Biomedical subjects
Publications and source records attributed to W Staib.
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Powdered calf uteri were lyophilized and sealed in a vacuum. The effects of lyophilization and the thermostability of the binding activity of uterine steroid hormone receptors were studied. The lyophilized powder was analyzed for its ability to bind estrogen, progestin, androgen, and glucocorticoid steroids. Lyophilization had no deleterious effects on the binding activity. Storage of the lyophilized powder at +20 C for more than 14 days did not reduce the binding activity. No reduction of the binding capacity was also observed when the storage temperature was at +30 C for 16 hours. However, a slight loss of specific binding sites was observed for 17 beta-estradiol (-12%) at the storage temperature of +40 C, but the other steroid binding proteins were more stable. When the storage temperature was at +60 C for 16 hours, a loss of about 24% was observed for the specific estrogen binding sites, whereas the binding capacity of progestin, androgen, and glucocorticoid receptors was reduced by only less than 10%. Storage at the high temperature of +80 C for 16 hours resulted in a loss of only 36% for the estrogen binding capacity and less than 30% loss for the other binding proteins. The freeze-dried uterine powders can be useful quality control specimens for steroid receptor assays.
The binding of methyltrienolone (R 1881, 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one), a highly active synthetic androgen, by cytosol preparations from human renal cell carcinoma was investigated. High-affinity, low-capacity binding components for R 1881 were detected in 3 out of 8 tumours analysed. The apparent dissociation constant of the R 1881-binder complexes was found to be in the range of 1.1-2.3 x 10(-9) mol/l. The number of binding sites in the positive tumours varied from 2.1 to 9.7 fmol/mg cytosol protein. Studies of binding specificity indicated a requirement for androgens. It is concluded that these binding components may be hormone receptors. If in consecutive studies this hypothesis holds true, progestins, most widely used in hormonal therapy of metastatic renal cancer, may possibly act on the tumour tissue by inhibiting the secretion of gonadotropins, thus lowering the blood levels of eventually growth-promoting.
In normal human liver from adult males and females cytoplasmatic components which bind dexamethasone specifically and with high affinity, were demonstrated via dextran-coated charcoal assay, agar gel electrophoresis, isoelectric focusing and sucrose gradient centrifugation. The apparent dissociation constant of the dexamethasone-binder complex was found to be 1.7 +/- 0.3 x 10-(8) mol/l. The binding capacity was limited to 67.5 +/- 5.3 fmol/mg of cytosol protein. The ligand specificity for binding to these components indicated the requirement for glucocorticoids. It was shown by means of agar gel electrophoresis that the dexamethasone-binding entities migrate to the receptor region of the gel. The isoelectric point of the binding components in human liver cytosol was found to be pH 6.3. Sedimentation in sucrose gradients revealed the bulk of these components to be in the 7S region. It is concluded that the specific dexamethasone-binding entities in human liver have all the properties of glucocorticoid receptors.
Metastases of human carcinoid tumors of bronchial and gastric origin have been analysed for glucocorticoid receptors using the fluorinated compound [3H] dexamethasone. In both tumors high concentrations of glucocorticoid-binding components were detected which based on several physicochemical characteristics were identified as glucocorticoid receptors in nature. The apparent dissociation constant of the dexamethasone-binder complex was found to be 1.8 x 10(-8) mol/l for the bronchial and 2.8 x 10(-8) mol/l for the gastric carcinoid tumor. Sedimentation in sucrose gradients disclosed the binding components to be macromolecules, sedimenting at about 7.S. By agar gel electrophoresis the molecular species binding dexamethasone were found to exhibit the same electrophoretic mobility as glucocorticoid receptors from well-known target organs. The ligand specificity for binding to these components indicated a requirement of active glucocorticoids as well as anti-glucocorticoids. A regulatory function of glucocorticoids in serotonin biosyntheses is discussed, which may be of potential clinical significance.
Based on the potential sensitivity of the growth of human renal cell carcinoma, tumour tissue from 88 patients was analysed for progestin receptors. With the dextran-coated charcoal assay, cytoplasmic components which bound the potent progestin R 5020 specifically and with high affinity could be detected in 42% of the carcinomas. The apparent dissociation constant of the R-5020-binder complex amounted to 3.1 +/- 1.2 X 10(-8) mol/l. Sedimentation in sucrose gradients revealed the bulk of these components to be in the 4-S region. The ligand specificity for binding to these sites indicated a requirement for progestins. Despite the enormous case material investigated, we were unable to confirm the presence of the high receptor concentrations reported in the literature.
In an attempt to elevate the concentration of progestin-binding components in human renal cell carcinoma, 24 patients were treated prior to tumour nephrectomy with various doses of oestrogens, potent inducers of progestin receptor synthesis. Low-dose oestrogen treatment was found to be insufficient to significantly induce progestin-binder synthesis in the tumour tissue. In the carcinoma of patients receiving high-dose oestrogen treatment, the average concentration of R-5020-binding sites did not differ from that of the low-dose group. However, 2 out of these patients (12.5%) exhibited R-5020-binder concentrations in the tumour tissue which exceeded the highest quantities of progestin-binding components found in 88 untreated controls previously analysed in our laboratory.
Cytosol preparations obtained from 9 different human renal cell carcinomas were investigated for the eventual presence of progestin-binding inhibitors which might offer an explanation for the previous failure to demonstrate high receptor levels in the tumour tissue. The inhibitory potency of these preparations was estimated by measuring the decrease of R 5020 binding to uterine progestin receptors in the presence of tumour cytosol. Interestingly, cytosol from human renal cell carcinoma was found to contain progestin-binding inhibitors. The average inhibition of R-5020-receptor interaction amounted to 46%. In only 1 out of 9 cases, binding was completely suppressed. In order to circumvent the inhibitory reaction potentially occurring in cell-free systems, R-5020-binding studies additionally were performed in cell suspension. Out of 7 carcinomas studied using this assay system, 5 did not contain any specific progestin-binding entities. In 1 tumour, receptor-atypical non-saturable binding was observed. Only in isolated cells prepared from 1 other carcinoma, could slight indications for the presence of low concentrations of progestin receptors be detected.
In order to investigate whether progestins may trigger tumour regression by a mechanism involving the glucocorticoid receptor, human renal cell carcinomas obtained from 15 patients were analysed for cytoplasmic glucocorticoid-binding components, using [3H] dexamethasone. The existence of glucocorticoid binders could be demonstrated in 10 out of 15 tumours studied. The average binding capacity was calculated and found to be 7.1 fmol/mg of cytosol protein. The apparent dissociation specificity experiments clearly cell-free system amounted to 1.9 X 10(-8) mol/l. The ligand specificity experiments clearly indicated that binding to these receptors is not restricted to glucocorticoids alone. Progesterone and aldosterone turned out to be moderate competitors for dexamethasone binding. Medroxyprogesterone acetate, the compound widely used in hormone therapy of advanced renal cancer in man, was demonstrated to be one of the strongest inhibitors of [3H] dexamethasone. It is concluded that binding of medroxyprogesterone acetate to glucocorticoid receptors might represent the primary mechanism of action of the compound in causing tumour regression.
An enzymatic procedure for the isolation of metabolically active tumour cells from human renal cell carcinoma is described. The cells were suspended by a multistep incubation procedure of the tissue in the presence of collagenase (10 mg enzyme/g tumour wet weight) dissolved in a calcium-free buffer solution. About 90% of the isolated tumour cells were viable, as judged by routine trypan blue staining. Electron microscopic examination revealed tumour cells in various stages of dedifferentiation. The cells had retained their capability of protein synthesis. In short term experiments the effects of 17 beta-oestradiol and progesterone on the incorporation of [U - C] L-leucine into cellular proteins was studied; Progesterone was found to exhibit a slight tumour antianabolic or catabolic action. A 17 beta-oestradiol-dependent modulation of the rates of protein synthesis was not observed.
Hydrolysis of 3H(G)-digitoxin by hydrochloric acid and human gastric juice is described. Incubation temperatures of 37 degrees C and 22 degrees C were chosen for electrolyte solutions, and 37 degrees C for the experiments with gastric juice. After dichloromethane extraction, the radioactive metabolites were separated by thin-layer chromatography, localized and quantified by a radiochromatogram scanner. The degradation products separated by TLC were digitoxigeninbis-digitoxoside, digitoxigenin-mono-digitoxoside, and digitoxigenin. After 10 min of incubation at pH 1, digitoxin amounted to 38.6% of the total radioactivity in electrolyte solution and 37.6% in gastric juice. After 60 min, the percentage of digitoxin decreased to 5.9% and 0%, respectively. After 120 min at 22 degrees C, amounts of unhydrolyzed digitoxin were: at pH 1 55%; and at pH 2 84%. After 10 min at pH 1, and 60 min at pH 2, digitoxin was hydrolyzed to such an extent that bioavailability should have been significantly reduced. Absorption of digitoxin (liquid) from the stomach was studied during gastroscopy in five patients. Among them, significant differences in absorption kinetics and bioavailability existed, as revealed by radioimmunological measurements of the digitoxin blood levels.
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Glucose metabolism of lymphoid cells isolated from thymus and spleen of Wistar rats, weighing 140-150 g, was found to be sensitive to insulin. The influence of insulin on glucose uptake by isolated cells from both lymphoid tissues displayed a significant sex specificity. Insulin at 0.1-10 nmol/1 stimulated glucose uptake of cells from male rats, whereas in cells from females, inhibition of glucose uptake was observed. However, lactate production was enhanced in lymphoid cells of both sexes. Cortisol (100 nmol/1) displayed a significant anti-insulin action on glucose uptake and lactate release by thymocytes of male rats. In contrast, in cells from females, cortisol and insulin both exhibited an inhibitory action on glucose uptake, whereas the hormones were found to antagonise lactate production. The sex specific stimualtory influence of insulin on glucose uptake by thymocytes of male rats was reversed and the hormone became inhibitory when animals were castrated. The action of insulin on glucose uptake was also shown to be age-dependent. In experiments with rats weighing either about 10 g or 40 g, sex-specific effects on glucose influx were found, that were similar to those of rats weighing 140-150 g. However, stimulation of glucose uptake was found with thymocytes from rats of both sexes weighing about 80 g. Comparable results were obtained with isolated thymocytes from immature humans (2 months-10 years old). Incubation of thymocytes from males with insulin (10 nmol/1) stimulated glucose uptake and lactate release, whereas insulin caused an inhibition of glucose uptake and an enhancement of lactate production in thymocytes from female. Age dependence and sex specificity of insulin action on glucose metabolism in lymphoid cells and tissues may explain the contradictory results reported by other authors.
A model of a pulsatile cardiovascular system is described including an electronic control, a hydraulic driving unit, a volume storage, a peripheral resistance and measuring equipment. Six different artificial heart valves, a designed aortic valve (Aachen pockett valve II) and a human aortic valve have been tested in the outflow position of the pump. Three of them are presently being used in clinical applications. Measurements of pressure and volume flow have been obtained for different frequencies n, pump volumes Vp and time ratios between pressure and suction phase. From the experimental results a comparative evaluation was deducted for different working conditions. In comparison to natural human aortic valve (NK) large volume flow is verified by the Aachen pocket valve II (AT), the Gott-Dagget value (GD), the Starr-Edwards valve (SE) and the Lillehei-Kaster valve (LK). The Gott-Daggett valve has the smallest back flow volume. The Smeloff-Cutter and in particular the Wada-Cutter valve have the largest regurgitation. This is to be expected since the Cutter valves are purposely so designed that they do not close tightly in order to avoid blood sedimentation. The human aortic valve, the Gott-Daggett valve and the Aachen pocket valve demonstrate the most favourable opening-closing characteristics. The human aortic valve achieves a negligible pressure loss, followed by the Björk-Shiley and the Lillehei-Kaster valve.
In normal human kidney from adult males cytoplasmic components which bound 17beta-estradiol specifically and with high affinity were demonstrated by dextran-coated charcoal assay, sucrose gradient centrifugation and agar gel electrophoresis. The dissociation constant of the oestradiol-binder complex amounted to 2.2 +/- 0.1 x 10(-9) mol/l. The binding capacity was limited to 34.0 +/- 9.7 fmol/mg of cytosol protein. Sedimentation in sucrose gradient revealed the bulk of these components to be in the 4S region. The binding entities could be clearly separated from sex hormone-binding globulin by agar gel electrophoresis. The ligand specificity for binding to these components indicated a requirement for oestrogens. The fact that an excess of aldosterone had no competitive effect on oestradiol binding suggests that the oestrogen-binding sites are independent of mineralocorticoid receptors. It is concluded that the specific binding components in human kidney have the properties of oestrogen receptors.
Specific binding of [3H]oestradiol-17beta by the cytosol fraction of human renal cell carcinoma was studied. The binding reaction displayed marked ligand specificity and high affinity of binding. Unlabelled oestradiol, oestriol and oestrone inhibited the binding of [3H]oestradiol-17beta to the cytosol binding sites, wehereas all other steroids tested turned out to be only weak or insignificant competitors for the oestrogen binding sites. Scatchard analyses suggested the existence of a single class of binding sites. The dissociation constant of the oestradiol-binding complex was found to be 2.51 +/¿.75 x 10(-9) mol/l. The number of binding sites was limited (17.5 +/- 3.8 fmoles per mg of cytosol protein). Sucrose gradient centrifugation revealed these binding components to be macromolecules either displaying a complex sedimentation pattern (peaks at 3.5 S, 4S, 5.7S and, in addition, high molecular weight aggregates) or sedimenting in the 4S region alone. By agar gel electrophoresis it could be demonstrated that the oestradiol-inding components migrated into the receptor region of the gel. Binding of [3H]oestradiol-17beta to these entities was markedly reduced, when the cytosol was heated (60 min at 45 degrees C) prior to the reaction with the labelled hormone. Since the specific binding components exhibit properties of oestradiol receptors in target tissues, a direct effect of oestradiol on human renal cell carcinoma is suggested.
By perfusion of the isolated human liver with collagenase and hyaluronidase a mixed suspension of various cell types was obtained. Pure parenchymal cells were prepared by differential centrifugation, pure non-parenchymal cells by the use of pronase and subsequent isopycnic centrifugation on metrizamide gradients (50-300 g/l). About 90% of the parenchymal and non-parenchymal cells were viable as judged by trypan blue staining. Non-parenchymal cells were not capable fo gluconeogenesis but at high rates. Parenchymal cells retained their ability to form glucose and to accumulate glycogen from fructose greater than lactate/pyruvate greater than alanine. Studies on binding of 125I-labelled insulin by isolated parenchymal cells were performed at 30 degrees C. The binding data may fit with a minimum of two classes of binding sites: (a) high affinity--low capacity sties (Kd approximately 6.6 nmol/l, capacity approximately 16 000 insulin molecules per cell) and (b) low affinity-high capacity sites (Kd approsimately 0.37 mumol/l, capacity approximately 646 000 molecules per cell).
The release of amino acids by skeletal muscle was studied in the isolated perfused rat hindquarter. Adrenalectomy depressed the formation of glutamine and alanine as well as the efflux of all other amino acids measured. Betamethasone--a synthetic glucocorticoid--caused a significant increase in the efflux of nearly all amino acids up to the level of normal controls. The release of amino acids was also increased in perfused hindquarters of diabetic rats. On the other hand, insulin exhibited a depressing effect on the release of amino acids by hindquarters of normal rats. The metabolic integrity of the muscle tissue was proved by measuring creatine phosphate, ATP, ADP and water content as well as by the significant insulin effect on glucose uptake and on [14C]leucine incorporation into muscle proteins.