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At least 775 records · Page 43Linked to original sources
PubMed2003

Use of an immortalized bovine mammary epithelial cell line (MAC-T) to measure the mitogenic activity of extracts from heifer mammary tissue: effects of nutrition and ovariectomy.

The objectives of the experiment were (1) to determine whether MAC-T cells would accurately mimic the previously observed proliferative responses of primary mammary epithelial cells (MEC) to mammary tissue extracts from high and low-fed heifers and (2) to determine whether mammary tissue extracts from ovariectomized (OVX) heifers would have lower mitogenic activity than intact controls. Addition of mammary tissue extracts to cell culture media of MAC-T cells plated on plastic or collagen-coated plastic to a range of concentrations between 1 and 8% resulted in dose-dependent increases in cell proliferation. Furthermore, mammary tissue extracts from low-fed prepubertal heifers aged 9 months, stimulated significantly more proliferation of MAC-T cells, as measured by 3H-thymidine incorporation into DNA than mammary tissue extracts from high-fed heifers (40.6 cpm x 10(3) per well versus 21.9+/-1.8 cpm x 10(3) per well). These observations suggested that MAC-T cells would be a suitable alternative to primary MECs for measuring the mitogenic activity of mammary tissue extracts. Conversely, no difference was observed in the mitogenic activity of mammary tissue extracts from OVX or control heifers. Possibly, MAC-T cells provide a good model for nutrition- but not ovarian-induced changes in mammary growth. Alternatively, that reduction of in vivo mammary development following OVX did not result in reduced mitogenic activity of the mammary tissue extracts emphasizes that heifer mammary development is the result of complex interactions between local growth factors and systemic hormones.

Animal Feed↗
PubMed1989

Application of sorbent extraction chromatography to the purification of diethylstilboestrol extracted from muscle tissue and determined by radioimmunoassay.

A chromatographic procedure is described for the purification of bovine muscle tissue extracts prior to the determination of diethylstilboestrol (DES) by radioimmunoassay. Sorbent extraction chromatography of tissue extracts on reversed-phase octadecyl (C18) columns gives adequate recovery of residue and a suitable sample for radioimmunoassay. This procedure, which is simple and rapid, provides an alternative to a more complex purification by high-performance liquid chromatography. Using this method, the limit of detection for DES in muscle samples is approximately 40 pg/g.

Animals↗
PubMed1981

Heparin facilitates the extraction of tissue fibronectin.

Extraction of fibronectin from two human tissues, lung parenchyma and placental villi, was facilitated by the incorporation of heparin into extraction media. The effect of heparin was additive to the effect of urea which is known to extract fibronectin. These experiments provide further evidence that fibronectin and glycosaminoglycans are associated in connective tissues and the use of heparin forms the basis for a simple method for extraction and quantitation of tissue fibronectin.

Dermatan Sulfate↗
PubMed1991

Measurement of insulin-like growth factor-II in physiological fluids and tissues. II. Extraction quantification in rat tissues.

The tissue distribution and developmental patterns of insulin-like growth factor-II (IGF-II) have not been investigated in rat tissues, primarily because of the lack of an efficient extraction method for IGF-II and a sensitive RIA. IGF-II was extracted from rat tissues by formic acid, and the extract was heated at an acidic pH and treated with acetone. The removal of binding proteins was demonstrated by fast protein liquid chromatography size exclusion column and the elimination of a dilutional bias in the RIA. Using rat IGF-II as standard, we optimized a RIA for the quantification of IGF-II in rat tissues. In adult rats, IGF-II was found in all 15 tissues examined, with the highest concentration in the pituitary, followed by kidney, seminal vesicles, intestine, and serum. This distribution is not only different from that of IGF-I, but also differs from that reported for IGF-II mRNA and IGF-II receptors, suggesting that the rates of synthesis and/or metabolism of IGF-II are tissue dependent. Developmentally, IGF-II levels fell postnatally in most tissues, a pattern similar to that of IGF-II mRNA and IGF-II receptor. This developmental pattern supports the hypothesis that IGF-II is important in early growth and development. A relatively homogeneous distribution was observed in the adult rat brain, a distribution also different from that reported for IGF-II mRNA. In the pituitary, the highest concentration was found in the posterior pituitary, followed by the intermediate and anterior pituitary. In conclusion, IGF-II is found in many tissues of adult rats. This observation supports an autocrine and/or paracrine roles for IGF-II.

Animals↗
PubMed1983

Radioimmunoassay of bile acids in tissue, bile, and urine.

Two commercially available (Abbott Labs.) radioimmunoassays for determination of conjugated cholic acid and sulfoglycolithocholic acid in serum have been modified for bile acid measurements in alcoholic tissue extracts, bile, and urine. The specificity of both radioimmunoassays has been determined with regard to 27 free and conjugated bile acids. After filtration, bile acids can be measured in urine and bile without prior extraction. Tissue is homogenized and the bile acids are extracted into methanol. Urinary excretion by 64 healthy humans was 2.09 (SD 1.09) mumol of conjugated cholic acid and 8.44 (SD 8.03) mumol of sulfated glycolithocholic acid per 24 h. In liver from 10 patients with various noncholestatic liver disease, the mean concentration of conjugated cholic acid was 32.4 (SD 15.9) nmol/g wet weight. In the liver of 27 male Wistar rats, the concentration of conjugated cholic acid was 41.3 (SD 11.7) nmol/g of tissue, of sulfoglycolithocholic acid 5.1 (SD 2.3) nmol/g of tissue.

Adult↗
PubMed1986

Use of Pico-Tag methodology in the chemical analysis of peptides with carboxyl-terminal amides.

A chemical method has been established for the detection of carboxyl-terminally amidated peptides in tissue extracts. Tissue was homogenized in an acidic medium designed to solubilize peptides while precipitating high-molecular-weight protein. The homogenate supernatant was in turn subjected to reversed-phase extraction with C18 Sep-Pak cartridges. The eluates were fractionated by reversed-phase high-performance liquid chromatography (RP-HPLC). Individual fractions were exhaustively digested with thermolysin, derivatized with phenylisothiocyanate (PITC), and then subjected to ethyl acetate extraction under basic conditions. The phenylthiocarbamyl (PTC)-amino acid amide derivatives were selectively taken up into the organic phase, while the other digestion products remained in the aqueous phase. The organic phase was analyzed by RP-HPLC on a Pico-Tag amino acid analysis column, monitoring eluates at 254 nm. PTC-amino acid amides were identified and quantitated by comparing their elution positions and peak areas, respectively, with those of standards. Their identities were confirmed by amino acid analysis, following hydrolysis with hydriodic acid. The technique was applied to extracts of bovine posterior pituitaries and a human medullary thyroid carcinoma. Vasopressin (-Leu-Gly-amide), oxytocin (-Gly-amide), Lys1 gamma 1-melanotropin (-Phe-amide), and various acetylated and non-acetylated forms of alpha-melanotropin (-Val-amide) were identified in the posterior pituitary extract. Various forms of calcitonin (-Val-Gly-Ala-Pro-amide) were detected in the tumour extract. For vasopressin and calcitonin the thermolytic digest resulted in di- and tetra-peptides, respectively, reflecting thermolytic cleavage at more favoured sites.

Amides↗
PubMed1994

Protein kinase C isoforms in human and rat colonic mucosa.

The protein kinase C (PKC) family of enzymes plays a key role in the regulation of cellular events, including cell proliferation and differentiation. Work from our laboratory has shown that the effects of dietary fat and fiber on colonic cell proliferation were positively correlated with membrane/cytosol PKC activity ratios (Chapkin et al., 1993, J. Nutr. 123, 649-655). The presence and subcellular distribution of specific PKC isoforms in rat and human colon were therefore determined in cytosolic and membrane extracts. Tissue extracts were probed with antibodies to individual PKC isoforms. PKC alpha, beta, delta, epsilon, and zeta were detected in both rat and human colonic mucosa, while PKC eta was detected in human colonic mucosa only. PKC alpha, beta, and zeta were predominantly localized in the cytosolic fraction, whereas the majority of PKC delta, epsilon, and eta were found in the membrane-associated fraction. Presence of mRNA for individual PKC isoforms was determined by reverse transcriptase PCR (RT-PCR). Using rat colonic mucosa, mRNA for PKC alpha, beta, delta, epsilon, eta, and zeta were detected by RT-PCR with identity confirmed by sequencing. The relative steady-state levels of PKC isoforms in human colon adenocarcinoma as compared with normal colonic mucosa were determined, with adenocarcinomas having higher amounts of cytosolic PKC beta, delta, epsilon, eta, and zeta. PKC isoforms were also detected in viable, exfoliated colonic cells isolated from human feces, demonstrating that this noninvasive method can be utilized to examine PKC expression in colonic cells. These results demonstrate that colonic mucosa expresses both calcium-dependent (classical) and calcium-independent (novel and atypical) PKC isoforms with distinct subcellular distributions for each. The dynamics of these PKC isoforms may have implications in the development of colon carcinogenesis.

Adenocarcinoma↗
PubMed1995

Mast cell tryptase levels in normal canine tissues.

Levels of canine tryptase from various tissues were quantified using a competition enzyme-linked immunosorbent assay (ELISA). The assay utilises an affinity-purified rabbit anti-tryptase antibody in the solid phase and alkaline-phosphatase conjugated tryptase together with unlabelled tryptase in the fluid phase. The assay will rapidly quantify 40-5000 ng ml-1 of tryptase in tissue extracts. Tissues from the skin, gut, liver and lung were studied, of which canine gut appeared to contain the highest levels of tryptase per milligram wet weight, which may suggest an important role for this enzyme at this site. This assay may prove valuable in assessing the role of mast cells in various disease states in the dog.

Animals↗
PubMed2005

Mycobacterial catalase-peroxidase is a tissue antigen and target of the adaptive immune response in systemic sarcoidosis.

Sarcoidosis is a disease of unknown etiology characterized by noncaseating epithelioid granulomas, oligoclonal CD4(+) T cell infiltrates, and immune complex formation. To identify pathogenic antigens relevant to immune-mediated granulomatous inflammation in sarcoidosis, we used a limited proteomics approach to detect tissue antigens that were poorly soluble in neutral detergent and resistant to protease digestion, consistent with the known biochemical properties of granuloma-inducing sarcoidosis tissue extracts. Tissue antigens with these characteristics were detected with immunoglobulin (Ig)G or F(ab')(2) fragments from the sera of sarcoidosis patients in 9 of 12 (75%) sarcoidosis tissues (150-160, 80, or 60-64 kD) but only 3 of 22 (14%) control tissues (all 62-64 kD; P = 0.0006). Matrix-assisted laser desorption/ionization time of flight mass spectrometry identified Mycobacterium tuberculosis catalase-peroxidase (mKatG) as one of these tissue antigens. Protein immunoblotting using anti-mKatG monoclonal antibodies independently confirmed the presence of mKatG in 5 of 9 (55%) sarcoidosis tissues but in none of 14 control tissues (P = 0.0037). IgG antibodies to recombinant mKatG were detected in the sera of 12 of 25 (48%) sarcoidosis patients compared with 0 of 11 (0%) purified protein derivative (PPD)(-) (P = 0.0059) and 4 of 10 (40%) PPD(+) (P = 0.7233) control subjects, suggesting that remnant mycobacterial catalase-peroxidase is one target of the adaptive immune response driving granulomatous inflammation in sarcoidosis.

Antigens, Bacterial↗
PubMed1978

Medium for the enumeration and isolation of bacteria from a Swine waste digester.

A habitat-simulating medium was developed for the enumeration and isolation of bacteria from a swine waste digester. A roll tube medium with growth factors for strict anaerobes from previously studied anaerobic ecosystems was used to evaluate the effects of deletion, addition, or level of digester fluid, digester fluid treated with acid or base, rumen fluid, fecal extract, anaerobic pit extract, tissue extract, carbohydrates, peptones, short-chain fatty acids, minerals, vitamins, N and P sources, reducing and solidifying agents, buffers, and gases on colony counts. Decreasing the agar concentration from 2.5 to 1.0% increased the counts twofold. Blending increased the counts 1.7-fold. With a medium (174) containing digester fluid, peptones, minerals, cysteine, sodium carbonate, and agar, colony counts were 60% of the microscopic count and improved yields 2.5 to 20 times those obtained with media previously used for digesters or developed for other anaerobic ecosystems. Colony counts continued to increase for up to 4 weeks of incubation. Medium 174 permits the enumeration of total, methanogenic, and, with deletion of reducing agent, aerotolerant bacteria. The results suggest that the predominant bacteria grow slowly and have requirements different from those of bacteria from other ecosystems.

Journal Article↗
PubMedDate not supplied

Solid-phase extraction cleanup for ivermectin in liver tissue.

Extraction of liver tissue with organic solvent produces coextractants with compounds of interest. The solid-phase extraction (SPE) cleanup of liver tissue developed for ivermectin removes nonpolar coextractants. Liver extract that has been reduced to dryness is reconstituted in 0.5 mL acetonitrile. The mixture is passed through 0.1 g C18 SPE column, and the eluate is collected. The column is eluted further with 2 mL acetonitrile. Combined eluates are derivatized with 1-methylimidazole and trifluoroacetic anhydride, and the ivermectin derivative is determined by liquid chromatography with fluorescence detection.

Animals↗
PubMedDate not supplied

Liquid chromatography-electrochemical detection of furazolidone and metabolite in extracts of incurred tissues.

One-day-old chicks were raised to maturity on a diet fortified with 0.0055% furazolidone. Analyses of tissue extracts by a liquid chromatographic-electrochemical detection screening procedure for nitro-containing drugs disclosed, in addition to the parent drug, an unidentified metabolite in the liver and breast tissue of the mature birds sacrificed while on the fortified feed. No evidence of residues of the drug or metabolite was found in birds removed from the medicated feed 48 h prior to sacrifice. In view of the rapid in vivo and postmortem metabolism of the parent drug in liver tissue, the metabolite can serve as an alternative means of detecting furazolidone residues in chicken tissues.

Animals↗
PubMed1975

Effects of extracts of rat tissues on the action of bleomycin.

The cell sap (105 000 times g supernatant) of various tissues of rats caused DNA degradation in the presence of bleomycin. The activity was fractionated into two peaks by column chromatography on Sephadex G-25. The activity in Peak A (excluded fraction) appeared to be due to some proteinaceous entity, while that recovered in Peak B (retarded fraction), constituting about 90% of the total activity, seemed to be due to ascorbic acid, judging by results of further gel filtration and the effect of treatment with ascorbate oxidase. Incubation of bleomycin with Peak A or B caused loss of the ability of the antibiotic to degrade DNA. It is proposed that the action of bleomycin on DNA, and its inactivation by tissue extracts, depend, at least in part, on the presence of ascorbic acid.

Animals↗
PubMed1980

Extraction of pineal and uterine tissue at different pH values: a preliminary report on the occurrence of a few groups of compounds in both tissues.

Extraction of pineal glands has been performed at different pH values. After extraction at pH +/- 4.9, 'porphyrin'-containing protein did not occur in the extracts, while at pH +/- 6.3 these proteins had passed over partly into the extract; at pH +/- 6.8 the proteins could no longer be detected in the residues, but a partial breakdown had taken place. The activity of pineal extracts depends on the method of extraction. In the watery extracts (pH +/- 6.3) both of pineal and of uterine tissue the presence could be shown among others of: porphyrins bound to protein, the indole compound 'bufotenine' also bound to protein, 'kynurenine', a 'basic peptide' containing amines and 'flavoproteins'. The hypothesis is proposed that in uterine endometrium, tryptophan can be metabolized in at least two pathways, one leading to kynurenine and one to bufotenine. Formation of bufotenine, probably a physiologically important compound, might be influenced by the oestrous cycle and conversely.

Animals↗
PubMed1979

Sequestration of adenosine in crude extract from mouse liver and other tissues.

Adenosine (1 microM) was incubated in the presence of dialyzed crude tissue extract from mouse liver and its degradation determined. At high concentration of tissue extract, a fraction of adenosine was not metabolized. This phenomenon, termed sequestration of adenosine, was shown to be affected in the same way by the same factors (pH, salt, reducing agent and adenine) as those affecting the protection of adenosine against deamination in the presence of the purified cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase from mouse liver (Saebø, J. and Ueland, P.M. (1979) Biochim. Biophys. Acta 587, 333--340). These data point to a role of this protein in the sequestration of adenosine in crude extract. The sequestration potency in crude extract could be determined by diluting the extract in the presence of a constant amount of adenosine deaminase added to the tissue extract. Under these conditions there was linearity of adenosine not available for degradation versus the concentration of tissue extract, and a total recovery of the sequestration potency of purified binding protein added to the crude extract was observed. The tissue level of the cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase in mouse liver was determined by two independent procedures based on the sequestration of adenosine and the hydrolysis of S-adenosylhomocysteine, respectively. The intracellular concentration was calculated to be 10 microM. The sequestration of adenosine in crude extract from mouse, rat, rabbit and bovine tissues was determined and showed requirements similar to those of the sequestration in mouse liver extract. The ability to sequester adenosine was high in liver and decreased in the following order: liver, kidney, adrenal cortex, brain, uterus, cardiac and skeletal muscle.

Adenosine↗
PubMed2000

PRMT1 is the predominant type I protein arginine methyltransferase in mammalian cells.

Type I protein arginine methyltransferases catalyze the formation of asymmetric omega-N(G),N(G)-dimethylarginine residues by transferring methyl groups from S-adenosyl-L-methionine to guanidino groups of arginine residues in a variety of eucaryotic proteins. The predominant type I enzyme activity is found in mammalian cells as a high molecular weight complex (300-400 kDa). In a previous study, this protein arginine methyltransferase activity was identified as an additional activity of 10-formyltetrahydrofolate dehydrogenase (FDH) protein. However, immunodepletion of FDH activity in RAT1 cells and in murine tissue extracts with antibody to FDH does not diminish type I methyltransferase activity toward the methyl-accepting substrates glutathione S-transferase fibrillarin glycine arginine domain fusion protein or heterogeneous nuclear ribonucleoprotein A1. Similarly, immunodepletion with anti-FDH antibody does not remove the endogenous methylating activity for hypomethylated proteins present in extracts from adenosine dialdehyde-treated RAT1 cells. In contrast, anti-PRMT1 antibody can remove PRMT1 activity from RAT1 extracts, murine tissue extracts, and purified rat liver FDH preparations. Tissue extracts from FDH(+/+), FDH(+/-), and FDH(-/-) mice have similar protein arginine methyltransferase activities but high, intermediate, and undetectable FDH activities, respectively. Recombinant glutathione S-transferase-PRMT1, but not purified FDH, can be cross-linked to the methyl-donor substrate S-adenosyl-L-methionine. We conclude that PRMT1 contributes the major type I protein arginine methyltransferase enzyme activity present in mammalian cells and tissues.

Animals↗