[Effect of aqueous saline extracts of mouse tissues on previously irradiated hereditary structures].
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Animals↗
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Variations of plasma and kidney renin contents during experimental acute renal hypertension induced by extracts from different tissues.
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Animals↗
[Studies of trophocytic function of lymphocytes. I. Biological actions of the extracts from lymphatic tissues].
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Animals↗
[Ferritin study in tumors of the female genitalia].
Ferritin levels in extracts of tumor tissue and serum of patients with gynecological malignancies were assayed by immunodiffusion methods using a standard system of testing for ferritin. A high blood-ferritin level (500-1,000 ng/ml) was found in 8 out 21 patients with ovarian cancer, 8 out of 24 patients with endometrial carcinoma. 2 out of 8 cases of mucinous cystadenoma and in 2 out of 4 cases of pyoovarium. It was lower in other malignancies of the female genital tract. There was no correlation between the ferritin level of blood serum and that of tumor tissue extracts. Although blood serum ferritin was found in very few healthy subjects (2.5%), it is suggested that blood-ferritin level test should be carried out in the course of examination of slowly-progressing purulent adnexitis and mass screening for tumors of female genitals.
Female↗
Low molecular weight proteinase inhibitors. III. Quantification and isolation of activity from benign and malignant human breast tissue samples.
Both normal and malignant breast tissue contain proteinase inhibitors. Extracellular proteins produced by tumors have been demonstrated to be qualitatively similar to their counterparts in normal tissue, but they are often present in significantly less quantity than in normal tissue. In our investigation, normal breast tissue extracts were shown to contain significantly greater inhibitory activity directed against the degradation of casein by trypsin in the isolated fraction of the extracts compared with extracts from malignant tissues. The molecular weight of this isolated fraction is approximately 17,000 daltons. Our observations suggest that malignancy is accompanied by a reduction in the net inhibitory activity of this proteinase inhibitor.
Breast↗
Presence of thyroxine in eggs and changes in its content during early development of chum salmon, Oncorhynchus keta.
In order to examine the role of thyroid hormones during salmonid development, techniques were developed for quantitative extraction of thyroxine from eggs, whole embryos, and alevins of chum salmon (Oncorhynchus keta) at various stages of development. Frozen eggs, embryos, alevins, or fry were homogenized in ice-cold methanol. The homogenate was centrifuged, and the supernatant was washed with a mixture of chloroform and 0.05% CaCl2. The aqueous layer was lyophilized, and the residue was redissolved in barbital buffer for thyroxine radioimmunoassay (RIA). Serial dilutions of the egg or tissue extracts gave inhibition slopes that were parallel to that of the thyroxine standard in the RIA. Immunoreactivity of the extracts coeluted with thyroxine standard in reverse-phase HPLC on an ODS column. Recovery of thyroxine from egg and tissue extracts was estimated from the recovery of 125I-labeled thyroxine added to the initial homogenates. Thyroxine content of eggs just after fertilization was 4-5 ng/egg, and this level was maintained until hatching. A decrease in thyroxine content was seen during yolk absorption. Total thyroxine increased to about 10 ng/fish, a level higher than that in the unfertilized egg, at the time of complete yolk absorption, and decreased within 10 days to a low level of 1 ng/fish. These findings are discussed in relation to the role of maternal thyroid hormones during early development and also to the onset of larval thyroid function.
Animals↗
Characterization of a novel neuroglandular antigen (NGA) expressed on abnormal human melanocytes.
Murine monoclonal antibodies (MAbs) were produced with reactivity to human malignant melanoma. Six MAbs, 3 of the IgGI (LS113, LS140, LS152) and 3 of the IgG2a (LS59, LS62, LS76) subclasses, were selected for their binding, with an identical pattern of reactivity, to a novel melanoma-associated antigen. As characterized by the enzyme-linked immunosorbent assay (ELISA), these MAbs were found to be positive on n-octyl-beta-D-glucopyranoside extracts of all 10 melanoma cell lines tested and on extracts of 22 metastatic melanoma tumors. The antibodies had minimal reaction with a panel of 14 normal adult tissue extracts. A degree of cross-reactivity was observed with 50% of 39 non-melanoma tumor extracts. The results obtained with the ELISA on cell line and tissue extracts were duplicated using the ABC method of peroxidase staining. The pattern of cross-reactivity, as demonstrated by the intense staining of paraffin-embedded and frozen tissue sections of normal, benign and malignant tissues, defines the recognized protein as a neuroglandular antigen (NGA). Immunoadsorbents made with the antibodies were used to purify the antigen shed from cultured melanomas. All 6 MAbs recognized this purified antigen while 5 other antimelanoma antibodies did not react with it. On gel electrophoresis this antigen is a highly glycosylated glycoprotein with a protein core of 21 kDa.
Animals↗
Quantitation of angiogenesis factor in bovine retina and tumour extracts by means of radioimmunoassay.
Using an antiserum raised against tumour angiogenesis factor (TAF) we have developed a radioimmunoassay for retina and tumour angiogenesis factor(s). This antiserum was previously shown to bind to both human and animal tumour extracts and to inhibit the angiogenesis induced by TAF in vivo. TAF from rat Walker tumour was used for iodination by the chloramine-T method. An excess of 125I-labelled TAF was incubated with TAF antibody in the absence (maximum binding) and presence (inhibition of maximum binding) of unlabelled tissue extract. A double antibody technique was used to separate free and bound TAF. Unlabelled human Wilms tumour TAF was used as a standard. The extent of inhibition of 125I-TAF-anti-TAF binding provided a measure of TAF in tissue extracts examined. Extracts of normal bovine retina, cornea, lung, aorta, lymph nodes, iris, vitreous humour, and human tumours and normal human pituitary and liver were assayed. Only bovine retina and human tumours were found to contain angiogenesis factor. These findings, together with our earlier results, suggest that angiogenesis factor from both bovine retina and human tumours induce angiogenesis in vivo and possess common antigenic determinants. The presence of angiogenesis factor in healthy retina and its relationship to neovascularisation in clinical conditions is discussed.
Angiogenesis Inducing Agents↗
The nonluteal porcine ovary as a source of angiogenic activity.
Nonluteal ovarian tissue extracts were tested for their ability to stimulate migration and proliferation of cultured bovine capillary endothelial cells. Stimulation of migratory and proliferative activity was found in the 105,000 X g supernatant of homogenates of either whole nonluteal porcine ovaries or isolated theca (follicular walls). Maximal proliferative and migratory activity was obtained with 1-10 micrograms homogenate protein. Neither follicular fluid nor extracts of granulosa cells showed significant activity when tested over a broad concentration range. The proliferative and migratory activities were associated with a heat-labile, nondialyzable, protease-sensitive fraction that was soluble in 40%, but not 60% (NH4)2SO4. The tissue extracts that effectively stimulated growth and migration of capillary endothelial cells in vitro also stimulated the formation of new capillary blood vessels in an angiogenesis assay performed on chick chorioallantoic membranes.
Allantois↗
Restorative chemotherapy in degenerative hip disease.
Twenty patients with degenerative joint disease (DJD) of the hip were treated for prolonged periods with an extract of cartilage and bone marrow. Although clinical improvement, lessened joint pain and increased function, occurred in a majority of the patients, the most significant changes were in four who had concomitant x-ray improvement. These patients had roentgenographic evidence of recovery of joint space and regeneration of the femoral head. Discussion is presented of possible mechanisms by which this tissue extract induces changes in chondrocyte metabolism. A theory is proposed to explain DJD as a multifactorial disorder in which liver impairment in processing growth hormone is the key malfunction. Treatment of DJD of the hip by injections of the tissue extract of cartilage and bone marrow is a rational and effective therapy without significant toxicity.
Aged↗
Abundant expression of immunoreactive endothelin 1 in mammary phyllodes tumor: possible paracrine role of endothelin 1 in the growth of stromal cells in phyllodes tumor.
Immunoreactive endothelin 1 (irET-1) concentrations were measured in extracts prepared from 4 phyllodes tumors and 14 fibroadenomas. irET-1 was detectable in all tissue extracts by specific radioimmunoassay, and the mean concentration of irET-1 was 18-fold and 27-fold higher in tissue extracts from phyllodes tumors than in those from intracanalicular fibroadenomas and pericanalicular fibroadenomas, respectively. Reverse-phase high-performance liquid chromatography coupled with radioimmunoassay in the extracts from phyllodes tumors revealed one major irET-1 component corresponding to human standard ET-1. Furthermore, immunocytochemical staining for ET-1 revealed that numerous ET-1-immunoreactive cells were seen in the epithelial cells but not in the stromal cells, suggesting that ET-1 is synthesized by the epithelial component of phyllodes tumors. A possible paracrine role of ET-1 in the growth of this rare tumor which is characterized by its prominent stromal cellularity is discussed.
Adenofibroma↗
Production of melanoma-associated antigen(s) by a defined malignant melanoma cell strain grown in chemically defined medium.
A human malignant melanoma cell strain, UCLA-SO-M14 (M14), was adapted to grow in serum-free, chemically defined medium (CDM). The 3 M KCl extract prepared from the CDM-grown cells (M-14-CDM) was assayed against leukocytes from melanoma patients, patients with other cancers, and normal donors by leukocyte migration inhibition (LMI). The leukocytes from 15 to 27 (56%) melanoma patients tested were LMI positive. In contrast, 4 of 18 (22%) other cancer patients and 5 of 30 (17%) normal donors leukocytes were LMI positive. One of 14 melanoma patients' leukocytes were LMI positive for a control 3 M KCI extract from autologous muscle. Comparative studies were performed with the M14-CDM extract and a 3 M KCI extract from a freshly biopsied tumor specimen from the donor of the M14 cell strain. Seven of 12 (58%) melanoma patients' leukocytes were LMI positive to the M14-CDM extract, but only 2 of 12 (17%) were LMI positive to the autologous melanoma tissue extract. Furthermore, only 100 to 300 mug protein of M14-CDM extract were required to educe delayed cutaneous hypersensitivity response in 6 of 8 (75%) melanoma patients and 0 of 5 lung cancer patients, but 500 mug protein from biopsied autologous melanoma tissue extract were needed to produce delayed cutaneous hypersensitivity response in 24 of 42 (57%) melanoma patients and 7 of 28 (25%) nonmelanoma cancer patients. These data suggest: (a) the M14-CDM cells synthesized melanoma-associated antigen(s) (MAA) in CDM; (B) the 3 M KCI extraction procedure effectively removed the MAA from the M14-CDM cells; (c) the M14-CDM cells were a more potent source of MAA than the surgical autologous melanoma specimen; and (d) the M14-CDM cells provided a continuous source of standard MAA.
Antigens, Neoplasm↗
Determination of serotonin and bufotenin as their dansyl derivatives.
A method is described which permits the determination of serotonin and bufotenin in the same tissue sample. It comprises the following steps: (a) tissue extraction with acetone-0.1 M hydrochloric acid (19:1); (b) reaction of the tissue extract with dansyl (Dns) chloride; (c) pre-separation of O-Dns-bufotenin from O,N-bis-Dns-serotonin and other Dns-amides on a small silica gel column (this step is dispensable if only serotonin or bufotonin is being determined); (d) TLC separation of O-Dns-bufotenin and O,N-bis-Dns-serotonin from other Dns derivatives; (e) quantitative evaluation of the separated compounds by fluorimetry for O-Dns-bufotenin and by fluorimetry or mass spectrometry for the serotonin derivative. The photometer response is linear within the range 0.1-300 nmole. With the mass spectrometric method, 2 pmole of O,N-bis-Dns-serotonin could be determined with a standard deivation of +/-9%. The recovery of the amines from tissue was better than 85%. Reserpine treatment of toads caused a concomitant decrease in serotonin and bufotenin in the brain, but not in the skin of the animals. Repletion of bufotenin in the brain occurs at a higher rate than the repletion of the serotonin pool.
Animals↗
Novel rapid tissue lysis method to evaluate cancer proteins: correlation between elevated Bcl-X(L) expression and colorectal cancer cell proliferation.
AIM: We optimized a rapid and efficient tissue lysis method using the MagNA Lyser (Roche, Germany). Using this novel method combined with immunoblot analysis, we investigated the correlation between abnormal Bcl-X(L) expression and clinicopathological characteristics in colorectal cancer. METHODS: Tissue samples from Sprague-Dawley rats were tested to determine optimal lysis conditions for use with MagNA Lyser. We next used the new method to extract tissue proteins from the tumor tissue of a colorectal cancer patient. The availability of extractable tissue proteins for proteomic study was demonstrated by two-dimensional (2D) gel electrophoresis and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In addition, we prepared tissue lysates from paired tumor tissues and adjacent nontumor tissues of 50 colorectal carcinoma patients. Ensuing immunoblot analyses were performed to detect the level of Bcl-X(L) expression. RESULTS: The optimal sample sizes processed were found to be around 200 mg, with oscillation frequency of 6500 r/min for 80 s. Test of the first human tissue lysate confirmed that the MagNA Lyser method was adequate for protein extraction and subsequent identification by current proteomic protocols. The method was also applicable to immunoblot analysis. Thirty of 50 (60%) colorectal patients exhibited higher level of Bcl-X(L) expression in their tumor tissues. Raised level of Bcl-X(L) expression correlated with patients' gender and tumor cell proliferation index (P = 0.037 and P<0.001, respectively), but was independent of clinicopathological characteristics and overall survival. CONCLUSION: We report a novel tissue lysis method applicable to proteomic and immunoblot analyses, which can facilitate the discovery and detection of cancer protein alterations.
Animals↗
[Stimulation of post-traumatic regeneration of rat spleen under conditions of gravitational stress].
Post-traumatic regeneration of the rat spleen was studied after resection of half the organ, under gravitation overloading (11 units) using spleen tissue extract prepared by Filatov's method. Gravitation overloading caused a decrease in the size of the spleen nodules, smooths their contours, increases the red pulp infiltration by the lymphocytes, reduces the number of labeled cells and the intensity of the label in the reactive centers of the spleen nodules, decelerates the capsule formation in the resection area. Application of the stimulant normalized the structure of white pulp, increased the number of labeled cells, and accelerated the capsule formation. The tissue extract used in gravitation overloading brought the restoration process nearer to the usual course of the posttraumatic spleen regeneration (by the character and periods of tissue differentiation development).
Animals↗
Analysis of excretory-secretory and somatic antigens of Gastrothylax crumenifer.
The excretory/secretory (ES) metabolic products released by Gastrothylax crumenifer (Trematoda: Digenea) during in vitro incubations and the somatic extract of the adult parasite were analysed using polyacrylamide gel electrophoresis (PAGE). Immunogenicity of ES and somatic extracts were evaluated by immunoblotting and ELISA using sera raised against ES and somatic antigens in rabbits. The electropherograms of ES and somatic extracts have been resolved into 38 and 41 polypeptides, respectively. The apparent molecular weights of these polypeptides range from < 29 to > 205 kDa. A total of 14 polypeptides were found to be common to both of the samples. The analysis of immunoblot results revealed 22 and 27 antigenic polypeptides in ES and somatic extracts respectively. Only 11 and 13 antigenic polypeptides were found specific to ES and tissue extract respectively. The molecular weights of these specific polypeptides were calculated to be < 14.4, 16, 20, 25, 33, 42, 119, 125 and > 205 kDa for metabolic products and < 14.4, 25, 30, 35, 78, 84 and > 205 kDa for the tissue extracts, respectively. Analysis of ELISA results revealed that a dilution of up to 1:3200 of the test sera could react with the ES product. Further, when the ES antigens were allowed to react with antisomatic extracts in hyperimmune sera the titre of IgG increased up to a dilution of 1:12800. The potential importance of these antigens in the immunodiagnosis of amphistomiasis is discussed.
Journal Article↗
Analysis of excretory-secretory and somatic antigens of Gastrothylax crumenifer.
The excretory/secretory (ES) metabolic products released by Gastrothylax crumenifer (Trematoda: Digenea) during in vitro incubations and the somatic extract of the adult parasite were analysed using polyacrylamide gel electro-phoresis (PAGE). Immunogenicity of ES and somatic extracts were evaluated by immunoblotting and ELISA using sera raised against ES and somatic antigens in rabbits. The electropherograms of ES and somatic extracts have been resolved into 38 and 41 polypeptides, respectively. The apparent molecular weights of these polypeptides range from <29 to > 205 kDa. A total of 14 polypeptides were found to be common to both of the samples. The analysis of immunoblot results revealed 22 and 27 antigenic polypeptides in ES and somatic extracts respectively. Only 11 and 13 antigenic polypeptides were found specific to ES and tissue extract respectively. The molecular weights of these specific polypeptides were calculated to be <14.4, 16, 20, 25, 33, 42, 119, 125 and > 205 kDa for metabolic products and <14.4, 25, 30, 35, 78, 84 and > 205 kDa for the tissue extracts, respectively. Analysis of ELISA results revealed that a dilution of up to 1:3200 of the test sera could react with the ES product. Further, when the ES antigens were allowed to react with antisomatic extracts in hyperimmune sera the titre of IgG increased up to a dilution of 1:12800. The potential importance of these antigens in the immunodiagnosis of amphistomiasis is discussed.
Animals↗
Purification of rat pro-atrial natriuretic factor: a simplified scheme using reversed-phase high-performance liquid chromatography.
A simple scheme for the rapid and efficient isolation of rat pro-atrial natriuretic factor (pro-ANF) has been developed. An isolated rat adrenal cell bioassay for ANF was established to optimize heart tissue extraction and chromatography conditions. This assay is based on the ability of ANF to inhibit angiotensin II-stimulated aldosterone secretion. IC50 values for ANF were approximately 320 pM. The protocol that was established consisted of extraction of rat atria in 5 N acetic acid containing protease inhibitors. The extract was lyophilized, resolubilized in 0.1% trifluoroacetic acid containing 1% (w/v) sodium chloride, and subjected to RP-HPLC. Extraction of small batches of atria (i.e., from 10 or 20 rats) resulted generally in a yield of 2 nmol per rat (i.e., approximately 30 micrograms). The identity and purity of the pro-ANF were confirmed by the determination of both the amino acid composition and the amino-terminal sequence. Purified pro-ANF was radioiodinated and the efficiency of the extraction and purification procedure was assessed by adding labeled peptide to the initial tissue extract. The structural integrity and overall recovery of radioactivity were determined by RP-HPLC. The purification scheme provides undamaged pro-ANF of high purity. Purified pro-ANF was compared with synthetic rat ANF in the rat adrenal glomerulosa cell and isolated rat aortic strip bioassays. The peptides were apparently equally active in the adrenal cell system and approximately threefold less potent in relaxing aortic strips. The apparent equipotency in the adrenal cell bioassay may be due to the conversion of pro-ANF to ANF-like peptides during the bioassay incubation.
Aldosterone↗