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Cultured endothelial cells from swine aorta possess the ecto-enzyme angiotensin-converting enzyme (E.C.3.4.15.1) in cell-associated form and also release it in soluble form into the culture medium. Using antibody to purified converting enzyme from swine kidney and incorporation of 3H-leucine, we examined the synthesis, degradation, and release of enzyme into the medium. 3H-leucine is incorporated into cellular converting enzyme, and later appears in the enzyme in the culture medium. The amount of cell-associated enzyme activity remains constant, and the amount of activity in the medium increases linearly during this period. These data show unequivocally that the appearance of enzyme activity in the cell culture medium is accompanied by synthesis of new enzyme protein. In pulse-chase experiments, the radioactivity disappeared from the cellular enzyme in two kinetic components with apparent half-lives of 1-2 hours and 20 hours, respectively. The appearance of the radiolabel in the medium enzyme corresponds very closely in rate and amount to the slow disappearance from the cells. There was no apparent uptake of labeled medium enzyme by the cells. The data suggest that the pool of active enzyme on the cell surface is the immediate precursor of the medium enzyme. The effects of culture conditions on the turnover of angiotensin-converting enzyme were also examined. The incorporation of 3H-leucine into both cell and medium enzyme was greater when cells were maintained in medium containing serum than when they were maintained in serum-free medium. The rate of degradation of the cellular enzyme was similar under the two culture conditions.
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Cultured chick pigment epithelial (PE) cells from stages 29 to 31 chick embryos grown in normal Eagle's minimum essential medium (MEM) exhibit marked colonial organization and differentiation. Individual cells appear epithelial and heavily pigmented. In contrast, cells grown in Ham's modified F-12 medium appear fibrocytic with colonial disorganization and little visible pigmentation. Biochemically, cells grown in F-12 medium lack receptors for both 3H-retinol and 3H-retinoic acid, although cells grown in MEM exhibit specific 3H-retinol binding. Pathways of glucose utilization differ significantly in cells grown in the two media, with considerably lower overall respiratory activity and lower pentose phosphate pathway activity seen with the F-12 medium. Thus different nutritional states can markedly affect PE cell characteristics in culture and possibly in vivo as well.
Variation in decimal reduction (D) time/value for attaining sterility assurance was reported in terms of difference between lot to lot and/or manufacturer to manufacturer of soybean casein digest (SCD) culture medium. The D value variation phenomenon itself due to SCD culture medium was reported by Graham et al; however, it was not sufficiently clarified what constituent(s) of SCD culture medium would mainly contribute to the variation of D value. If it is not well clarified, the reproducible sterility assurance cannot be successfully attained. The authors studied individually the constituent of SCD broth (SCDB), SCD agar (SCDA) and SCD broth plus 1.5% agar (SCDB/A). As a result, the culture medium constituent causing D value variation among SCD culture medium was determined as the difference of calcium (Ca) amount as well as the presence and the absence of K2HPO4 in SCD culture medium. Additionally, it was clarified D value differed significantly between SCDB and SCDA. D value using the former was around 1 min lower than that using the latter. This phenomenon can be explained from the formation of insoluble CaHPO4 in SCDB culture medium, resulting in insufficiency of usable Ca to B. stearothermophilus in SCDB culture medium.
The culture medium administered to C57Bl/6 mice 18 h and 8 h before a single irradiation (9 Gy) had a radioprotective effect and clearly influenced postirradiation changes in haemopoiesis. Haemopoiesis recovery appeared to be faster in culture medium-pretreated animals than in those irradiated without such pretreatment. By 12-15 days after irradiation, the thymus cortex appeared to be repaired, on day 21 a multiple increase in extramedullary erythropoiesis, myelopoiesis and megakaryocytopoiesis in the red pulp was found and later, on day 28, the lymphopoiesis in the white pulp of spleen was restored. The rate of haemopoiesis proliferation of predominantly myeloid cells which reached a control level on day 28 following irradiation. Consequently, the regenerative processes in blood-forming organs were accompanied by considerable reticulocytosis and complete recovery of neutrophil and platelet counts in the peripheral blood as seen on day 21. Despite a slower rate complete recovery of the total leukocyte count was reached by day 180 after irradiation.
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Melanoma cells produce tissue plasminogen activator (t-PA) that plays an important role in tumor invasion and metastasis. The production of t-PA by normal human uveal melanocytes has not been reported previously. In order to explore this possibility, we studied the production of t-PA by cultured human uveal melanocytes and compared that with the production by cultured human uveal melanoma cells and epidermal melanocytes. Human adult uveal melanocytes were isolated and cultured from donor eyes. The cells were cultured in serum-free medium for 48 h and the conditioned medium then collected for the plasminogen activator (PA) activity assay. Free PA activity was tested in an amidolytic assay using a t-PA standard curve. PA type was identified by fibrinography and antihuman t-PA and urokinase plasminogen activator (u-PA) blocking antibodies. Free PA activity was found in the conditioned medium of normal melanocytes and melanoma cells. The predominant PA activity was t-PA. Normal uveal melanocytes produced more t-PA (3.23 +/- 0.73 IU/105 cells/24 h) than that of epidermal melanocytes (1.25 IU/105 cells/24 h) but much less than uveal melanoma cells (11.0 +/- 3.39 IU/105 cells/24 h). Western blot analysis revealed that most t-PA in conditioned media were one-chain t-PA with molecular weight of 69 kDa. Our study indicates that uveal melanocytes may contribute to the free t-PA activity previously found in aqueous humor and choroidal eye cup superfusions. Therefore, this function of uveal melanocytes may play a role in intraocular matrix remodeling, fibrinolysis and aqueous humor outflow.
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Detection and quantitation of extracted proteoglycans, by staining with the dye Alcian blue on cellulose acetate followed by dissolution of the stained cellulose acetate strips in dimethyl sulfoxide containing 0.5% (v/v) sulfuric acid for absorbance measurement, is described. It is shown that, in the present system, the dye uptake by the proteoglycan is dependent only on the glycosaminoglycan content of the proteoglycan. The method is applied to the quantitation and characterization of proteoglycans and glycosaminoglycans, which have been extracted from radiolabeled bovine ankle cartilage and from mononuclear cell supernatant and which have been separated by DEAE-Sephacel column chromatography. The high sensitivity of the method allows detection of proteoglycans in 25-microliters samples of solutions containing as little as 1 microgram of glycosaminoglycan per milliliter of solution.
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It has been shown that pretreatment of mice with a five-fold condensed culture medium before irradiation increased the number of multipotential haemopoietic stem cells in the spleen. The degree of cyto- and radioprotection is dependent on both the time of administration and the dose of the culture medium. The administration of culture medium in twofold doses in a volume of 1 ml intraperitoneally 18 h and 8 h before irradiation with a dose of 9 Gy protected 95% of C57Bl/6 mice. On the other hand, no therapeutic effect of different doses of the culture medium was found. Our observation suggests that the culture medium can mediate a radioprotective effect. The possible mechanisms participating in this effect are discussed.
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