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At least 37 records · Page 2Linked to original sources
PubMed1993

Determination of c-erbB-2 protein in primary breast cancer tissue extract using an enzyme immunoassay.

The c-erbB-2 protein in breast cancer tissue extract was determined by using an enzyme-immunoassay (EIA) to see whether the quantitative determination of the oncoprotein correlates with the results of immunohistochemistry and other prognostic factors. Primary breast cancer from 104 patients was assayed for c-erbB-2 protein with an EIA that used two monoclonal antibodies directed against the extracellular domain of the protein. Pelleted tissue homogenate prepared routinely for hormone receptor assay was used as the starting material. The mean quantity of c-erbB-2 protein was 695 unit/mg protein (range 23 to 5939), and this correlated well with the results of immunohistochemical staining (P < 0.00001). It was found that 17.3% (18/104) of all tumors contained amounts of c-erbB-2 protein exceeding 1000 units/mg protein. All tumors with negative or weakly positive staining contained the oncoprotein as less than 1000 units/mg protein. The content of c-erbB-2 protein was correlated with the histologic grade (P = 0.0022), mitotic index (P = 0.0002) and degree of nuclear atypia (P = 0.013). It was inversely correlated with progesterone receptor (P = 0.006) and less strongly with estrogen receptor status (P = 0.016). Values of hormone receptor concentration and c-erbB-2 protein content showed a hyperbolic relationship that suggested biological interactions between c-erbB-2 protein and steroid hormone receptors. We conclude that c-erbB-2 protein in tissue extracts of primary breast cancer can be determined reliably by EIA, and it seems feasible to explore further the advantages of introducing EIA as a routine laboratory examination for providing additional information about the biological aspects of breast cancer.

Adult↗
PubMed1983

Measurement of factor VIII CAg by immunoradiometric assay in human tissue extracts.

Samples of normal tissues taken from three patients within 24 hours of death were physically disrupted, washed and extracted. Centrifuged supernatants were tested for factor VIII coagulant antigen (VIII CAg) and factor VIII related antigen (VIII RAg) by radioimmunoassays using homologous and heterologous antisera respectively. VIII RAg was readily washed out by isotonic saline whereas VIII CAg required 1.5 M sodium chloride for optimal extraction from tissue homogenates. Highest levels of VIII CAg were detected in some lymph nodes followed by lung, liver and spleen. There was rapid loss of VIII CAg from most tissues in the absence of proteolytic inhibitors.

Antigens↗
PubMed1978

Selective inhibition of leukocyte adherence in women with breast cancer using a variety of tissue extracts.

Leukocyte adherence inhibition (LAI) tests for the assessment of tumor immunity in vitro have been based on the assumption that the adherence of the leukocytes is inhibited by specific interaction with an antigenic tumor extract. However, loss of leukocyte adherence could depend on several non-immunological factors including disease state of the leukocyte donor and the presence of a variety of agents, proteins or tissue extracts. We have studied LAI , therefore, using different species and concentrations of serum protein or tissue extracts. Our results show that leukocytes from patients with a variety of diseases adhere to glass in a rather consistent fashion. High concentrations of serum protein or tissue extract inhibited leukocyte adherence. 39 breast cancer patients and 38 female controls were studied with a total of 23 solubilized extracts from breast carcinoma, other tumor and non-tumor tissues. Breast cancer patients showed selective LAI in the presence of 200 microgram per test tube of several breast carcinoma extracts.

Antigens, Neoplasm↗
PubMed1978

[Tissue extracts of malignant human lymphomas in the leukocyte migration test].

Water-soluble lymph node extracts and sera from 23 patients with benign and malignant lymphomas and tissue extracts from liver, spleen, kidney, and muscle of one patient without any lymphatic disease were analyzed using the direct leukocyte migration test (LMT). The leukocytes were obtained from healthy persons. 10 of the lymphomas and 3 of the tissue extracts varied the leukocyte migration. In most cases of chronic nonspecific lymphadenitis, in 2 cases of Hodgkin's disease, and in extracts of liver, spleen and muscle inhibitory effects were found. Stimulating effects could be demonstrated in 2 cases of immunocytoma and in 4 cases of Hodgkin's disease. The results indicate that in cases of stimulation the target cells might be influenced by a pathological migration inhibitory factor.

Cell Migration Inhibition↗
PubMed1970

Production of both prolactin and growth hormone by clonal strains of rat pituitary tumor cells. Differential effects of hydrocortisone and tissue extracts.

Several established clonal strains of rat pituitary cells which produce growth hormone in culture have been shown to secrete a second protein hormone, prolactin. Prolactin was measured immunologically in culture medium and within cells by complement fixation. Rates of prolactin production varied from 6.6 to 12 microg/mg cell protein per 24 hr in four different cell strains. In these cultures ratios of production of prolactin to growth hormone varied from 1.0 to 4.1. A fifth clonal strain produced growth hormone but no detectable prolactin. Intracellular prolactin was equivalent to the amount secreted into medium in a period of about 1-2 hr. Both cycloheximide and puromycin suppressed prolactin production by at least 94%. Hydrocortisone (3 x 10(-6)M), which stimulated the production of growth hormone 4- to 8-fold in most of the cell strains, reduced the rate of prolactin production to less than 25% of that in control cultures. Conversely, addition of simple acid extracts of several tissues, including hypothalamus, to the medium of all strains increased the rate of production of prolactin six to nine times and decreased growth hormone production by about 50%. We conclude that multifunctional rat pituitary cells in culture show unusual promise for further studies of the control of expression of organ-specific activities in mammalian cells.

Animals↗
PubMed1984

Distribution of growth hormone-releasing hormone-like immunoreactivity in human tissue extracts.

A specific RIA for human pancreatic tumor GH-releasing hormone [hpGRH(1-44)NH2] was developed using an antiserum which recognizes the region (Met27-Leu44)NH2 of hpGRH(1-44)NH2. This RIA was used to measure GH-releasing hormone-like immunoreactivity (GRH-LI) in various human tissue extracts. The highest concentration of GRH-LI was detected in extract of pituitary stalk with moderate amounts found in hypothalamus and optic chiasm but none in the cerebrum, cerebellum, medulla oblongata, spinal cord, and anterior pituitary. In peripheral organs appreciable quantities of GRH-LI were found in the pancreas, whereas extracts of thyroid, lung, stomach, duodenum, ileum, colon, adrenal, and kidney contained very low concentrations of GRH-LI. No GRH-LI was detected in liver and spleen extracts. Gel permeation chromatographic analysis of the tissue extract from the hypothalamus revealed only one peak which eluted at the same position as that of 125I-hpGRH(1-44)NH2. Similar analysis of an extract from the optic chiasm showed one peak at the same position as that of 125I-hpGRH(1-44)NH2 and another peak which eluted after hpGRH(1-44)NH2. In contrast, an extract from the pancreas contained only one peak which eluted before 125I-hpGRH(1-44)NH2, indicating a possible precursor form of hpGRH(1-44)NH2. Limited trypsin digestion of the GRH-LI material from the pancreas, followed by gel permeation chromatographic analysis, yielded a major peak eluting at the same position as that of 125I-hpGRH(1-44)NH2. These results suggest that the GRH-LI detected in the hypothalamus most likely corresponds to hpGRH(1-44)NH2 in structure and that the GRH biosynthesized in the hypothalamus is transported to the stalk median eminence and stored there for release into the portal vessels.

Adult↗
PubMed1984

Determination of pseudouridine in tRNA and in acid-soluble tissue extracts by high-performance liquid chromatography.

A high-performance liquid chromatographic method to measure pseudouridine and other nucleosides in hydrolyzed unfractionated tRNA and in acid soluble tissue extracts is described. The method is based on the following steps: tRNA extraction and hydrolysis by a mixture of ribonuclease A, snake venom phosphodiesterase and bacterial alkaline phosphatase; nucleoside purification (in the case of acid soluble tissue extract) by affinity chromatography on a phenyl-boronate gel column; nucleoside separation and quantitation by high-performance liquid chromatography on an octadecylsilane column by a reversed polarity gradient elution. The procedure allows a very accurate quantitation of pseudouridine and some other nucleosides, and its sensitivity is such that only 20 micrograms of tRNA are required. The method has been utilized to compare the pseudouridine content of hydrolyzed tRNA extracted from normal and lymphomatous murine thymus, as well as the pseudouridine content in acid soluble extracts from the same tissues.

Animals↗
PubMedDate not supplied

[Method of measuring binding constants and the number of specific cyclic AMP receptor sites in tissue extracts].

A method for evaluation of binding constants and of amount of cAMP binding sites in crude tissue extracts was developed. The method is based on equilibrium binding of 3H-cAMP by proteins with subsequent ultrafiltration. Hydrolysis of cAMP and its unspecific sorption by proteins were eliminated under the conditions selected. Rat spleen cytosole contained 3.57 +/- 0.34 pmol of cAMP binding sites per mg of protein with dissociation constant of protein-cAMP complex (1.68 +/- 0.28).10(-8) M. As shown by studies on kinetics, binding constants and specificity of binding, the method permitted to evaluate quantitatively cAMP-dependent protein kinases in crude tissue extracts and to estimate their affinity to cAMP.

Animals↗
PubMed1982

Immunodetection of insulin after transfer from gels to nitrocellulose filters. A method of analysis in tissue extracts.

This report describes the development of a rapid method for detection of nanogram quantities of insulin in tissue extracts after electrophoresis. Following electrophoresis the proteins are transferred to nitro-cellulose filters and treated with a photoreactive crosslinking agent. Filter bound insulin is detected by antiinsulin antibody and 125I-protein A, followed by autoradiography. The photoaffinity crosslinking is simple, rapid, and stable, and does not require reactive binding sites on derivatized paper. Under these conditions insulin maintains its immunoreactivity yet can be washed extensively to reduce nonspecific background; as little as 10 ng can be visualized. The method has proven to be useful for rapid analysis of qualitative as well as quantitative differences in immunoreactive insulin in tissue extracts.

Animals↗
PubMed1992

Homogeneous trinder-coupled assay for the determination of glucose-6-phosphatase activity in tissue extracts.

We describe an automated, homogeneous, glucose oxidase-coupled method for the determination of glucose-6-phosphatase activity in tissue extracts. The method is based on measurement of the rate of glucose formation by the Trinder reaction, in which the end product is a quinoneimine dye which absorbs maximally at 505 nm and has a molar extinction coefficient of 5700. The incubation mixture contains 20 microL of tissue extract, 25 microL of 0.5 M phosphate buffer, pH 7.0, 175 microL of Trinder/glucose-6-phosphate reagent, and 30 microL of distilled water. After a delay period of 15 min, to exhaust any glucose endogenously present in the extract, glucose production from glucose-6-phosphate is monitored at 505 nm for 5 min in a centrifugal analyzer. The Km was 13 mM over a 10-fold range in glucose-6-phosphate concentration and the reaction was linear up to about 250 U/L. Within-run CV of the assay at activities of 48 and 190 U/L ranged between 2.5-5.0%. The between-run CV at 190 U/L was 5.1%.

Animals↗
PubMed1989

A simple enzymic method to separate [3H]inositol 1,4,5- and 1,3,4-trisphosphate isomers in tissue extracts.

A novel method to separate [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 in tissue extracts is described. It is based on the selective metabolism of Ins(1,3,4)P3 by a crude cerebral supernatant in a Mg2+-free buffer followed by separation of [3H]inositol trisphosphates using conventional anion-exchange chromatography. Evaluation of the assay was performed using [3H]Ins(1,3,4)P3 standards and tissue extracts containing different proportions of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3. Parallel h.p.l.c. separations of extracts established the selective and complete metabolism of [3H]Ins(1,3,4)P3 under the above conditions and demonstrated that the enzymic method provides an accurate estimate of the trisphosphate isomers in rat cerebral cortex, parotid gland and bovine tracheal smooth muscle.

Animals↗
PubMed1994

Identification of ethanolamine in rat and gerbil brain tissue extracts by NMR spectroscopy.

Some NMR resonances which have previously been observed but not identified in mammalian brain tissue extracts have been shown to arise from ethanolamine. This conclusion is drawn from a systematic study of the perchloric acid extracts of rodent brain tissue in which several NMR experiments were used to assign the peaks unambiguously. The extraction procedure used in this work gave samples with highly reproducible spectra, and ethanolamine was observed in all our extract samples. A localized increase in the concentration of ethanolamine was seen in the spectra of extracts produced from a cerebral infarct induced by occlusion of the middle cerebral artery in the rat.

Animals↗
PubMed1987

Study of metabolism of psychotomimetic indolealkylamines by rat tissue extracts using liquid chromatography.

The use of a series of liquid chromatographic techniques involving cation-exchange, reverse-phase and normal-phase chromatography has permitted the separation and characterisation of a number of metabolites of the psychotomimetic indolealkylamines N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine which were isolated following incubation of these compounds with rat tissue extracts. In liver, kidney and brain tissue extracts the routes of metabolism identified included oxidative deamination, N-demethylation, O-demethylation and N-oxidation. The quantitative significance of individual routes of metabolism in these tissues was assessed using N,N-dimethyltryptamine as a substrate.

Animals↗
PubMed1995

Detection and characterization of the prostate-specific membrane antigen (PSMA) in tissue extracts and body fluids.

The prostate-specific membrane antigen (PSMA) glycoprotein is recognized by the monoclonal antibody (MAb) 7E11-C5.3 as a predominant 100 kDa and minor 180 kDa component in LNCaP cell line extracts and its expression has been shown by immunohistochemistry to be highly restricted to prostate epithelium. The aim of the present study was to utilize Western blot analysis to determine if PSMA could be detected in human tissue extracts and body fluids and if so, which molecular forms were present. PSMA was detected as 120 and 200 kDa bands in normal, benign and malignant prostate tissues and seminal plasma. Further analysis demonstrated that the larger molecular form of PSMA may be a dimer of the lower m.w. species. The PSMA glycoprotein was not detected in the majority of non-prostate tissue extracts examined except for a low yet significant amount in normal salivary gland, brain and small intestine, suggesting that PSMA may not be as prostate-specific as originally thought. Since the prostate-specific antigen (PSA) has been shown to be maximally shed into the serum in high-grade and metastatic prostate carcinomas, it was surprising that PSMA could not be detected in serum by Western blot analysis even in patients with actively progressive metastatic disease. Second generation antibodies generated against different epitopes may be required to determine if PSMA is shed into serum. Our results support the hypothesis that PSMA is a novel prostate biomarker.

Animals↗
PubMed1982

Growth from regenerating goldfish retinal cultures in the absence of serum or hormonal supplements: tissue extract effects.

The minimal requirements for the regeneration of optic nerve fibers in vitro were established in a serum-free retinal explant preparation. This serum-free preparation was developed as a prerequisite for testing the growth-promoting activity of tissue extracts prepared from the primary target of regenerating fibers. Explants taken from goldfish retinas 14 days after a prior optic nerve crush were capable of long-term survival and regenerated neurite outgrowth without serum or hormonal supplements. Serum-free conditions for explant outgrowth required only a basic Leibovitz (L-15) media containing 0.6% methyl cellulose (MC). Explants were also capable of neurite outgrowth in L-15 media alone when culture dishes were preplated with MC. MC treatment permitted both the regeneration of neurites in serum-free L-15 and a significant increase in the rate and extent of neurite outgrowth when combined with 10% fetal calf serum (FCS). Explants grown in L-15 with both MC and FCS produced a 2.5-fold increase in the length of neurite outgrowth over MC alone and a 1.5-fold increase in the length of neurite outgrowth over FCS alone. MC activity which permitted minimal serum-free regeneration and optimal serum supplemented regeneration was determined to be substrate related. Retinas were dissociated to determine if ganglion cells, like the intact explant, were capable of survival and neurite regeneration in serum-free conditions. These cells survived and extended long neurites when grown in L-15 with FCS or with FCS and MC, but they did not survive in serum-free L-15 with MC. The minimal serum-free conditions for explant survival and neurite regeneration were used as a model system to test the growth-promoting activity of crude tissue extracts prepared from the goldfish brain. Extracts prepared from the primary target region, the optic tectum, stimulated a significant 2.5-fold increase in the length of regenerating neurites. The optic tectal extract (OTex) stimulated outgrowth with significantly high specific activity when compared with extracts of identical protein concentrations prepared from the cerebellum (Cex). At a minimal protein concentration of 150 micrograms/ml, the OTex stimulated a 1.5-fold increase in neurite outgrowth above Cex. These results indicated that a serum-free culture preparation had been established for optic nerve regeneration. This culture system has proven to be an extremely sensitive bioassay model without the masking effect of a serum supplement. Serum-free cultures may be used in further studies to determine the role neurotrophic factors may play in a widely used model of successful central nervous system (CNS) regeneration.

Animals↗
PubMed1991

Determination of released CD4 and CD8 antigens in the suction-blister fluid and horny-tissue extract in patients with psoriasis.

The amounts of soluble CD4 and CD8 antigens were measured in suction-blister fluid and extracts of horny tissue in patients with psoriasis. The levels of soluble CD4 and CD8 in the suction-blister fluid of lesional skin in psoriasis was significantly higher than from normal skin and the uninvolved skin in psoriasis. Levels of soluble CD4 and CD8 in the extracts of horny tissue in psoriasis were significantly higher than those from non-psoriatic skin.

Adolescent↗
PubMed1976

Absence of collagen stimulating factors for L929 cells in granulation tissue extracts.

We have attempted to find collagen synthesis stimulating activity in polyvinyl sponge granulation tissue extracts and inflammatory fluid. The cells studied in culture were mouse L929 cells grown in monolayers. A prior study by another group reported such factors to be present in healing wound extracts and in extracts of CHHl3 injured liver. We have been unable to find such stimulating activity for L929 cells in granuloma tissue fluid. In addition we have examined the procedures used by the prior group and have found major problems in their methodology which are discussed below.

Animals↗