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At least 1,009 records · Page 56Linked to original sources
PubMed2003

Low-attomole electrospray ionization MS and MS/MS analysis of protein tryptic digests using 20-microm-i.d. polystyrene-divinylbenzene monolithic capillary columns.

This work explores the use of 20-microm-i.d. polymeric polystyrene-divinylbenzene monolithic nanocapillary columns for the LC-ESI-MS analysis of tryptic digest peptide mixtures. In contrast to the packing of microparticles, capillary columns were prepared, without the need of high pressure, in fused-silica capillaries, by thermally induced in situ copolymerization of styrene and divinylbenzene. The polymerization conditions and mobile-phase composition were optimized for chromatographic performance leading to efficiencies over 100000 plates/m for peptide separations. High mass sensitivity (approximately 10 amol of peptides) in the MS and MS/MS modes using an ion trap MS was found, a factor of up to 20-fold improvement over 75-microm-i.d. nanocolumns. A wide linear dynamic range (approximately 4 orders of magnitude) was achieved, and good run-to-run and column-to-column reproducibility of isocratic and gradient elution separations were found. As samples, both model proteins and tissue extracts were employed. Gradient nano-LC-MS analysis of a proteolytic digest of a tissue extract, equivalent to a sample size of approximately 1000 cells injected, is presented.

Amino Acid Sequence↗
PubMed1984

Distribution and chromatographic characterisation of gastrin and cholecystokinin in the rat central nervous system.

Two tissue extraction techniques and two radioimmunoassays were used to study the distribution of gastrin and cholecystokinin in rat brain. Small amounts of gastrin were found in extracts of neurohypophysis, but in neither ice-cold 90% methanol nor in boiling water-acetic acid extracts of the other 33 brain areas studied. Cholecystokinin was found in equivalent amounts in both types of extract of 31 areas. The distribution was similar to that in previous studies. The components of cholecystokinin immunoreactivity were characterised in 10 rat CNS tissues using four tissue extraction methods in conjunction with gel filtration and ion-exchange chromatography. The results demonstrated that gastrins were present only in the neurohypophysis and that in all other rat CNS tissues the main molecular component was indistinguishable from the sulphated octapeptide of cholecystokinin. Minor immunoreactive components were observed in all types of extract of all tissues with the properties of the desulphated octapeptide and the C-terminal tetrapeptide amide, suggesting they are genuine tissue components, not extraction artefacts. Large molecular forms of cholecystokinin were not detected in any tissue. The results emphasise the necessity of using two or more extraction methods and two or more chromatography systems in such a study.

Animals↗
PubMed2001

Electrophoretic characterization of heat-stable squamous cell carcinoma antigen.

The aim of this study was to investigate the heat stability of squamous cell carcinoma (SCC) antigen, a tumor-associated serine proteinase inhibitor (serpin), in tumor tissue extract by electrophoretic methods. After heat treatment at 70 degrees C for 2 h, the tumor tissue extract showed a single main protein band of 45 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which reacted with a monoclonal antibody specific for SCC antigen. The heat-stable SCC antigen was separated by two-dimensional electrophoresis (2-DE) into four spots with pI 6.4-5.9 and Mr 44500-45 000 of SCC antigen-1. Furthermore, the SCC antigen-1 still showed its inhibitory activity against a cysteine proteinase, papain, by gelatin zymography. These results suggest that heat treatment of protein sample at 70 degrees C for 2 h may be a useful method for a partial purification of SCC antigen-1 which can inhibit lysosomal cysteine proteinases such as cathepsin L, S, and K.

Antigens, Neoplasm↗
PubMed2002

A spectrophotometric assay of gamma-glutamylcysteine synthetase and glutathione synthetase in crude extracts from tissues and cultured mammalian cells.

An assay of gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GS) in crude extracts of cultured cells and tissues is described. It represents a novel combination of known methods, and is based on the formation of glutathione (GSH) from cysteine, glutamate and glycine in the presence of rat kidney GS for the assay of gamma-GCS, or from gamma-glutamylcysteine and glycine for the assay of GS. GSH is then quantified by the Tietze recycling method. Assay mixtures contain the gamma-glutamyl transpeptidase (GGT) inhibitor acivicin in order to prevent the degradation of gamma-glutamylcysteine and of the accumulating GSH, and dithiothreitol in order to prevent the oxidation of cysteine and gamma-glutamylcysteine. gamma-GCS and GS levels determined by this method are comparable to those determined by others. The method is suitable for the rapid determination of gamma-GCS GS in GGT-containing tissues and for the studies of induction of gamma-GCS and GS in tissue cultures.

3T3 Cells↗
PubMed2001

Comparison of photosensitizer (AIPcS2) quantification techniques: in situ fluorescence microsampling versus tissue chemical extraction.

A noninvasive in situ fluorescence-based method for the quantification of the photosensitizer chloroaluminum disulfonated phthalocyanine was compared to the highly accurate but nonreal time ex vivo spectrofluorometry method. Our in vivo fluorescence technique is designed to allow real-time assessment of photosensitizer in tumor and normal tissues and therefore temporally optimal light delivery. Laser-induced fluorescence was used to measure photosensitizer concentration from multiple microscopic regions of tissue. Ex vivo chemical extraction was used to quantify photosensitizer concentration in the same volume of tissue. The amount of photosensitizer in the vascular and/or parenchymal compartments of skeletal muscle and liver was determined by quantifying fluorescent signal in vivo, ex vivo and after blood removal. Confocal microscopy was used to spatially document photosensitizer localization 30 min and 24 h after delivery. While a linear correlation can exist between the fluorescence intensity measured by our fiber-optic bundle system and actual tissue concentration, temporal changes to this calibration line exist as the photosensitizer changes its partitioning fraction between the blood (vasculature) and the tissue parenchyma. In situ photosensitizer fluorescence microsampling (dosimetry) systems can be performed in real time and linearly correlated to actual tissue concentration with minimal intertissue variance. Tissue-specific differences may require temporal alterations in the calibration.

Animals↗
PubMed1975

Erythrocytosis due to erythropoietin-producing uterine fibromyoma.

Erythrocytosis has been observed to occur infrequently in association with uterine fibromyomas. Study of this case revealed erythrocytosis prior to surgery and a remission was noted after myomectomy. Elevated erythropoietin activity in this patient's serum was demonstrated. The erythropoietin activity in the tumor tissue extract was 10 times higher than in the control tissue extract. We postulate that the tumor itself was responsible for the erythrocytosis observed. The mechanisms proposed to explain polycythemia in such a tumor were revised and our conclusion is that the tumor produced the erythropoietin autonomously and is not subjected to feedback control mechanism.

Adult↗
PubMed1987

High-performance liquid chromatography analysis of 1-aminocyclopropane-1-carboxylic acid using o-phthaldialdehyde precolumn derivatization.

A simple, rapid, direct method for the HPLC analysis of 1-aminocyclopropane-1-carboxylic acid (ACC) as its o-phthaldialdehyde derivative is described. The method is sensitive to about 1 pmol and can be used on plant tissue extracts with no cleanup. It will prove valuable in plant extracts where the chemical conversion of ACC in the tissue extracts to ethylene is variable, or when analyzing the specific radioactivity of ACC produced from radiolabeled precursors.

Amino Acids↗
PubMed1992

A fraction of murine placental extract enhances IgA production in cultured splenocytes.

During the second half of murine pregnancy there is a characteristic increase in the number of spontaneous immunoglobulin-secreting cells in the maternal spleen (IgM, IgG and IgA), the uterus-draining lymph nodes (IgG) and Peyer's patches (IgA). There are indications that signals originating from the feto-placental unit are of importance for the generation of at least some of these changes. To evaluate further the role of placental factors as regulators of maternal Ig-secretion, placental extract (PE) was separated into eight fractions by gel-chromatography and each fraction was screened for its effect on splenic (1) background DNA-synthesis, (2) background Ig-secretion and (3) Ig-secretion in lipopolysaccharide (LPS) activated B-cells (anti-Thy1.2 + complement-treated splenocytes). Similarly prepared extracts of fetuses, maternal spleen and maternal liver served as controls. All tissue extracts and splenocytes were syngeneic to avoid reaction to allogeneic determinants. Placental extract fractions did not differ significantly from the other tissue extracts in affecting background lymphocyte activity. However, fraction number 3 from placental extract (PE3, corresponding to an approximate molecular weight of 18-70 kDa) was found strongly to increase the number of IgA-secreting cells (P less than or equal to 0.001) in LPS-activated B-cells. This effect was absent or much less pronounced in the corresponding controls. Blocking of PE3-activity by antibodies against rat prolactin indicated that the enhancing activity may be attributed to proteins of the prolactin/placental lactogen/growth hormone family. This assumption was further strengthened by experiments demonstrating that prolactin exerted preferential enhancement of IgA secretion when added to LPS-activated B-cell cultures in vitro. The possible role of placental prolactin-like molecules as regulators of maternal IgA secretion is discussed.

Animals↗
PubMed2000

Induction of telomerase activity in fibroblasts from bleomycin-injured lungs.

Bleomycin-induced lung injury causes increased fibroblast numbers in the lung and pulmonary fibrosis. Studies of fibroblasts isolated from such injured lungs have revealed evidence of increased intrinsic proliferative capacity, but the mechanism is unknown. Telomerase catalyzes the addition of telomeric DNA repeats onto chromosomal ends, which is associated with increased cellular life span or immortality. To examine whether telomerase might play a role in regulating fibroblast proliferative capacity in pulmonary fibrosis, lung fibroblasts were isolated from rats treated with endotracheal injections of phosphate-buffered saline or bleomycin. At selected time points, the rats were killed and lung fibroblasts isolated. The isolated cells and lung tissue were then used in experiments for measurement of telomerase activity. The results show undetectable telomerase activity in fibroblasts isolated from control uninjured lungs, or in the control lung tissue extracts. Similar results were obtained in cells and lung tissue from Days 1, 3, and 28 bleomycin-injured lungs. However, significant telomerase activity was detected in fibroblasts and tissue extracts isolated from Days 7, 14, and 21 bleomycin-treated rat lungs, with maximal activity observed in the Day 14 samples. Analysis of the isolated cells for telomerase messenger RNA or reverse transcriptase expression, combined with alpha-smooth-muscle actin expression by immunohistochemistry, revealed that telomerase expression localized primarily to nonmyofibroblasts. These findings suggest that in addition to elevated growth factor expression, the injured lung fibroblast population may contain cells with increased life span, which could contribute to the observed overall increase in lung fibroblast numbers.

Animals↗
PubMed1979

Radioimmunoassay for pantothenic acid in blood and other tissues.

We describe a radioimmunoassay for pantothenic acid in biological tissues. D-Pantothenic acid was conjugated with bovine serum albumin by use of a bromoacetyl derivative of pantothenic acid, and antibody to this antigen was raised by injecting it into the foot pads of rabbits. For the radioimmunoassay, a 100-fold dilution of the resulting antiserum was incubated with radiolabeled pantothentic acid. The antibodies were precipitated and dissolved, and the radioactivity of the solution was measured in a liquid scintillation counter. Between 5 and 125 ng of pantothenic acid can be detected in 75 muL of tissue extract. Validation included recovery and precision studies, parallelism with tissue extracts, and competitive binding studies. Results of the radioimmunoassay and those of microbiological assay with use of Lactobacillus plantarum correlated well (r = 0.80).

Aged↗
PubMed1976

Fructose metabolism in the rat endometrium under normal and experimental conditions.

Fructose-1,6-diphosphate (FDP) hydrolysis, which was measured in tissue extracts from mature rat endometrium, was maximal during late dioestrus and early pro-oestrus and reflected the variations in the plasma levels of ovarian steroids. Treatment of ovariectomized animals with oestradiol or medroxyprogesterone acetate increased FDP hydrolysis when compared with control animals, although oestradiol was the more effective. FDP hydrolysis was greater in extracts of endometrial tissue from uterine horns containing a silk IUD or of deciduomal tissue from pseudo-pregnant rats, in which the peak was on Day 9, than in extracts of endometrium from the control contralateral horn. These findings are interpreted as linking glycogen metabolism in normal endometrium, IUD-containing horns, and decidual tissue to the role of FDP in gluconeogenesis and the promotion of glycogen storage.

Animals↗
PubMedDate not supplied

Thin layer chromatographic/bioautographic method for identification of antibiotic residues in animal tissues.

An analytical method for the identification of the residues from 14 commonly used antibiotics is presented. The technique is based on selective tissue extraction followed by thin layer chromatography (TLC)/bioautography. Antibiotic residues are extracted from the tissues with methanol and methanol-HCl (98 + 2). The methanol extract is further extracted with chloroform to isolate groups of antibiotics. The extracts are spotted onto TLC plates and developed in suitable solvent systems. Developed plates are placed on set medium seeded with Bacillus subtilis and a bioautograph is produced. The locations of zones of inhibition are used to identify antibiotic residues. Recoveries of antibiotics were quantitative, while the effect of naturally inhibiting components of the matrix was minimized. The sensitivity of the method can be adjusted through minor modifications, which allows its use in routine regulatory analysis.

Animals↗
PubMed1988

Regulation of glycogen synthase in muscle and adipose tissue during fasting and refeeding.

Using immunoblot analysis, we examined the electrophoretic mobility of glycogen synthase from rat skeletal muscle and adipose tissue. Extracts from muscle freeze clamped in situ contained at least three forms of synthase with different electrophoretic mobilities. Extracts from adipose tissue also contained multiple forms but lacked the form with greatest mobility found in the muscle extracts. Phosphorylation at multiple sites of glycogen synthase is known to deactivate the enzyme and retard its electrophoretic mobility in sodium dodecyl sulfate gels. These results suggest that there is very little or no dephosphorylated glycogen synthase in adipose tissue and that phosphorylated forms of glycogen synthase synthesize adipose tissue glycogen. Relative to control, it is known that fasting decreases and refeeding increases glucose incorporation into glycogen in rat epididymal adipose tissue but not skeletal muscle incubated in vitro in the presence of insulin. Fasting did not change the electrophoretic pattern of muscle synthase but decreased the relative amount of adipose tissue forms with greater mobility. Refeeding increased above control the relative amount of adipose tissue synthase with greater mobility. These results indicate that changes in the phosphorylations that retard mobility contribute to the effects of fasting and refeeding on adipose tissue glycogen metabolism.

Adipose Tissue↗
PubMed1994

Post-translational processing of the neurotensin/neuromedin N precursor in the central nervous system of the rat--I. Biochemical characterization of maturation products.

Neurotensin and neuromedin N are two biologically active related peptides which are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences which are thought to represent putative processing sites occur in the precursor molecule. Of these sites, the three that are closest to the C-terminus flank and separate neurotensin and neuromedin N. The fourth precedes a neuromedin N-like sequence. The present studies were aimed at determining the extent to which each of these four dibasic sites is cleaved and at identifying and quantifying the intermediate and mature products to which this cleavage gives rise in extracts from whole rat brain, hippocampus and globus pallidus. This was achieved by means of radioimmunoassays specific for sequences of the neurotensin/neuromedin N precursor that are adjacent to the dibasic processing sites used in combination with high pressure liquid chromatography and arginine-directed trypsin digestion of tissue extracts. In all tissue extracts, it was found that the three most C-terminal dibasic processing sites in the neurotensin/neuromedin N precursor are processed to a similar extent, whereas the dibasic site that precedes the neuromedin N-like sequence is processed to a lesser extent. As reported previously, the globus pallidus was shown to contain proportionally lower levels of neuromedin N than other brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Effects of intravenous infusion of human prostate tissue substances in dogs.

Symptoms of postresection syndromes following transurethral prostatectomy have been associated with a transient increase in serum acid phosphatase due to intraoperative absorption of prostate tissue substances. Factors associated with postoperative syndromes include intraoperative absorption of irrigant solution, intraoperative blood loss, and intraoperative absorption of prostate tissue substance. An animal model was used in this study to determine the isolated physiologic effects of intravenous infusion of a saline prostate tissue extract using an infusion schedule comparable to that of transurethral prostate resection. Twenty-four animals received either normal saline infusion (control) or saline prostate tissue extract infusion (experimental). The experimental group showed a significant decrease in fibrinogen, platelet count, white blood count, activated clotting time, and plasma volume. These results suggest that the isolated effects of intravascular absorption of prostate tissue substances are due to disseminated intravascular coagulation, most likely resulting from tissue thromboplastin and activation of plasminogen.

Absorption↗
PubMed1984

Detection and partial characterization of Crohn's disease tissue specific proteins recognized by Crohn's disease sera.

To identify immunoreactive proteins in Crohn's disease (CD) tissue, we examined intestinal tissues from eight patients with CD, seven patients with ulcerative colitis (UC) and four normal colon specimens from patients with colon carcinoma. Tissues were homogenized in 0.05 M Tris-buffered saline containing 2 mM phenyl-methyl-sulphonyl fluoride, and the cell free supernatants were incubated with sera from eight patients with CD, five with UC and five normal volunteers. Immune complexes formed in vitro were precipitated with pansorbin, washed with several detergents and analysed by SDS-polyacrylamide gel electrophoresis. When CD tissue was immunoprecipitated with CD sera, three major proteins of 160,000, 120,000 and 110,000 daltons were detected. Partial enrichment of these proteins were achieved when immune complexes formed in vitro were precipitated with polyethylene glycol followed by protein A-Sepharose 4B chromatography. Affinity gel chromatography and autoradiographic studies with purified serum IgG from CD patients further confirmed the presence of these immunoreactive proteins in CD tissue extracts. When similarly examined, these proteins were absent from UC and control tissue extracts after incubation with CD, UC or control sera. These studies suggest that CD tissue contains several proteins which are specifically recognized by CD patients' sera. Characterization of these immunoreactive proteins may provide an important lead in understanding the pathogenesis of CD.

Antibody Specificity↗
PubMed1991

Blood and bone marrow eosinophilia in malignant tumors. Role and nature of blood and tissue eosinophil colony-stimulating factor(s) in two patients.

Eosinophilia of the blood and bone marrow may be encountered in patients with disseminated malignancies. It is unrelated to the histologic type of the tumor but usually reflects its aggressiveness and prognostic outlook. Its pathogenesis is controversial. The presence of eosinophil colony-stimulating factor(s) in serum and malignant tissue extracts was evaluated in two cases of malignancies accompanied by marked blood and bone marrow eosinophilia. When compared with controls, serum and tumor tissue extracts stimulated the growth of G, M, and Eo colonies in semisolid cultures of human bone marrow. Such stimulating effect was inhibited by the addition of antibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3. Thus, the colony-stimulating factor(s) play a role in the pathogenesis of the eosinophilia associated with some malignant tumors, and these factors appear to include GM-CSF, interleukin-3, and probably others.

Adenocarcinoma↗
PubMed1990

Plasma and tissue concentrations and molecular forms of somatostatin in calves infected with Sarcocystis cruzi.

The effects of parasitic infection on plasma and tissue content of immunoreactive somatostatin (SRIF) were studied in 4-mo old male calves inoculated with the protozoan Sarcocystis cruzi. Because feed intake significantly decreased (70%) in infected calves around day 28 postinfection (pi), concomitant with the asexual replication of S. cruzi and outward expression of clinical signs, the relative contributions of infection and associated reduction in nutrition on plasma SRIF were evaluated. Treatment groups were: noninfected ad libitum fed (C), infected (250,000 S. cruzi oocysts per os) ad libitum fed (I) and noninfected calves pairfed to the level of intake of each infected calf (PF). Mean plasma concentrations of SRIF (pg/ml) on day 30 pi were: C, 224 +/- 22; I, 742 +/- 150; PF, 246 +/- 31 (effect of infection P less than .05). In another study, SRIF was measured in plasma and in pancreatic, duodenal, jejunal and ileal tissue extracts from normal and S. cruzi infected calves. Plasma and tissue samples were collected on day 42 pi. Mean plasma SRIF were 2.5 times higher in infected than control calves. Plasma insulin and insulin-like growth factor 1 was lower in infected v control calves (P less than .02). Plasma glucagon was similar between groups. Duodenal (P less than .05) and jejunal (P less than .02) SRIF content was higher in infected than control calves. Chromatography of tissue extracts on Sephadex G-50 revealed that the increase in SRIF was accounted for, in part, by molecular forms larger than cyclic SRIF-14. Data suggest that peripheral SRIF is increased in calves during S. cruzi infection. The increase in SRIF is not solely related to plane of nutrition. Altered levels of gut SRIF(s) may be associated with perturbed metabolic regulation in parasitized animals through direct effects on the gut.

Animals↗