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At least 55 records · Page 3Linked 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↗
PubMed1997

Isolation of myelin basic protein from whole tissue extracts by selective pH-dependent solubilization.

A previous study of the selective solubility of myelin basic protein (MBP) of tissue extracts at pH 9.0 has raised issues of its quantitative recovery, and the differential solubility of its charge isomers. The pH-dependent solubility of proteins of acid extracts of delipidated tissue of bovine spinal cord was therefore reexamined. MBP of whole extracts was completely soluble up to pH 8.0 only, and less so by 25% at pH 9.0, and 43% at pH 10.0. The proteins other than MBP were virtually insoluble between pH 5.0 to 6.0, and 9.0 to 10.0. The solubility of the main charge isomers I to III of MBP of 18.5 kDa was found not to be affected by pH. Either pH 5.0 or 9.0 is therefore suitable for the selective isolation of MBP from whole tissue extracts, only pH 5.0 providing for the complete recovery of MBP. The pH-dependent solution behaviour was also examined following the separation of proteins of whole extracts by anion exchange chromatography at pH 10.4. Purified MBP and several related minor cationic components of lower molecular weight were soluble throughout. In contrast, the anionic proteins were only partly soluble between pH 4.0 to 10.0, i.e. by 4 to 20%. The results are consistent with specific protein-protein interactions of the proteins of whole extracts, either enhancing the solubility of non-MBP proteins, e.g. at pH 7.0, or impairing that of MBP between pH 8.0 to 10.0.

Animals↗
PubMed2000

Ion-pairing high-performance liquid chromatographic method for the detection of N-acetylaspartate and N-acetylglutamate in cerebral tissue extracts.

An ion-pairing high-performance liquid chromatographic method for the determination of N-acetylaspartate and N-acetylglutamate using a C-18 column and a UV detection at 210 nm wavelength, by means of a diode array detector, is presented. A buffer containing 2.8 mM tetrabutylammonium hydroxide, 25 mM KH(2)PO(4), 1.25% methanol, pH 7. 00, is utilized for the isocratic separation of these N-acetylated amino acids, at a flow rate of 1 ml/min and a column temperature of 23 degrees C. The suitability of this chromatographic separation (without additional chromatographic steps prior to HPLC assay) to monitor variations both of N-acetylaspartate and of N-acetylglutamate in perchloric acid brain extracts from rats subjected to the impact acceleration model of diffuse brain injury is also reported. According to the data presented, this HPLC method allows the separation of the two N-acetylated amino acids considered from the many possible interfering compounds, commonly present in extracts of cerebral tissue, which have high extinction coefficients at 210 nm wavelength. Values of N-acetylaspartate and N-acetylglutamate determined by this method showed that cerebral trauma negatively affects both compounds, according to the severity of trauma itself.

Animals↗
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↗
PubMed1996

Tissue extract from dopamine-depleted striatum enhances differentiation of cultured striatal type-1 astrocytes.

The effects of tissue extract from dopamine (DA)-depleted striatum (lesion extract, L-ext) on morphological and electrophysiological natures of cultured striatal astrocytes were investigated. L-ext treatment suppressed the proliferation of type-1 astrocytes. They became fibrous in a concentration-dependent manner. These changes were not observed in type-2 astrocytes. By whole cell patch-clamp recording, two kinetically and pharmacologically distinct voltage-activated potassium currents, A current and delayed rectifier, were identified. L-ext treatment enhanced both currents in type-1 astrocytes, but only A current in type-2. Data suggest that in tissue extract from DA-depleted striatum, there are increased trophic activities that promote the differentiation of type-1 astrocytes.

Animals↗
PubMed2006

Procedures for the quantitative protein determination of urokinase and its inhibitor, PAI-1, in human breast cancer tissue extracts by ELISA.

The determination of the protein content of urokinase-type plasminogen activator (uPA) and its inhibitor, PAI-1, in breast cancer tissue extracts is used clinically to identify patients at risk to experience disease recurrence (metastasis) or early death. The serine protease uPA, in concert with its inhibitor PAI-1, promotes tumor cell adhesion, migration, and proliferation, as well as extracellular matrix degradation and, thus, facilitates tumor cell invasion and metastasis. The various technical steps to recover uPA and PAI-1 protein from archived breast cancer tissues and to quantitatively determine uPA and PAI-1 protein content in tumor tissue extracts by enzyme-linked immunosorbent assay (ELISA) are described in detail. The technical steps involved require fresh-frozen breast cancer tissue, a dismembrator machine (ball mill) to pulverize the tissue in the frozen state, detergent (Triton X-100) containing Tris-buffered saline to extract uPA and PAI-1 from the pulverized breast cancer tissue, an ultracentrifuge to separate the detergent fraction from cellular debris, uPA and PAI-1 ELISA kits, protein determination reagents, and a 96-well spectrophotometer (ELISA reader) to assess uPA, PAI-1, and total protein in the detergent extract. The uPA/PAI-1 ELISAs and the protein determination format described are robust and highly sensitive. In addition to the macromethod of tissue disintegration, we present a simple but sensitive micro-extraction procedure using cryostat sections or core biopsies as the source of breast cancer tissue. Such a technique allows rapid and quantitative determination of uPA and PAI-1, even in small breast cancer specimens.

Animals↗