The influence of myo-inositol on phosphatidylglycerol synthesis by rat type II pneumonocytes.

Type II pneumonocytes isolated from adult rat lung were incubated in a serum-free medium containing [14C]glycerol and the incorporation of 14C into glycerophospholipids was measured. After 24 h, more than 80% of the 14C incorporated into total lipids or into phosphatidylcholine and approx. 90% of the 14C incorporated into phosphatidylglycerol after 24 h was recovered in the glycerophosphoester moieties of these molecules. Supplementation of the incubation medium with foetal-bovine serum (10%, v/v) did not alter the incorporation of [14C]glycerol by type II pneumonocytes after 24 h into either a total lipid extract or phosphatidylcholine. In the presence of foetal-bovine serum, however, the incorporation of 14C into phosphatidylglycerol was decreased and the incorporation of 14C into phosphatidylinositol was increased. In the absence of foetal-bovine serum, the incorporation of 14C into phosphatidylglycerol was decreased progressively as the concentration of myo-inositol in the incubation medium was increased. The range of concentration (0.04-0.50 mM) over which myo-inositol had the greatest influence on [14C]glycerol incorporation into phosphatidylglycerol by type II pneumonocytes in vitro encompassed the concentration range measured in foetal-rat serum late in gestation. At 4 days before birth, the concentration of myo-inositol in foetal-rat serum was 0.36 mM and decreased to 0.23 mM 1 day before birth. The concentration of myo-inositol in adult rat serum increased from 0.03 mM to 0.06 mM during pregnancy. Isolated rat type II pneumonocytes were found to take up myo-inositol by a saturable process. A half-maximal rate of myo-inositol uptake occurred at a concentration of myo-inositol of 0.29 mM. The results of this investigation are consistent with the hypothesis that late in gestation there is a decreasing availability of myo-inositol to the foetal lungs and that this favours the biosynthesis of phosphatidylglycerol for surfactant at the expense of phosphatidylinositol biosynthesis.     

Retinoid metabolism in cultured human retinal pigment epithelium.

Uptake, esterification and release of all-trans-retinol in primary cultures of human retinal epithelium were studied. Cultured cells were supplemented with 3H-labelled 11,12-all-trans-retinol, using fatty-acid-free albumin as the carrier. This led to incorporation of retinal and the formation of all-trans- and 11-cis-retinyl palmitate. The metabolism of the all-trans ester was monitored in a medium containing various concentrations of foetal-bovine serum (FBS). In 20% (v/v) FBS, the ester was hydrolysed, and all-trans-retinol was released into the culture medium. In the absence of FBS, little ester was hydrolysed and no retinol was found in the medium. Dialysed or heat-inactivated FBS or fatty-acid-free albumin was as effective as FBS in provoking ester hydrolysis and retinol release. The concentration-dependency of this effect on FBS was matched by the corresponding concentrations of albumin alone. A linear relationship was also found between interphotoreceptor retinoid-binding protein and retinoid release. Haemoglobin, which does not bind retinoids, is ineffective in this capacity. It is concluded that lipid-binding substances, mainly albumin, in FBS act as acceptors for retinol and drain the cultured cells of this molecule. The release of the retinol is coupled to the hydrolysis of retinyl esters in the cell, so that there is little or no net hydrolysis of ester if there is no acceptor for retinol in the culture medium. This effect explains why cultured human retinal epithelial cells are depleted of their stores of retinoids when maintained in medium supplemented with FBS.     

The effect of retinoic acid on proteoglycan biosynthesis in bovine articular cartilage cultures

The addition of retinoic acid to adult bovine articular cartilage cultures produces a concentration-dependent decrease in both proteoglycan synthesis and the proteoglycan content of the tissue. Total protein synthesis was not affected by the presence of retinoic acid, indicating that the inhibition of proteoglycan synthesis was not due to cytotoxicity. The proteoglycans synthesized in the presence of retinoic acid were similar in hydrodynamic size, ability to form aggregates with hyaluronate, and glycosaminoglycan composition to those of control cultures. However, the presence of larger glycosaminoglycan chains suggests that the core protein was substituted with fewer but longer glycosaminoglycan chains. In cultures maintained with retinoic acid, a decreased ratio of the large proteoglycan was synthesized relative to the small proteoglycan compared to that measured in control cultures. In cultures maintained with retinoic acid for 1 day and then switched to medium with 20% (v/v) fetal calf serum, the rate of proteoglycan synthesis and hexuronate contents increased within 5 days to levels near those of control cultures. Within 2 days of switching to medium with 20% (v/v) fetal calf serum, the relative proportions of the proteoglycan species were similar to those produced in cultures maintained in medium with 20% (v/v) fetal calf serum throughout. The rate of proteoglycan synthesis by bovine articular cartilage cultures exhibited an exponential decay following exposure to retinoic acid, with estimated half-lives of 11.5 and 5.3 h for tissue previously maintained in medium alone or containing 20% (v/v) fetal calf serum, respectively. The addition of 1 mM benzyl beta-D-xyloside only partially reversed the retinoic acid-mediated inhibition of proteoglycan synthesis. This indicates that the inhibition of proteoglycan synthesis by retinoic acid was due to both a decreased availability of xylosylated core protein and a decreased capacity of the chondrocytes to synthesize chondroitin sulfate chains.     

Small cooperative activity of old rat hepatocytes may depend on composition of the intercellular medium

Ultradian oscillations of protein synthesis were used as a marker of hepatocyte synchronous cooperative activity producing a common rhythm in vitro; amplitude of the rhythm defines expression of the cell cooperation. Dense synchronous and sparse non-synchronous rat hepatocyte cultures on slides in a serum-free incubation medium 199 supplemented with 0.2 mg/ml albumin and 0.5 microg/ml insulin have been studied. The amplitude of the rhythm averaged approximately 2x in dense cultures of young (3 month old) rats than in old (2 year old) rats. But some cultures of young rats had the amplitude patterns similar to cultures of old rats, and vice versa. Addition to the medium of either 0.3 microM bovine brain gangliosides or 2 microM phenylephrine resulted in increase of the oscillation amplitude in dense cultures of old rats to the level inherent in young ones. Addition to the medium of 10% rat blood serum in non-synchronous sparse cultures from young rats resulted in detection of a protein synthetic rhythm. Although after serum from young rats, the rhythm expression was high, the rhythm after serum from old rats had been given was weak. Addition of gangliosides to old-rat serum resulted in synchronization of sparse cultures with amplitudes inherent of young-rat serum. The data tend to the conclusion that cell cooperation depends to a greater extent on the composition of the medium rather than on the age of the cell or animal.     

Rapid isolation of metaphase chromosomes containing high molecular weight DNA

Normal rat kidney cells were cultured in medium supplemented with normal fetal bovine serum (FBS) or FBS depleted of fibronectin. The cell surface fibronectin of these cultures was visualized by indirect immunofluorescence using species-specific antisera for either rat fibronectin or bovine fibronectin. Anti-rat-fibronectin revealed fibrillar structures on the cells grown in either normal medium or fibronectin-depleted medium. Anti-bovine fibronectin revealed similar fibrillar networks, but only on the cells grown in medium containing bovine fibronectin. Staining in each case was abolished by absorption with the homologous antigen. It appears that exogenous fibronectin was incorporated into the same structures as endogenous fibronectin. This finding suggests that circulating fibronectin may serve as a building block for the assembly of extracellular matrix, possibly by cells which are incapable of synthesizing it.     

Development of rat embryos in culture media containing different concentrations of normal and diabetic rat serum.

In vitro culture of rodent embryos has been extensively used in the search for teratologic agents, with possible relevance to diabetic pregnancy. However, the high concentrations of rat serum added to the culture medium (approximately 75%) have raised concern that the teratogenic effects of some compounds may be attenuated or masked in this culture system and thereby forced the addition of pharmacological concentrations of the compounds (e.g., D-glucose and beta-hydroxybutyrate) to the medium. This issue has been examined in the present study where the effects of different concentrations of rat serum on growth and differentiation of rat embryos were recorded in cultures supplemented with increased concentrations of D-glucose and beta-hydroxybutyrate. The embryonic development was also evaluated after culture in medium supplied with serum from diabetic rats. Compared with normal rat serum, the diabetic serum had an elevated glucose concentration as well as markedly increased levels of triglycerides and branched amino acids, indicating a potentially rich supply of major nutrients for the cultured embryos. Lowering the serum concentration in the culture medium from 80% to 50% yielded progressively retarded embryonic growth but no increased rate of other morphological malformations. At 40% serum concentration, however, there was a sharp rise in the incidence of somatic malformations, in addition to the prevailing growth retardation. When the embryonic growth and development were compared at 50% and 80% serum concentrations, increased D-glucose or beta-hydroxybutyrate concentrations caused similar degrees of embryonic dysmorphogenesis. Also, the uptake of each compound by the embryos exposed to elevated levels of the two agents were similar in 50% and 80% serum cultures. There was, therefore, no protection against the teratogenic and growth-retarding effects of increased D-glucose or beta-hydroxybutyrate offered by high serum concentrations in the culture medium (i.e., 80% vs. 50%). Embryos cultured in 50% or 80% diabetic rat serum at 30 mmol/L or 50 mmol/L D-glucose concentration showed similar rates of somatic malformations as did embryos exposed to the same proportion of normal rat serum at similar glucose concentrations. By contrast, the diabetic rat serum amplified the general retarding effects of high D-glucose levels, yielding lower protein levels and somite numbers in embryos from diabetic serum culture than in embryos cultured in normal rat serum.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of retinoic acid on proteoglycan turnover in bovine articular cartilage cultures

This paper describes proteoglycan catabolism by adult bovine articular cartilage treated with retinoic acid as a means of stimulating the loss of this macromolecule from the extracellular matrix of cartilage. Addition of retinoic acid (10(-12)-10(-6) M) to adult bovine articular cartilage which had been labeled with [35S]sulfate for 6 h after 5 days in culture, resulted in a dose-dependent increase in the rate of loss of 35S-labeled proteoglycans from the matrix of the tissue. Concomitant with this loss was a decrease in the proteoglycan content of the tissue. Incubation of cultures treated with 1 microM retinoic acid, at 4 degrees C, or with 0.5 mM cycloheximide, resulted in a significant decrease in the rate of retinoic acid-induced loss of proteoglycans and demonstrated cellular involvement in this process. Analysis of the 35S-labeled proteoglycans remaining in the matrix showed that the percentage of radioactivity associated with the small proteoglycan species extracted from the matrix of articular cartilage explants labeled with [35S]sulfate after 5 days in culture was 15% and this increased to 22% in tissue maintained in medium alone. In tissue treated with 1 microM retinoic acid for 6 days, the percentage of radioactivity associated with the small proteoglycan was 58%. Approximately 93% of the 35S-labeled proteoglycans released into the medium of control and retinoic acid-treated cultures was recovered in high density fractions after CsCl gradient centrifugation and eluted on Sepharose CL-2B as a broad peak with a Kav of 0.30-0.37. Less than 17% of these proteoglycans was capable of aggregating with hyaluronate. These results indicate that in both control and retinoic acid-treated cultures the larger proteoglycan species is lost to the medium at a greater rate than the small proteoglycan species. The effect of retinoic acid on proteoglycan turnover was shown to be reversible. Cartilage cultures maintained with retinoic acid for 1 day then switched to medium with 20% (v/v) fetal calf serum for the remainder of the culture period exhibited decreased rates of loss of 35S-labeled proteoglycans from the matrix and increased tissue hexuronate contents to levels near those observed in tissue maintained in medium with 20% (v/v) fetal calf serum throughout. Furthermore, following switching to 20% (v/v) fetal calf serum, the relative proportions of the 35S-labeled proteoglycan species remaining in the matrix of these cultures were similar to those of control cultures.     

Improved response of isolated liver cells to glucagon in the presence of rat serum.

The stimulation by glucagon of gluconeogenesis from 10 mM lactate and urea formation in intact isolated rat liver cells is enhanced from about 33 % to 61 % and 92 %, respectively, by the addition of rat serum (30 % v/v) to the incubation medium. This effect is exerted by (a) dialysable, heat-labile serum component(s), different from known stimulators of gluconeogenesis, such as fatty acids, lysine, ammonium chloride or calcium, which do not improve the response of hepatocytes to glucagon. The factor(s) could provide a useful tool for the study of glucagon action(s) on liver metabolism.     

Non-neuronal cells of rat hypothalamus in dissociated cell culture

Summary The neurophysin that is biosynthesised in association with the neurohypophysial hormone vasopressin (vasopressin-neurophysin) affects the growth and DNA synthesis of rat hypothalamic non-neuronal cells in culture. Over a narrow range of concentrations vasopressin-neurophysin stimulated growth, as assessed by increase in cell numbers, about five-fold, in conditions where fetal calf serum concentration was limiting (0.2% fetal calf serum). Maximum stimulation occurred in the presence of 20 to 30 ng vasopressin-neurophysin per ml of medium. DNA synthesis was increased by a factor of three in the presence of 30 ng vasopressin-neurophysin per ml of medium. At least two populations of non-neuronal hypothalamic cells were present in the cultures, and these were both affected by vasopressin-neurophysin.This study allows the suggestion that neurophysin may be acting as a growth-regulating factor at its release site, playing a part in the interactions of neurones and glial cells in the hypothalamo-neurohypophysial system.     

Studies of the Numbers and Morphology of the Intracellular Form of Leishmania donovani Grown in Cell Culture*

SYNOPSIS. Macrophages were infected in vivo with the intracellular form of Leishmania donovani (LDs), harvested from the previously saline-stimulated peritoneal cavities of hamsters and explanted into Leighton tubes containing removable coverslips. Serum from either rabbit, chicken, human, calf, hamster or cotton rat blood was used as the 40% component of a Hanks' BSS₆₀ serum₄₀ medium used to maintain these Leighton tube cultures at 37 C. After varying lengths of time coverslips were removed from tubes, stained with Giemsa, and the parasites per infected macrophage, total number of hamster cells and total number of parasites on each coverslip were counted. Maerophages constituted more than 90% of the explanted cells on the coverslips. When cotton rat serum was used as a component of the medium, fibroblastic overgrowth of the coverslips followed. Some similarities and differences in the numbers of macrophages, fibroblasts and parasites were noted with regard to the serum used as part of the medium. Except for cotton rat serum, the serum component of the medium used apparently did not influence, to any great degree, the morphology of either the macrophages or parasites therein. Thus, vacuolarization and granularization of macrophages did not appear to be very distinctly correlated with the time of sampling or the type of serum in the medium used for maintenance nor could any morphologic variations of the LDs be ascribed to these factors. When cotton rat serum, but not any of the other sera, was the serum component of the medium, leptomonads were noted in the overlay fluid of the cultures after 6 days. Under these conditions of cell culture, fibroblasts could not be infected with LDs although macrophages on the same coverslip were heavily parasitized.     

INHIBITION OF MALIGNANT CELL PROLIFERATION BY CULTURE MEDIA CONDITIONED BY CARDIAC OR SKELETAL MUSCLE

The present work is an attempt to explain the high resistance of muscles to cancer development. We used primary cultures of rat skeletal and cardiac muscle, and examined the effect of the supernatant of these cultures (conditioned medium; CM) on proliferation of cancer cells. The results demonstrated that CM inhibited the proliferation of several types of malignant cells by more than 50%, without a significant inhibition on normal cells. Cell cycle analysis revealed that CM increased the number of cells in S and G2 phases, suggesting a cytostatic effect of CM. For defining the biological properties of the factor(s) which are present in the CM, skeletal muscle cultures were grown in chemically defined medium (serum free medium). The concentrated sample was applied to a Sephadex G-50 column and three fractions were obtained. Only one fraction showed inhibitory activity. Four protein bands were observed in this fraction, as revealed by SDS-PAGE. We suggest that some, or all of these proteins are responsible for inhibition of tumor cell replication.     

Variation in the length of the lag phase following serum restimulation of mouse 3T3 cells

We have investigated the length of the lag phase (time taken for the first cells to enter S phase) and the kinetics of entry into DNA synthesis after serum restimulation of Swiss mouse 3T3 cell cultures that were allowed to become quiescent under different conditions. Cells were allowed to reach quiescence as a confluent monolayer in medium containing 10% (v/v) calf serum. Alternatively, when serum was reduced to 1% (v/v), cultures became quiescent at about 30% confluency and there was little cell to cell contact. The results show that the lag, or prereplicative phase becomes longer as the time spent in the quiescent state increases. This is the case in both confluent and non-confluent cultures. The rate of entry of cells into the S phase, however, remains the same under all conditions.     

Glucose Transport in Astrocytes: Regulation by Thyroid Hormone

Primary cultures of astrocytes from newborn rat brain showed evidence of a substrate-saturable process for glucose transport. The system shows a relatively high affinity for the substrate, with an apparent Km of approximately 1 mM. Maintenance of the cells in medium containing thyroid-hormone-free serum for 3, 6, or 9 days resulted in significantly reduced rates of hexose transport. Addition of exogenous triiodothyronine to the transport incubation medium of these "hypothyroid" cells markedly increased the net rate of 2-deoxyglucose uptake within 60 s to values equal to or above those of control cultures (cells maintained in normal serum). These findings support a key role for thyroid hormone in the transport of glucose across plasma membranes of brain cells and demonstrate the presence of this regulatory system in astrocytes.     

Cellular activities associated with the transition of chick embryo fibroblasts from stationary to proliferation state

Chick embryo fibroblasts on the 5th day of culture in proteinfree medium were stimulated to accelerated growth by supplementation of the medium with ATP (50 mumol/l, insulin (0.16 I.U./ml) or chick serum (5% v/v). Kinetics of the entry of cells into the S phase and later into the logarythmic phase of growth were found to be different in cultures treated with these three factors in spite that the final saturation densities reached after 30 days of culture were similar. No direct correlation between cell spreading and the cell transition from the stationary to the proliferation state was found. The proliferating cells showed the higher rate of locomotion than in stationary cultures. The initial protein-free medium, supporting the long survival of chick embryo fibroblasts and their susceptibility to growth accelerating factors, was further simplified by replacement of ADA buffer with EDTA (0.4 mmol/l).     

Production of progestin-stimulatory factor(s) by enriched populations of rat T and B lymphocytes

We have previously demonstrated that a progestin-stimulatory factor(s) (PSF) is present in the supernate of concanavalin A-activated rat splenocytes. In the absence of FSH, PSF evokes dose-dependent increases in both progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) production by undifferentiated rat granulosa cells while basal estrogen production is unaffected. Because splenocytes are composed predominantly of T and B lymphocytes, we sought to determine if PSF production is restricted to one of these specific cell types. To accomplish this, dispersed splenocytes were prepared from the spleens of adult female rats. Macrophages were removed by standard adherence procedures, and highly enriched populations of T and B lymphocytes were obtained by negative selection panning utilizing monoclonal antibodies to T and B lymphocytes. Purity of the T- and B-cell populations (typically greater than 85-95%) was assessed by single-color flow cytometry. Enriched populations of T cells, B cells, or unseparated lymphocytes were then seeded into tissue culture flasks and stimulated with 2.5 micrograms/ml concanavalin A. Following a 48-h incubation at 37 degrees C, cytokine-rich fractions were prepared from the conditioned medium of these cultures by ammonium sulfate fractionation. The presence of PSF was assessed by the ability of the resulting fractions to specifically stimulate progestin production by undifferentiated rat granulosa cells in vitro. Granulosa cells were treated with increasing doses (ranging from 0-30% v/v) of supernatants from cultures of T cells, B cells, or unseparated splenocytes for 48 h. Media were removed and progesterone, 20 alpha-OH-P, and estrogen levels were quantified by RIA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Retinoic acid can change normal differentiation of rat egg-cylinders cultured in vitro

The modified organ culture of rat egg cylinders provides favorable conditions for 2 weeks for the differentiation of main tissue types. To study the effect of retinoids on early rodent differentiation, retinoic acid (RA) was added in various concentrations to serum-supplemented or serum-free medium. Explant survival decreased when RA was added to serum-free medium. Although the cartilage was well differentiated even in cultures deprived of serum, RA inhibited chondrogenesis in all cultures without or with serum. The frequency of columnar epithelium was higher and its folds more often present when RA was added to the medium. Keratinization of squamous epithelium depended on the RA concentration added to the medium, and was almost absent when the concentration was high. Other tissues often present in serum-supplemented medium (such as neuroblasts and myotubes) were not affected by RA, a result that differs from those obtained in other experimental systems.     

Partial differentiation of rat egg cylinders in serum-free and protein-free medium

Modified organ cultures of rat egg cylinders were grown for 2 weeks in Eagle's MEM without serum or with serum added at different times. Explant survival was decreased only in cultures grown for the entire 2 weeks in serum-free medium, whereas the explant growth was impeded in all but the cultures grown for 2 weeks in 50% MEM plus 50% serum. Differentiation of epidermis and cartilage in the cultures deprived of serum for the entire 2-week period was comparable to that in fully serum-supplemented medium, whereas other differentiated tissues were rare or absent. In explants cultivated without serum for only the first week, neuroblasts were scarce.     

Identification of media supplements that improve the viability of primarily cell cultures ofCrassostrea gigas oysters

Media supplements have been investigated for their influence on the viability of primary cell cultures from the heart ofCrassostrea gigas oysters. Soluble factors of vertebrate origin were tested, belonging to five families of supplements that had proven to increase the viability of insect and mammal cell cultures. Using two-level complete factorial assays, factors and mutual interactions were screened within each family with a MTT reduction assay. Results pointed out the positive influence of hormones, growth factor, antioxidants and lipids on the mitochondrial metabolism of oyter's heart cells. Consequently, a new concentrated complex supplement was developed. At 10% (v/v) final concentration in modified Leibovitz L-15 medium, it increases by 30% the cellular viability of one-week old cultures as compared with non-supplemented medium, a similar improvement as the one obtained with 10% (v/v) fetal calf serum. Combined with fetal calf serum, this new supplement doubles the cellular viability of one-week old cultures and allows networks of cardiomuscular cells to be maintained functional over three monthsin vitro.Abbreviations MTT-3 (4,5-dimethylthiazol;-2-yl)2,5-diphenyl-tetrazolium bromide - FCS fetal calf serum     

Thyroid hormone and apotransferrin regulation of growth hormone secretion by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium

Summary Growth hormone (GH) production by GH₁ rat pituitary tumor cells in iron restricted serum-free defined medium requires apotransferrin (apoTf) and triiodothyronine (T₃). As measured by radioimmunoassay, apoTf plus T₃ induced GH levels 2 to 4-fold above controls. Deletion of either apoTf or T₃ arrested GH secretion. ApoTf/T₃ defined medium regulated GH production as effectively as whole serum. Because glucocorticoids enhance GH secretion in serum containing cultures, the effects of dexamethasone were evaluated in apoTf/T₃ defined medium. The steroid hormone showed no enhancing effects unless the cells were exposed to serum prior to incubation in apoTf/T₃ defined medium. Even under these conditions, the response to dexamethasone remained T₃ dependent. These observations indicate that a yet to be characterized serum factor(s), other than apoTf, regulates the reponse to the steroid hormone. This is the first report of thyroid hormone regulation of GH secretion by rat pituitary tumor cells under completely serum-free chemically defined conditions.     

In vitro cultivation of Dipetalonema viteae third-stage larvae: evaluation of culture media, serum, and other supplements

Third-stage larvae of Dipetalonema viteae obtained from the tick vector developed to the fourth stage in several cell-free culture systems. Survival and development of larvae in a number of commercially available cell culture media, supplemented with serum and other defined and undefined components, were compared. All cultures were gassed with 5% carbon dioxide in nitrogen. Best survival, growth and development were obtained in stationary cultures containing 1:1 (v/v) mixtures of NCTC 135, either RPMI 1640 or Iscove's Modified Dulbecco's Medium, and a supplement of 20% non-heat-inactivated fetal bovine serum. The importance of the medium composition and physical environment of the culture system, for the survival, growth and development of D. viteae was demonstrated.