electric current for 1 h at 4 degrees C.

Centrifugation↗
PubMed1991

Preparation of representative homogenates of biological tissues: effect of salt on protein extraction.

A technique for the preparation of representative homogenates of tissues is described. For most soft tissues and biological fluids (liver, kidney, semen) more than 80% of protein was recovered after homogenization in isotonic buffer. With tissues that are harder to homogenize, such as heart, spleen, and skeletal muscle, however, only approximately 40-50% of protein was extracted in this medium. Inclusion of salt or salt plus detergent during the homogenization increased recovery of the protein to levels close to those recorded with soft tissues. The increase represented a true recovery and was not an artifact induced by salt/detergent. This situation is parallel to previously reported results for lipid-rich biological tissues.

Animals↗
PubMed1979

Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media.

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. (Na+ + K+)-stimulated, Mg2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca2+-stimulated, Mg2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell segments in an amber low-speed (800 x g) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at 100,000 x g for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of (Na+ + K+)-stimulated ATPase compared with the other fractions, but it had essentially no Ca2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate. Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined bu a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basememt membrane, could not be detected in this fraction.

Adenosine Triphosphatases↗
PubMed1977

Correlation between microsomal (Na+ + K+)-ATPase activity and [3H]ouabain binding to heart tissue homogenates.

ATP plus Mg2+ plus Na+ supported [3H]ouabain binding to canine left ventricular tissue homogenates and microsomal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity from the same tissue were measured. A linear relationship was found between the initial velocity of [3H]ouabain binding to tissue homogenates and microsomal (Na+ + K+)-ATPase activity from the same tissue in the presence and absence of in vivo bound digoxin. In vivo bound digoxin reduced both measurements. With tissue from digoxin-free hearts, a linear relationship was also obtained between the initial velocity and the maximum level of [3H]ouabain binding to tissue homogenate. Binding of [3H]ouabain to whole tissue homogenate is a convenient method for estimating (Na+ + K+)-ATPase activity in small left ventricular biopsy samples.

Adenosine Triphosphatases↗
PubMed1978

Characteristics of fatty acid oxidation in rat liver homogenates and the inhibitory effect of malonyl-CoA.

Experiments were carried out to study the control of fatty acid oxidation and ketogenesis in rat liver homogenates. In contrast to findings with the perfused liver, rates of fatty acid oxidation were high and equal in liver homogenates from fed and fasted animals. No difference in apparent Km values for oleate, ATP, coenzyme A or carnitine could be detected in the two types of homogenate. Over the concentration range 20--40 micron, malonyl-CoA inhibited oleate oxidation by 50--75%. The fact that the inhibitory effect could be removed by pre-treatment with alkali or fatty acid synthetase indicated that the inhibitory molecule was malonyl-CoA rather than a contaminant. The effect was readily reversible and appeared to be competitive with oleyl-CoA. Malonyl-CoA also inhibited oleate oxidation in homogenates of heart and kidney cortex but this is unlikely to have physiological relevance since, in contrast to liver, neither tissue contains an active cytosolic pathway for the generation of malonyl-CoA and the synthesis of fatty acids.

Acyl Coenzyme A↗
PubMed1982

Relative acylglycerol acyltransferase activities in homogenates of enzymically dispersed rat jejunal villus and crypt cells.

The relative acyltransferase activities were compared in homogenates of rat jejunal villus and crypt cells isolated by differential scraping and hyaluronidase dispersion. The contributions of the monoacylglycerol and phosphatidic acid pathways to the higher acylglycerol and phospholipid biosynthesis were assessed using 2-oleoyl-sn-[3H] glycerol and [I-14C] palmitic acid as tracers. The stereochemical course of the diacylglycerol biosynthesis was determined by stereospecific analysis. Using 2-oleoyl-sn-glycerol as a tracer, the villus cells exhibited for times higher diacylglycerol and 19 times higher triacylglycerol biosynthesis than crypt cells on an equivalent protein basis. Furthermore, while the villus cell homogenates yielded a preponderance (75%) of the 1, 2-diacyl-sn-glycerols, the crypt cell homogenates formed essentially racemic proportions of 1, 2- and 2,3-diacyl-sn-glycerols. Both villus and crypt cell homogenates exhibited comparable acyl acceptor and acyl donor concentration dependence and the same cofactor requirements. It is unlikely that these acyltransferase activities in the crypt cell preparation are due to contamination with the villus cells, because then more comparable proportions of the enantiomeric diacylglycerols and triacylglycerols would have been anticipated. It is concluded that the crypt cells possess intrinsic monoacylglycerol and to a much lesser extent diacylglycerol acyltransferase activities, which are acquired prior to the development of a distinct brush border and which probably do not require dietary stimulus for induction.

Acyltransferases↗
PubMed1986

An accurate and sensitive assay of [14C]octanoate oxidation and its application on tissue homogenates and fibroblasts.

A procedure was developed to assay [14C]octanoate oxidation from the production of both 14CO2 and 14C-labeled acid-soluble products. Octanoic acid and its CoA and carnitine esters were eliminated from the acid-soluble products by alkaline hydrolysis of the esters and acidification and binding of the acid to Lipidex 1000. The method was evaluated with homogenates of various rat tissues and human muscles and with human fibroblasts. 14CO2 production was variable and comprised less than 3% of the total oxidation products with homogenates and 26 +/- 19% with fibroblasts. As compared to palmitate, oxidation rates of octanoate were higher in rat liver and heart homogenates, of the same magnitude in muscle homogenates, but lower in fibroblasts. The proportion of antimycin-insensitive oxidation was much lower with octanoate than with palmitate. Using the assay a case of medium-chain acyl-CoA dehydrogenase deficiency could be indicated.

Adolescent↗
PubMed1975

DNA replication by a possible continuous-discontinuous mechanism in homogenates of Physarum polycephalum containing dextran.

Nuclear DNA synthesis in homogenates of Physarum is greatly stimulated by the presence of dextran in the homogenizing medium. In this cell-free system, the DNA precursor is incorporated approximately equally into two classes of DNA intermediates. On of these is similar in size to that observed previously in the intact organism, i.e. its sedimentation rate in alkaline sucrose density gradients increases, presumably by chain elongation, as the organism progresses through the S phase. The other class (approx. 10 S) is similar to 'Okazaki' fragments. Thus, nuclear DNA synthesis in homogenates of Physarum may occur by a continuous-discontinuous mechanism. Substantial DNA-synthetic activity is obtained by the addition of dextran to dextran-free homogenates. Maximal activity in this system requires the presence of both the nuclear and post-nuclear supernatant fractions. It is possible that a partial separation and recombination of a DNA polymerase and the endogenous template is effected by this procedure.

Cell Division↗
PubMed1982

Receptor independent stimulatory effect of noradrenaline on Na,K-ATPase in rat brain homogenate. Role of lipid peroxidation.

The effect of different adrenoceptor agonists on Na,K-ATPase activity and lipid peroxidation of rat brain homogenate was studied. Drugs which enhanced Na,K-ATPase activity--noradrenaline, adrenaline and oxymethazoline--were found to inhibit endogenous membrane lipid peroxidation. Other drugs--phenylephrine, xylazine and clonidine--which did not cause any change in the enzyme activity did not influence lipid peroxidation either. No increase of Na,K-ATPase activity by noradrenaline could be detected after preincubation of the homogenate for 5 min at 37 degrees. During this time endogenous lipid peroxidation of considerable extent could be observed. It is concluded that there is no correlation between the adrenoceptor agonist feature of noradrenaline and its stimulatory effect on Na,K-ATPase activity of rat brain homogenate. However, it seems likely that in rat brain homogenate the increase of Na,K-ATPase activity and inhibition of endogenous lipid peroxidation by noradrenaline are related.

Adrenergic Agonists↗
PubMed1986

Decarboxylation of 1,2,3,4-tetrahydro-beta-carboline-1-carboxylic acids in brain homogenate and catalysis by pyridoxal-5'-phosphate.

[Carboxyl-14C] labelled 1,2,3,4-tetrahydro-beta-carboline-1-carboxylic acid (I) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-1-carboxylic acid (II) were synthesized and their decarboxylation was studied in mouse brain homogenate and buffer. The decarboxylation rates of (I) and (II) in the homogenate were about 6-fold and 4-fold, respectively, as compared with the rates in phosphate buffer. The increase could not be prevented by preheating the homogenate, but was partially abolished by addition of 1 mM EDTA. The decarboxylation was increased dose-dependently when pyridoxal-5'-phosphate was included in the buffer, 400 microM being sufficient to exceed the rate in homogenate for both (I) and (II). Mass spectrometric examination of the decarboxylation products indicated that both (I) and (II) were degraded mainly to corresponding 1,2,3,4-tetrahydro-beta-carbolines, but some 3,4-dihydro analogues also were detectable. In conclusion, the results outline a way through which these pharmacologically active beta-carbolines are readily formed under conditions that may be regarded as physiological.

Animals↗
PubMed1988

Hydrolysis of angiotensin I by peptidases in homogenates of rat lung and aorta.

The hydrolytic cleavage of angiotensin I has been studied in homogenate preparations of rat lung and aorta using gradient elution HPLC to monitor the formation of peptide products. Fresh crude homogenate preparations produced a rapid breakdown of angiotensin I to largely unidentifiable fragment peptides. Neither His-Leu nor angiotensin II was observed in these preparations even in the presence of captopril (20 microM) and the amino-peptidase inhibitors, puromycin, amastatin and bestatin. However, in freeze-thawed homogenates, angiotensin II and His-Leu were detectable together with the tetrapeptide, angiotensin (1-4). The addition of captopril (20 microM) reduced the amount of angiotensin II produced but did not completely block its formation. Higher concentrations of captopril or the addition of enalaprilat or EDTA did not further reduce the amount of angiotensin II produced. In the presence of captopril a peptide corresponding to des-Leu(10)angiotensin I was formed in relatively large amounts (equivalent to 40% of angiotensin I catabolized). Homogenates purified by concanavalin A affinity chromatography gave a clean hydrolysis of angiotensin I to angiotensin II and His-Leu which was completely blocked by captopril. These results suggest an ACE-like activity in rat lung and aorta that is not sensitive to converting enzyme inhibitors.

Angiotensin I↗
PubMed1981

Histamine-activated adenylate cyclase in brain homogenates of several species.

Histamine has been shown to activate cyclic AMP synthesis in brain slices and homogenates of certain species, although less is known about species differences in brain homogenates. Dutch Belted and New Zealand White rabbit brain homogenates contained a histamine-responsive adenylate cyclase similar to that of the guinea pig. In contrast, adenylate cyclase of gerbil and hamster brain exhibited little or no stimulation by histamine. Male rat hypothalamic homogenates contained adenylate cyclase, but also exhibited minimal stimulation by histamine, in disagreement with some recent reports. Detailed studies of the conditions of assay failed to resolve this discrepancy.

Adenylyl Cyclases↗
PubMed1986

Features of ligand binding in homogenate and section preparations.

The binding of [3H]N-methyl scopolamine (NMS) to muscarinic binding sites in rat forebrain was compared in two types of preparations: homogenates and slide-mounted sections. Under standard assay conditions, [3H]NMS bound to the muscarinic binding site with an apparent Kd that was almost one order of magnitude lower in homogenates (Kd = 0.15 nM) than sections (Kd = 1.25 nM). In addition, the muscarinic agonist carbachol inhibited [3H]NMS binding more effectively in homogenates (Ki = 5 microM) than sections (Ki = 100 microM). The higher Kd observed in sections was dependent upon the thickness of the section since the apparent Kd decreased as the thickness of the section was reduced. A slower equilibration rate may account, in part, for the higher apparent Kd seen in thicker sections. The results support previous evidence that certain ligands exhibit different binding profiles in homogenate and section preparations.

Animals↗
PubMed1981

Assay of adenylate cyclase in homogenates of control and Duchenne human skeletal muscle.

The wide range of values reported for activity of adenylate cyclase (AC) in human skeletal muscle prompted re-evaluation of conditions used for homogenization and assay. Adenylate cyclase activity in the same normal muscle differed with different techniques of homogenization. In pH 7.5 isotonic Tris buffer, basal and catecholamine-activated activities declined rapidly in homogenates kept at 4 degrees C. Loss of basal activity was prevented by addition of a chelator of divalent cations. Loss of response to isoproterenol was prevented by addition of guanylnucleotides. Enzyme activity was maximal at 37 degrees C and pH 7.6. Enzyme activity was lower when theophylline was used to prevent degradation of labelled 3',5' cyclic adenosine monophosphate (cyclic AMP) than when unlabelled cyclic AMP was used to this purpose. Basal activity increased with increased MgCl2 concentration up to 50 mmol/l, but isoproterenol-activated activity was maximal at 4 mmol/l MgCl2. AC was inhibited by exogenous adenosine, but addition of adenosine deaminase to the assay mixture did not increase AC activity. Based upon these observations, standardized procedures of homogenization and assay were devised and used to measure AC activity in muscles of boys with Duchenne muscular dystrophy: basal and isoproterenol-stimulated activities were abnormally low.

Adenosine Triphosphate↗
PubMed1984

[3H]p-Aminoclonidine binding to multiple alpha 2-adrenoceptor sites in homogenates of cat frontal cortex and cat spinal cord.

We have investigated the characteristics of [3H]p-aminoclonidine binding to homogenates of cat frontal cortex and of cat spinal cord. At 37 degrees C, saturable [3H]p-aminoclonidine binding displayed rapid association and dissociation kinetics. Scatchard plots of data obtained from either tissue were consistently biphasic. Computer-assisted nonlinear least squares regression analysis based upon a one ligand/two binding site model was employed to resolve these plots into two linear components. The results of this analysis indicate that, for homogenates of either tissue, the KD of [3H]p-aminoclonidine binding, to the high and low affinity binding sites respectively, is approximately 0.5 nM and 9 nM. Mean Bmax values (fmol/mg protein), for the high and low affinity binding sites respectively, of 28 and 149 for homogenates of cat frontal cortex and 7.1 and 56 for homogenates of cat spinal cord were obtained. The results of competitive inhibition experiments, where 0.13 nM [3H]p-aminoclonidine was used, indicate that catecholamines compete for [3H]p-aminoclonidine binding sites in a stereoselective fashion, and that these sites possess a pharmacology characteristic of alpha 2-adrenoceptors. The results of Hill analysis of the competition data suggest that alpha 2-adrenoceptor agonists and alpha 2-adrenoceptor antagonists possess selective affinity for these two alpha 2-adrenoceptor binding sites. The two populations of saturable [3H]p-aminoclonidine binding sites appear to correspond to those alpha 2-adrenoceptor ligand binding sites that have been referred to as the 'super high' and 'high' affinity state of the alpha 2-adrenoceptors.

Animals↗