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PubMed1994

Gelatinase/type IV collagenases in the loosening of total hip replacement endoprostheses.

Gelatinase/Type IV collagenases, namely, 72 kDa matrix metalloproteinase (MMP)-2 type and 92 kDa MMP-9 type, were analyzed to evaluate the role of extracellular matrix degradation in 17 cases of loosening of total hip endoprostheses. Zymographic and densitometric analyses revealed elevated production of MMP-2 and induction of MMP-9 in tissue extracts from both the interface tissues between bone and implants and the pseudocapsular tissues around the loose endoprostheses when compared with those of 8 control noninflammatory knee synovial tissues. The level of MMP-9 was higher in the interface tissues than in the pseudocapsular tissues. MMP-9 activity was not detected in the control samples. Although differences in the type of prosthetic fixation (cemented versus cementless) or the type of alloy (cobalt-chromium-molybdenum versus titanium-aluminum-vanadium) existed, they shared a similar potential to stimulate tissues to produce MMP-2 and MMP-9. These findings suggest a role for MMP-2 and MMP-9 type gelatinase/Type IV collagenases in the degradation of extracellular matrix of periprosthetic tissues, where they may cause weakening of the connective tissue bed and the loosening of total hip replacement endoprostheses. The pseudocapsular tissues could contribute to the loosening via the production and release of matrix metalloproteinases into the synovial fluid. Alternatively, induction of matrix metalloproteinases in such tissues may reflect remodeling of the pseudocapsular connective tissues.

Aged↗
PubMed1996

Prognostic value of urokinase plasminogen activator and plasminogen activator inhibitor-1 in renal cell cancer.

PURPOSE: Urokinase type plasminogen activator and its inhibitor plasminogen activator inhibitor are associated with invasion and formation of metastases in tumors. In a prospective study urokinase and plasminogen activator inhibitor-1 content in renal cell cancer and benign renal tissue was correlated with the traditional factors of TNM staging and grading as well as ploidy and actual clinical outcome of the patients. MATERIAL AND METHODS: A total of 152 patients, who underwent transperitoneal tumor nephrectomy for renal cell cancer, was followed for a mean of 23.9 months. Urokinase and plasminogen activator inhibitor-1 from the tumor tissue and corresponding benign renal tissue were quantified from detergent extracted tissue samples (1% triton-X = 100 in TBS) and measured with an enzyme-linked immunosorbent assay. RESULTS: Urokinase content correlated with the development of distant metastases (log rank 4.32, p = 0.037). Cutoff value was 0.84 ng/mg. A group of 11 patients were considered to be at high risk for metastases (9 events) based on urokinase greater than 0.84 ng/mg., while 94 patients were considered to be at low risk (5 events) with urokinase less than 0.84 ng/mg. Plasminogen activator inhibitor-1 significantly correlated with the prevalence of distant metastases (log rank 5.17, MO, 10.04 versus M1, 23.79, p = 0.02) and the development of new metastases postoperatively (MO, 10.85 versus M1, 27.36, p = 0.001). Cutoff value was 12 ng/mg. protein. A group of 41 patients were considered at high risk for relapse (6) based on plasminogen activator inhibitor-1 greater than 12 ng/mg. protein compared to 55 patients with plasminogen activator inhibitor-1 less than 12 ng/mg. protein with only 1 relapse during followup. CONCLUSIONS: Plasminogen activator inhibitor-1, the specific inhibitor of urokinase is a strong and independent prognostic factor in predicting early relapse of renal cell carcinoma. High and low risk groups for disease-free survival can be discriminated by plasminogen activator inhibitor-1 antigen content in the tumor tissue.

Carcinoma, Renal Cell↗
PubMed1997

High level of urokinase-type plasminogen activator is a new prognostic marker in patients with gastric carcinoma.

BACKGROUND: Prognosis of gastric carcinoma is related to invasion and metastasis. Evidence has accumulated that invasion and metastasis in solid tumors require the action of tumor-associated proteases, which promote the dissolution of the surrounding tumor matrix and the basement membrane. The serine protease urokinase-type plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), appear to have a major function in these processes. Recent reports have demonstrated that expression of these proteolytic enzymes is elevated in breast and colon carcinoma and that it can be associated with invasiveness and poor prognosis. Therefore, the authors evaluated whether the expression and activation of uPA and PAI-1 might be of clinical value as a tumor/biologically defined risk factor in patients with gastric carcinoma. METHODS: Enzyme-linked immunoadsorbent assays were used to test for uPA antigens and PAI-1 in tissue extracts of normal and cancerous tissue from 160 gastric carcinoma patients who were enrolled in the Yonsei Cancer Center Study Group. RESULTS: Both uPA and PAI-1 levels were significantly higher in cancerous tissues than in normal tissues (uPA: 9.4 +/- 8.7 vs. 5.3 +/- 3.1 ng/mg protein cytosol; PAI-1: 10.9 +/- 9.1 vs. 5.8 +/- 2.9 ng/mg protein cytosol), (P < 0.001, respectively). Both high uPA and PAI-1 levels were associated with differentiation of the tumor (P = 0.04 and P = 0.004, respectively), and a high PAI-1 level was associated with lymph node metastasis at an advanced stage (P = 0.003 and P = 0.04, respectively). There was a correlation between the levels of uPA and PAI-1 expression in cancerous tissues (correlation coefficient = 0.57). In univariate analysis, a high level of uPA or PAI-1 was associated with a short relapse free survival, but in multivariate analysis only a high level of uPA was an independent prognostic parameter for a short relapse free survival for gastric carcinoma patients. CONCLUSIONS: These data indicate that uPA is a new independent variable for the identification of high risk gastric carcinoma patients. Therefore, therapy targeting uPA can be applied as a new biologic treatment modality for these individuals.

Adult↗
PubMed1983

Species and tissue distribution of cholecystokinin/gastrin-like substances in some invertebrates.

Twenty-six species of invertebrates representing eight phyla were surveyed for the presence of cholecystokinin/gastrin-like (CCK/gastrin-like) peptides by radioimmunoassay of various tissue extracts. This is the first report of the presence of CCK/gastrin-like peptides in representatives of the phylum Ectoprocta, the arthropodan classes Crustacea and Merostomata, and in the nervous systems of the gastropod mollusc Aplysia californica and the oligochaete annelid Lumbricus terrestris. It has been proposed that CCK/gastrin evolved in the invertebrates as a neural peptide and was subsequently exploited by the vertebrates as a regulatory peptide in both the nervous system and the gastrointestinal endocrine system. The present results indicate that some gastropod molluscs, a merostomatan arthropod, and an annelid have detectable CCK/gastrin in both nervous and gut tissue. However, extractable CCK/gastrin was found only in gut tissue and not in the central nervous system of a crustacean arthropod. The tissue origin of the extracted CCK/gastrin in Bugula (phylum Ectoprocta) was not determined. Final resolution of the question of the nervous versus gut endocrine cellular origin of CCK/gastrin in invertebrates awaits further investigation. CCK/gastrin-like peptides are widely distributed among the invertebrates, which thus provide a rich source of comparative material for study of these regulatory substances.

Animals↗
PubMed1994

A liver factor increasing glucose uptake in rat hindquarters.

The effect of the liver on glucose uptake in muscle was studied in the isolated rat hindquarter, perfused with and without an isolated rat liver included in a recirculating system. Glucose uptake in the hindquarter was 0.75 +/- 0.10 mumol.min-1 x 100 g bw-1 (mean and SEM), and increased to 1.30 +/- 0.12 during the period when the liver was included in the perfusion (N = 17; p = 0.0001). In experiments where tissue extracts were added to the perfusate during the second period, raw liver extracts increased the glucose uptake from 0.87 +/- 0.11 to 1.25 +/- 0.10 (N = 6; p = 0.003). Deproteinized liver and kidney extracts increased glucose uptake similarly. When the liver and hindquarter were perfused together during the first period, the glucose uptake in the hindquarter was 1.55 +/- 0.16 mumol.min-1 x 100 g bw-1 and decreased gradually during the period of isolated perfusion to 1.27 +/- 0.16 (N = 4; p = 0.15). In control experiments where tissue free extract fluid was added to the perfusate during the second period, or where the hindquarter was perfused either alone or with the liver during both periods, glucose uptake decreased slightly from 0.87 +/- 0.18 to 0.80 +/- 0.13 (N = 9; p = 0.2). In conclusion, a factor from the liver may increase the glucose uptake in muscle tissues. This factor is present in extracts from the liver and kidney, it is probably not a protein, but its nature is not known. It is speculated that glucose intolerance in patients with liver disease may be related to a lack of this factor.

Animals↗
PubMed1988

Inositol is a required nutrient for keratinocyte growth.

Bovine hypothalamus is known to contain a growth-promoting activity for human epidermal keratinocytes. By sequential purification, the substance was isolated and found to be myo-inositol. The identity of the substance as myo-inositol was confirmed by ion modified partition, gas liquid, thin layer chromatography, by mass spectrometry, and quantitative bioassay. The inositol content of the crude hypothalamic extract and of an active acetone precipitate (the first step in the purification) was determined to be sufficient to account for their observed bioactivity. At an optimal concentration of 55 microM (10 micrograms/ml), myo-inositol approximately tripled keratinocyte yield compared to paired cultures in basal medium containing 0.3 microM, although this yield was only half that produced by a crude saline extract of hypothalamus, suggesting that there are additional growth-promoting activities in the tissue extract. No other skin-derived cell type tested was stimulated by supplemental inositol. These results establish that the inositol requirement for cultured human keratinocytes is markedly higher than for any other normal or malignant cell type investigated to date, and expand the list of brain-derived phospholipid precursors known to stimulate epithelial proliferation in vitro. These data suggest that inositol may subserve quantitatively or qualitatively different functions in the keratinocyte than in other cell types.

Cells, Cultured↗
PubMed1987

Tissue plasminogen activators in breast cancer.

Increased levels of tissue fibrinolytic activity have been detected in some malignant tumours and they have been implicated in metastatic spread. We have investigated tissue plasminogen activator (tPA) and urokinase (UK) in 26 breast carcinomas and 13 benign breast biopsies. Tissue extracts were analysed for overall fibrinolytic activity on fibrin plates and by fibrin-overlay zymography after electrophoresis on SDS-PAG. Supernatants of the extracts were analysed by an antigenic immunoassay (ELISA) and a functional bioimmunoassay (BIA) using polyclonal antibodies. Total ELISA and BIA results correlated (P less than 0.001) and all the tissues contained similar tPA levels. Malignant extracts contained significantly increased UK compared with benign extracts (1.60 +/- 0.37 iu, 0.36 +/- 0.16 iu; P less than 0.002). Zymography showed no high molecular weight inhibitor complexes and UK was almost exclusively confined to the malignant tissues (P much less than 0.02). The results suggest that malignant transformation of breast tissue is associated with the significantly increased production of UK. This may be responsible for the characteristics of malignancy or it may be a growth factor.

Breast Neoplasms↗
PubMed1968

Rapid, sensitive spectrophotometric method for quantitative determination of sulfatides.

A rapid, sensitive spectrophotometric method for the analysis of sulfatides is described. The assay method is based upon the formation of a colored complex between the cationic dye azure A and (anionic) sulfolipids; the complex is extractable by a solution of chloroform-methanol 1:1. With the exception of some of the phospholipids, the reaction is specific for the sulfolipids. Cardiolipin was the most reactive of the non-sulfolipid compounds that were tested. Except for sphingosine and the mono- and dihexose derivatives of sphingosine, a wide variety of lipids did not interfere with the formation of color by standard sulfatides. Sulfur-containing amino acids, sugar sulfates, and sulfated polymers neither produced color nor interfered in its formation by sulfatide. Chloride and nitrate ions produced relatively little color. The method is much more sensitive (lower limit, about 0.002 micromole) than most methods currently employed for the analysis of sulfatides and has a high degree of precision. It is applicable to the analysis of sulfolipids in tissue extracts and gives values similar to those obtained by previously published procedures. The reliability of the method is increased when it is applied to partially purified lipid solutions. Sulfatides added to extracts of rabbit brain tissue were quantitatively accounted for by the assay. In addition, the method can be applied to samples obtained directly from thin-layer plates.

Animals↗
PubMed1972

Serotonin and inhibition in Limulus lateral eye.

The response to light of one ommatidium is reduced or suppressed by simultaneous illumination of neighboring ommatidia. The mechanism of this lateral inhibition may be chemical synaptic transmission, based on the physiological findings of a number of investigators and on the following evidence. The fine structure of the neuropil of the lateral plexus exhibits numerous clear vesicles (ca. 400 A), dense-core vesicles (ca. 700-1400 A), Golgi regions, and other morphological features of neurochemical synapses. The indolealkylamine, serotonin (5-HT), even in nanomolar concentrations, has a potent inhibitory action. An initial, potent inhibitory dose of 5-HT produces a long lasting densensitization to subsequent doses. The desensitization affects lateral inhibition evoked by light stimulation of neighboring receptors, i.e. crossed-desensitization. Eye tissue extracts contain 5-HT and melatonin (MLT) at a level greater than 1 microg/g wet tissue and perhaps as high as 20-30 microg/g, as determined by two-dimensional thin-layer chromatography (TLC) and o-phthaldialdehyde fluorescence assay techniques. Subcellular fractionation on sucrose gradient indicates a peak in 5-HT and MLT content associated with an intermediate density fraction. 5-HT may be an inhibitory transmitter for lateral inhibition. One pathway for metabolism of 5-HT in the lateral eye may be via N-acetylserotonin and melatonin.

Animals↗
PubMed1982

Albumin concentration in normal and pathological human thyroids.

1. The concentration of thyroid albumin was determined in 54 normal and pathological human thyroid tissue specimens obtained at surgery from 34 subjects who had received 125I prior to thyroidectomy. 2. The saline-soluble proteins were fractionated by gel filtration on Sephadex G-200 and the absorbance of the effluent containing albumin and hemoglobin was measured at 280 and 414 nm. The serum albumin content of the blood contaminating the tissue extract was calculated on the basis of hemoglobin determination (A414) and hematocrit values. This "intravascular albumin" was present in all extracts studied but accounted for less than 10% of the total thyroid albumin in normal tissue and even less in pathological specimens. "Extravascular albumin" was taken as the difference between total and "intravascular" albumin. 3. The amount of extravascular albumin in normal tissue (N = 20), 14.9 +/- 1.9 mg/g thyroid, was significantly lower than in adenomas (hot nodules, N = 7, 32.0 +/- 4.1; cold nodules, N = 16, 24.0 +/- 2.7), papillary carcinomas (N = 9, 51.1 +/- 16.7) and Hashimoto's thyroiditis (N = 2, 36.1 and 176.3). 4. In vivo labelling by 125I of the albumin fraction was demonstrated by Sephadex G-200 gel filtration and by cellulose acetate electrophoresis. Although the ratio of concentration of total albumin/thyroglobulin determined by electrophoresis was similar for 4 normal (0.263 +/- 0.046) and 5 pathological tissues (0.268 +/- 0.059), the relative distribution of radioactivity (total albumin/thyroglobulin) was significantly higher for pathological tissue (0.091 +/- 0.007) than for normal tissue (0.052 +/- 0.023), P less than 0.05. 5. The data suggest that most of the extravascular thyroid albumin is found within the follicle, and demonstrate that there is a significant increase in the concentration of extravascular thyroid albumin associated with several thyroid disorders.

Albumins↗
PubMed1998

In vitro inhibition of human malignant brain tumour cell line proliferation by anti-urokinase-type plasminogen activator monoclonal antibodies.

A brain tumour-associated marker, urokinase (UK), was investigated using rabbit anti-UK polyclonal and murine anti-UK monoclonal antibodies, which were prepared by immunization with low molecular weight UK (LMW-UK) and high molecular weight urokinase (HMW-UK) synthetic peptide respectively. The polyclonal antibody cross-reacted with both LMW-UK and HMW-UK, whereas the murine MAbs were specific for HMW-UK. These immunological probes were used to study urokinase in glioma extracts, tissues, sera and cell lines that had been prepared from primary cultures of freshly dissected gliomas. Radioimmunoassays showed that glioma extracts had much higher level (5- to 44-fold) of UK than normal human brain extracts. This result was confirmed by immunoblotting of electrophoresis gels of glioma and human brain extracts. Immunohistochemical study using anti-UK MAb demonstrated much higher levels of UK in glioma tissue than normal brain tissue. Immunohistochemical study using anti-UK MAbs localized UK on the cell surface of glioma cells. Anti-UK MAbs inhibited the proliferation of AA cell lines and GB cell lines (50% to > 90%) and exerted minor effects (< or = 20%) on normal human liver, intestine and lymphocyte cell lines. Taken together, these results suggest that anti-UK MAbs may have therapeutic potential for human gliomas and cancer metastasis.

Antibodies, Monoclonal↗
PubMed1989

Steroidogenesis in Fundulus heteroclitus. II. Production of 17 alpha-hydroxy-20 beta-dihydroprogesterone, testosterone, and 17 beta-estradiol by various components of the ovarian follicle.

Fundulus heteroclitus prematurational follicles (1.3-1.4 mm) were dissected into various components and cultured in vitro to examine the type of cells involved in the synthesis of steroids upon F. heteroclitus pituitary extract (FPE) stimulation or addition of exogenous precursors (25-hydroxycholesterol or pregnenolone). Culture media and follicular tissue extracts were assayed for 17 alpha-hydroxy-20 beta-dihydroprogesterone (17 alpha-OH.20 beta-DHP), testosterone (T), and 17 beta-estradiol (E2) content using specific radioimmunoassays. Complete removal of the follicle wall (denuded oocytes) eliminated steroid accumulation induced by FPE treatment. Removal of the theca/epithelium layer (defolliculated oocytes) did not affect the steroidogenic response (17 alpha-OH,20 beta-DHP, T, E2 production) of the follicles to FPE or exogeneously added precursors (25-hydroxycholesterol or pregnenolone). Isolated theca/epithelium layers secreted only T. Isolated follicular preparations that did not contain the oocyte (theca/epithelium layers or follicle cells) secreted higher levels of steroids to the culture media than did intact follicles. We conclude from these results that (1) the follicle cells (granulosa cells) are the primary source of the various steroids produced by the F. heteroclitus ovarian follicle in response to FPE stimulation: (2) the synthesis of 17 alpha-OH,20 beta-DHP and E2 does not require the involvement of two cell types as shown in other teleosts; (3) the theca/epithelium layer is able to produce T but lacks the aromatase activity necessary for E2 synthesis; and (4) steroids synthesized in the follicle wall are both secreted to the medium and accumulated in the oocyte.

20-alpha-Dihydroprogesterone↗
PubMed1982

Lipoprotein lipase activity in human and guinea-pig placenta.

Human and guinea-pig placental activity was measured in tissue extracts and in medium in which tissue fragments have been incubated. Lipase activity was estimated from measured rates of release of 3H-labelled fatty acids from a 3H-labelled triacylglycerol substrate. Whole tissue lipase activity was 1.6 +/- 0.3 units/g (mean +/- SEM, mumol of unesterified fatty acid release/min) in the human, and 87.1 +/- 7.3 units/g in guinea-pig placental tissue. 70% of the human and 56% of the guinea-pig placental activity was lost on treating the tissues with cold acetone. Sodium chloride (0.75 M) decreased activity by 50% in extracts from both species. Lipase activity was released into media when placental fragments were incubated. The addition of heparin increased the release of lipase from human placenta but not significantly from that of the guniea-pig. Insulin had no effect on placental lipase activity from either species. It is concluded that the placentas of both species contain at least two lipases. One of these, which constituted a large of the total, had the characteristics of lipoprotein lipase.

Animals↗
PubMed1974

Antimitotic agents and tumor regression.

The selective action of nonantigenic tissue extracts on malignant cells was demonstrated using tissue culture techniques. The mitotic index of three strains of neoplastic cells was significantly reduced by the use of extracts which previously had proven effective in adversely affecting the development of spontaneous neoplasms. Under the same conditions the division rate of normal cells in tissue culture was not affected by the extracts. The malignant cells used were the Krebs 2, Sarcoma 180 and the L1210 leukemia. The normal cells were the L929 fibroblasts and epithelial-like cells from bone marrow and corneal tissue.

Animals↗
PubMed2003

Development and validation of a method for the confirmation of halofuginone in chicken liver and eggs using electrospray tandem mass spectrometry.

A method is described for the quantitative confirmation of halofuginone (HFG) residues in chicken liver and eggs. This method is based on LC coupled to positive ion electrospray MS-MS of the tissue extracts, prepared by trypsin digestion of the tissues followed by liquid-liquid extraction and final clean-up using Solid Phase Extraction (SPE). The [M+H](+) ion at m/z 416 is monitored along with four transitions at m/z 398, 138, 120 and 100. The method has been validated according to the draft EU criteria for the analysis of veterinary drug residues at 15, 30 and 45 microg kg(-1) in liver and 5, 15 and 50 microg kg(-1) in eggs. The new analytical limits, CCalpha and CCbeta were calculated for liver and were 35.4 and 43.6 microg kg(-1), respectively.

Calibration↗
PubMed1994

Endothelin in the ovine uterus during the oestrous cycle and early pregnancy.

Endothelin, which has potent vasoconstrictor and mitogenic actions, was measured by radioimmunoassay in tissue extracts of sheep endometrium and myometrium and was found to be present in similar amounts in both tissues during the oestrous cycle and in increasing amounts during the first 20 days of pregnancy (250-630 pg g-1 wet weight). Immunoreactive endothelin extracted from endometrium eluted at the same position as standard endothelin-1 on reverse-phase HPLC. Immunohistochemical techniques demonstrated that during the oestrous cycle endothelin immunoreactivity was very low in caruncular and intercaruncular stroma, luminal epithelium, outer and inner glandular epithelium, myometrium and blood vessels until after day 12 (oestrus: day 0). Staining increased in all but the inner glands to day 16 and the most intense staining was found in intercaruncular luminal epithelium and outer glands and in myometrium, although endothelin in tissue extracts did not change over this period. During early pregnancy (days 4-20), staining in intercaruncular areas and in myometrium increased slightly from day 4 to day 12 to a maximum which was maintained from day 15 to day 20. Intensity of staining in caruncles increased only from day 15, particularly in the epithelium. Immunoreactive endothelin was also present in the trophoblast cells of the embryo on day 20 of pregnancy. Strong endothelin immunostaining was observed in uteri from ovariectomized ewes, particularly in epithelial cells and in blood vessels. The intensity of immunostaining in epithelium and epithelial cells and in blood vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗