THE NUTRITION OF ANIMAL TISSUES CULTIVATED IN VITRO : III. USE OF A DEPLETION TECHNIQUE FOR DETERMINING SPECIFIC NUTRITIONAL REQUIREMENTS

1. Cultivation of freshly explanted chick embryonic heart tissues in Hanks's salt solution for 3 to 4 days has been shown to create a general state of nutritional deficiency in the cultures. Provided the depletion is not prolonged beyond 4 days, the cultures subsequently revive and survive to a normal period in synthetic medium M 150. 2. Paper chromatographic studies on the culture medium have shown that the amino acid metabolism of the depleted cultures is restored to a normal pattern within a few days in medium M 150. 3. By the use of the nutritional deficiency technique, a coenzyme A requirement for this type of culture has been established. 4. The application of these findings to tissue cell nutrition and the possible hazards of using serum, or other uncharacterized additions, either to establish cultures, or as part of the experimental medium, are discussed.     

Flower Induction by Nitrogen Deficiency in Lemna paucicostata 6746

Lemna paucicostata 6746, a short-day plant, produced flowerbuds even under continuous light when cultured in nitrogen-deficientmodified Hoagland medium with 1% sucrose for 3 days or morefollowed by culture on nitrogen-rich medium (either nitrateor ammonium). Flowering was also induced by culture on mediumcontaining 20–100 µM nitrate as the sole nitrogensource for 10 days or more, but not on medium with a low ammoniumconcentration. However, if plants cultured on medium containing5–20 µM ammonium as the sole nitrogen source for10 days were grown in a nitrogen-rich medium for a further 4days, they produced flower buds. Thus, nitrogen deficiency caninduce day length-independent flowering in Lemna paucicoslata6746, but nitrogen is required for the manifestation of flowering. (Received January 31, 1986; Accepted April 24, 1986)     

The morphogenic effect of gonadotropic hormones on the Leydig cells of the boar testis. II. Effects of administration of chorionic gonadotropin after hypophysectomy. Effects in vivo and in organ culture

The longer ago the hypophysectomy has been performed, the more marked is Leydig cell atrophy in the testis. The effects of HCG on cellular morphology have been observed in vivo and in organ culture; qualitative quantitative and ultrastructural aspects were studied. In vivo, the effects of a daily injection of gonadotropin on the testis of 2 boars hypophysectomized 3 1/2 months ago are shown. Markedly atrophied cells are strongly stimulated by HCG during the 15 first days (the cell and nucleus recover nearly to standard size, with the typical histological and ultrastructural appearance with all the cell organelles which characterize a functional steroid cell). Then after 1 1/2 month injection it decreases again to the initial state (very small size cytoplasm strongly reduced with very low organelle content). The number of the Leydig cells is maintained during the first 15 days, then it progressively decreases. The effects of HCG on the testicular tissue of 4 boars were studied in organ culture. Interstitial tissue with a greater or lesser degree of atrophy was examined experimentally (1 month, 3 months and 4 months after hypophysectomy) in order to prove a possible irreversibility of the effects of hypophysectomy. In each case, cell changes were studied according to the duration of the culture. Control cultures without HCG in the medium were set up simultaneously. 1 month and/or 3 months after hypophysectomy, the Leydig cells in culture progressively recover the size and the histological and ultrastructural appearances of a typical Leydig cell. After 16 days of culture, the stimulation is highest, as in vivo. The number of Leydig cells is maintained. From the 17th day stimulation decreases and the cell enters a new atrophy phase. In the anhormonal control medium the atrophy continues as long as the culture is maintained, and the number of Leydig cells decreases. 4 months after hypophysectomy, stimulation in culture is still possible during the first 10 days (proved by the same tests); however the size of the cell remains small compared to the normal; then it atrophies again quickly. In this case the hormone does not maintain the number of the Leydig cells. In the control cultures, slight response of the cell is observed, but this effect is limited and disappears a few days later; the number of the cells rapidly decreases. It has been shown that markedly atrophied Leydig cells can highly be stimulated during the first 2 weeks under the influence of HCG as well in vivo as in organ culture. The lability of the effect is not yet explained. 4 months after hypophysectomy, stimulation is not so effective.     

Organ culture of mammalian testis. III. Inhibin secretion.

Mouse testes were cultured for 19--20 days at either 31 or 37 degrees C with a change of medium every 4 days. After treatment with charcoal and dextran T, the recovered testis media were incubated with rat anterior pituitary cells, and secretions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were estimated by radioimmunoassay 3 days later. FSH release was significantly lowered when pituitary cells were grown with media of testes cultured 31 degrees C compared to cultures grown with fresh medium or with media of testes cultured at 37 degrees C for more than 4 days. LH secretion was normal in one experiment and reduced in the other with the media of testes cultured at 31 degrees C. Treatment of testicular media by heat or trypsin reduced the inhibiting activity. After 8 days at 37 degrees C, both germinal and Sertoli cells were damaged in the testis cultures, while at 31 degrees germinal cells alone were destroyed, Sertoli cells remained normal. These studies suggest that (1) a substance which responds to the definition of inhibition (protein--preferentially acting on FSH) is secreted in the medium of testis culture; (2) inhibin is produced by Sertoli cells; (3) inhibin is secreted only if the temperature is inferior to 37 degrees C.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.     

Murine B-cell stimulatory factor-1 (BSF-1)/interleukin-4 (IL-4) is a multilineage colony-stimulating factor that acts directly on primitive hemopoietic progenitors

Interleukin-4 (IL-4), which was originally identified as a B-cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL-4 on early hemopoetic progenitors in methylcellulose culture. IL-4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5-fluorouracil (5-FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI-3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL-4. To test whether or not IL-4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL-3 and IL-4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL-4 was significantly smaller than that supported by IL-3. When colony-supporting abilities of IL-4 and IL-3 were compared using day-4 post-5-FU spleen and day-2 post-5-FU marrow cells, IL-4 supported the formation of fewer blast cell colonies than did IL-3. IL-4 and IL-6 revealed synergy in support of colony formation from day 2 post-5-FU marrow cells. These results indicate that murine IL-4 is another direct-acting multilineage colony-stimulating factor (multi-CSF), similar to IL-3, that acts on primitive hemopoietic progenitors.     

Catabolism of newly synthesized decorin by explant cultures of bovine ligament.

The catabolism of newly synthesized decorin by explant cultures of bovine collateral ligament was investigated. The tissue was placed in explant culture for 6 days then incubated with radiolabeled sulfate for 6 h and replaced in culture for 5 days to allow for the loss of the radiolabeled large proteoglycan. The metabolic fate of the remaining radiolabeled decorin present in the matrix of the tissue over the next 9-day period was determined. It was shown that this pool of decorin was lost from ligament explant cultures either directly into the culture medium or taken up and degraded within the cells of the tissue. The intracellular degradation of the radiolabeled pool of decorin by ligament explant cultures was shown to result in the generation of [35S]sulfate. This process required metabolically active cells and involved the lysosomal system since sulfate generation was inhibited when cultures were maintained at 4 degrees C or in the presence of either 10 mM ammonium chloride or 0. 05 mM chloroquine. The inhibition of intracellular processing of decorin resulted in an increase in the rate of loss of this proteoglycan into the medium of the cultures. The inhibition of intracellular degradation of decorin was reversible on incubation of the explant cultures at 37 degrees C or removal of ammonium chloride from the culture medium. After removal of the ammonium chloride from the culture medium the rate of intracellular catabolism was greater than that observed in cultures maintained in medium alone, which suggested that there was an intracellular accumulation of native and/or partially degraded material within the cells.     

Application of a serum-free medium for the growth of Vero cells and the production of reovirus

Two strains of reovirus (serotype 1 Lang/TIL and serotype 3 Dearing/T3D) were propagated in Vero cells grown in stationary or agitated cultures in a serum-free medium, M-VSFM. Solid microcarriers (Cytodex-1) were used to support cell growth in agitated cultures with a normal doubling time of 25 h. Cell yields of 1 x 10(6) cells/mL were obtained from an inoculum of 2 x 10(5) cells/mL in 4 days in microcarrier cultures. The growth profile and cell yield was not significantly different from serum-supplemented cultures. The virus titer increased by 3-4 orders of magnitude over a culture period of 150 h. The maximum virus titer in stationary cultures reached >1 x 10(9) pfu/mL for both strains of reovirus in M-VSFM. M-VSFM also supported high viral yields in microcarrier cultures. Both the specific productivity and final viral yield was higher in M-VSFM than serum-supplemented cultures. The high viral productivity suggests that this is a suitable system for the production of reovirus as an oncolytic agent for human therapeutic use.     

Increase of L-serine dehydratase activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh.

Hormonal regulation of L-serine dehydratase [L-serine hydro-lyase (deaminating), EC 4.2.1.13] was studied in primary cultures of adult-rat hepatocytes. The hepatocytes were isolated by collagenase perfusion and maintained in culture on collagen-gel/nylon-mesh substrata. L-Serine dehydratase activity was measured with [14C]threonine as substrate. The enzyme activity in hepatocytes of normal adult rats was low and declined rapidly in culture in L-15 medium containing 0.1 micro M-insulin and even more in the presence of glucose. L-Serine dehydratase activity in hepatocytes of rats with streptozotocin-induced diabetes was initially 20-fold higher than that of normal rats, but fell rapidly to a low value by 4 days in culture. Hormonal regulation of the enzyme activity was manifested by treatment of the cultured hepatocytes with insulin (0.1 micro M), glucagon (0.3 micro M), dexamethasone (10 micro M) and combinations of these hormones. Either glucagon or dexamethasone in the absence of insulin enhanced the activity of L-serine dehydratase, but failed to do so in the presence of insulin. Treatment with both hormones resulted in a 2-3-fold increase in enzyme activity in culture on days 3 and 4. Under conditions in which the enzyme activity was enhanced, glucose production by the cultured hepatocytes was concomitantly increased. Glucose production resulted in part from gluconeogenesis from pyruvate and not entirely from glycogenolysis. The gluconeogenic conditions of culture resulted in a decrease in cellular lipids in the cultured hepatocytes, as evidenced by ultrastructural studies.     

The Effect of Indole-3-Butyric Acid and Riboflavin on the Morphogenesis of Adventitious Roots of Eucalyptus ficifolia F. Muell. Grown In Vitro

Shoot explants from seedling-derived culture of Eucalyptus ficifoliaF. Muell. cultured on a rooting medium free from indole-3-butyricacid (IBA) develop a root system (Type I) consisting of a fewcomparatively long roots and only small amounts of callus. IBAat 5.0 µM in a rooting medium free from riboflavin inducesthe development, on the shoot explants, of a compact root system(Type II) consisting of callus and many short roots. Riboflavinwhen exposed to light, is able to photo-oxidize IBA; the degreeof photo-oxidation depends on the photon fluence of the lightreceived. The rooting response of the cultures reflects thedegree of photo-oxidation of IBA: concentrations of IBA fromabout 10^–4M to 10^–6M in the rooting medium induceformation of the Type II root system whilst photo-oxidationof the auxin to concentrations of about 10^–8M or lowerinduces the formation of the Type I root system. Thus, exogenousriboflavin and exogenous IBA are linked in a distinct light-induced,riboflavin-mediated change in root morphogenesis. The anatomyof root development in the Type I and Type II root systems wasstudied and factors affecting the development were defined.Characteristics of riboflavin and IBA breakdown in various lightregimes were determined and related to root morphogenesis. Theresults and their implications are discussed. Key words: Auxin photo-oxidation, Riboflavin, Root morphogenesis, Tissue culture     

Metabolic active-high density VERO cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding

The control of cell death occurring in high density cultures performed in bioreactors is an important factor in production processes. In this work, medium nutrient removal or feeding was used to determine at which extension apoptosis could be, respectively, involved or prevented in VERO cell cultures on microcarriers. Glutamine and galactose present in the VERO cell culture medium was consumed after, respectively, 6 and 12 days of culture. Kinetics studies showed that fresh medium replacement and, to some extent, galactose or glutamine depleted-fresh medium replacement provided a nutritional environment, allowing the VERO cell cultures to attain high densities. Galactose was shown to be a more critical nutrient when cultures reached a high density. In agreement with that, VERO cell cultures supplemented with galactose and/or glutamine were shown to confirm previous findings and, again at high densities, galactose was shown to be a critical nutrient for VERO cell growth. These observations also indicated that in VERO cell cultures, for feeding purposes, the glutamine could be replaced by galactose. The inverse was not true and led, at high densities, to a decrease of cell viability. In the absence of glutamine and galactose, apoptosis was observed in VERO cell cultures by cytofluorometry, Acridine orange staining or light and electron microscopy, reaching high levels when compared to cultures performed with complete medium. VERO cells apoptosis process could be prevented by the galactose and/or glutamine feeding and, at high densities, galactose was more efficient in protecting the cultures. These cultures, prevented from apoptosis, were shown to synthesize high levels of measles virus following infection. Our data show that apoptosis prevention by glutamine/galactose feeding, led to high productive and metabolic active VERO cell cultures, as indicated by the high cell density obtained and the virus multiplication leading to higher virus titers.     

Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration.

A recently developed sandwich culture system, in which hepatocytes are sandwiched between two layers of collagen, has been shown to be capable of maintaining long-term expression of hepatocellular function (J. C. Y. Dunn et al., Biotechnol. Prog. 7, 237-245, 1991). The development of an adequate technique for the cryopreservation of hepatocytes in such a stable culture configuration would ensure a ready supply of hepatocytes for use in bioreactors or bioartificial liver support devices. This report describes the effects of exposing hepatocytes in sandwich culture to different concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) at 22 degrees C on Day 7 of culture. Cell function, morphology, and cytoskeletal organization were followed for 14 days after exposure. Hepatocellular morphology and albumin secretion remained normal when cultures were exposed for up to 120 min to predicted final Me2SO concentrations up to 1.33 M. Exposure for less than 60 min to equilibrium concentrations of up to 3.33 M Me2SO did not adversely affect cell morphology or albumin secretion rate, but at the highest concentration (3.33 M), increase of the exposure time to 60 or 120 min resulted in dramatic, irreversible cell damage and loss of function. Actin filament organization was shown to be undisturbed when the cells were exposed to 1.33 M Me2SO for 60 min, but was irreversibly disrupted by exposure to 3.33 M for 120 min. Based on these results, a simple and safe procedure is suggested for the addition of Me2SO to hepatocytes in a sandwich culture configuration and its subsequent removal, which will be valuable for studies on hepatocyte cryopreservation.     

Studies on the biosynthesis of quinones in fungi. Incorporation of 6-methylsalicylic acid into fumigatin and related compounds in Aspergillus fumigatus I.M.I. 89353

1. Orsellinic acid has been detected as a metabolite of Aspergillus fumigatus. 2. The other principal aromatic components of the medium are fumigatin and the quinol, fumigatol. Fumigatol has been shown to be dihydrofumigatin after oxidation to the quinone followed by acetylation. 3. (14)C-labelled 6-methylsalicylic acid can be hydroxylated in A. fumigatus to form orsellinic acid and decarboxylated to give m-cresol. 4. (14)C-labelled 6-methylsalicylic acid is incorporated into fumigatin and fumigatol (1.0-1.5%), but the conversion does not occur until about 2-3 days after supplementation of the medium. At this stage of growth, the organism has already synthesized approx. 20 times as much fumigatol as fumigatin and this ratio is reflected in the much lower specific activity of the quinol. 5. Supplementation of the medium with either orsellinic acid or orcinol, in addition to (14)C-labelled 6-methylsalicylic acid, greatly decreases the latter's incorporation into fumigatin. At the same time, the cultures containing these substances are stimulated to produce another quinone with relatively high specific activity. 6. 6-Methylsalicylic acid has not been detected in the medium of normal cultures. The results indicate that 6-methylsalicylic acid itself is not a direct precursor of fumigatin and fumigatol but that it is converted into a true intermediate, probably after hydroxylation to orsellinic acid. 7. Supplementation of the medium with 6-methylsalicylic acid (15-25mg./200ml.) greatly affects the metabolism of A. fumigatus. Growth is inhibited and the synthesis of fumigatol is markedly depressed in these cultures. The inhibitory effects may possibly be related in some way to the production of m-cresol.     

Proton motive force, energy recycling by end product excretion, and metabolic uncoupling during anaerobic growth of Pseudomonas mendocina.

Batch cultures of Pseudomonas mendocina, grown in rich medium with glucose excess, showed metabolic differences dependent upon whether the growth conditions were aerobic or anaerobic, with or without added electron acceptor. Under anaerobic conditions in the absence of nitrate, P. mendocina reached the stationary phase of growth after 2 or 3 days, followed by a stationary phase of 4 to 5 days. Under these conditions, a mixed-type fermentative metabolism (formic, lactic, and acetic acids) appeared. A fivefold-higher specific rate of glucose consumption and eightfold-higher production of organic acids, compared with aerobic cultures, were shown by this microorganism growing anaerobically in the absence of exogenous electron acceptors. The gradients of organic acid produced by P. mendocina under these conditions reached a maximum (lactate, 180 mV; formate, 150 mV; acetate, 215 mV) between days 2 and 3 of culture. The proton motive force (delta p) decreased during growth from -254 to -71 mV. The intracellular pH remained alkaline during the culture, reaching a steady-state value of 7.9. The gradients of organic acids apparently contributed to the generation of a delta p, which, according to the Energy Recycling Model (P. A. M. Michels, J. P. J. Michels, J. Boonstra, and W. N. Konings, FEMS Microbiol. Lett. 5:357-364, 1979), would produce an average energy gain of 1 or 1.5 mol of ATP equivalents per mol of glucose consumed with H+/ATP stoichiometry of 3 or 2, respectively. Low YATP and Yglucose values were observed, suggesting that an uncoupled metabolism exists; i.e., ATP produced by catabolic processes is not directly used for biomass synthesis. This metabolic uncoupling could be induced at least in part by organic acids and the ATP wastage could be induced by a membrane-bound ATPase involved in intracellular pH regulation.     

Short- and long-term exposure of articular cartilage to curcumin or quercetin inhibits aggrecan loss

The aim of this study was to determine if curcumin and quercetin inhibit induced aggrecan loss from bovine articular cartilage explants given that these polyphenols have been shown to suppress the expression of matrix-degrading enzymes. The kinetics of loss of ³⁵S-aggrecan and the loss of total aggrecan in cartilage explants maintained in catabolic medium containing either 1 μM retinoic acid or 50 ng/ml interleukin (IL)-1α were studied in the presence of either 1–25 μM curcumin or 10–50 μM quercetin. The reversibility of catabolism of ³⁵S-aggrecan was also studied in catabolically stimulated cultures treated with 25 μM curcumin or 50 μM quercetin for the initial 4–5 days of culture followed by 10–15 days of culture in catabolic medium in the absence of either polyphenol. Curcumin and quercetin suppressed ³⁵S-aggrecan and total aggrecan loss from the explants in a dose-dependent manner. When the exposure of explants to curcumin or quercetin was limited to the first 4–5 days of culture, the suppression of ³⁵S-aggrecan loss was maintained in the extended culture period when the tissue was stimulated with either retinoic acid or IL-1α. Quercetin suppressed IL-1α-stimulated expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4. Curcumin suppressed retinoic acid stimulated expression of ADAMTS-5, and both polyphenols suppressed basal expression of ADAMTS-5. The ability of curcumin and quercetin to protect cartilage from stimulated aggrecan loss and to maintain this protection posttreatment may, at least in part, be due to the suppression of gene expression of ADAMTS-4 and -5.     

Serum substitute in epithelial cell culture media: nonfat dry milk filtrate.

A number of milk types and milk fractions were investigated as possible substitutes for serum in cell culture media. A filtrate of reconstituted nonfat dry milk showed promise. Culture fluids containing 5% of the nonfat dry milk filtrate were used to propagate primary and continuous cell cultures, and the cell growth from these cultures was compared with that of cells grown in a serum-containing medium. The nonfat dry milk filtrate-supplemented medium supported the growth of all epithelial cells tested, but two fibroblast-type cultures failed to replicate. Cells grown in the medium containing the milk filtrate grew slowly for 2 to 3 days and then propagated to confluency in 6 to 8 days. Viable cell counts of 9 days were comparable to those of serum-grown cells that had been propagated for 7 days. Cells grown in the milk filtrate could be split 1 to 4 when subcultures were prepared. Cell growth could be stimulated by refeeding on days 2 to 3 or by the addition of 30 microM 2-mercaptoethanol to the growth medium. Virus susceptibility and titer comparisons with poliovirus 1, coxsackievirus B2, echovirus 7, and herpes simplex virus indicated that approximately the same data were obtained when either the nonfat dry milk filtrate-treated or the serum-treated cells were studied. The nonfat dry milk filtrate is inexpensive, is easily prepared, and is a substitute for serum in epithelial cell culture media.     

Reduced lysyl oxidase activity in skin fibroblasts from patients with Menkes'' syndrome.

Lysyl oxidase activity against both collagen and elastin substrates has been examined in the culture medium of skin fibroblasts derived from unrelated patients with Menkes' syndrome and from control subjects. The medium of three Menkes' fibroblast lines showed 3--30% of the activity present in the medium of control fibroblasts, against a purified collagen substrate. Lysyl oxidase activity in the culture medium of two of the Menkes' fibroblast lines was also examined by using a crude aortic-elastin substrate and was similarly decreased in comparison with that in the medium of control fibroblasts. Lysyl oxidase activity in the medium of a fourth fibroblast line, derived from a foetus with Menkes' syndrome, was 42% of that in the medium of control fibroblasts derived from a 1-day-old baby against a collagen substrate, and 26% of that in control fibroblast medium against an elastin substrate. The copper content of the cell layers of the Menkes' fibroblast cultures was elevated in comparison with normal fibroblast cultures, as has previously been reported to be characteristic of such cells. It is suggested that the decrease in lysyl oxidase activity would help to explain the connective tissue defects observed in Menkes' syndrome, and that this reduction, in conjunction with the elevated concentrations of cellular copper, would support the hypothesis that a functional intracellular copper deficiency exists in Menkes' syndrome.     

Estradiol and raloxifene decrease the formation of multinucleate cells in human bone marrow cultures

Estrogen (E2) deficiency is responsible for increased bone turnover in the postmenopausal period, and it can be prevented by estrogen replacement therapy. The way estrogen acts on bone cells is not fully understood. Human bone marrow cell cultures may be a reliable model for studying the action of steroids on osteoclastogenesis in vitro. We examine the effects of estradiol and Raloxifene, a selective estrogen receptor modulator, on human primary bone marrow cells cultured for 15 days. 17beta-estradiol and Raloxifene significantly decreased the number of tartrate-resistant acid phosphatase multinucleate cells from osteoclast precursors on day 15. Estrogen receptor alpha (ER-alpha) mRNA was present in bone marrow mononuclear cells cultured for 5 days, but there was no estrogen receptor beta (ER-beta) mRNA, suggesting that this effect was mediated by ER-alpha. 15-day cultures no longer contained ER-alpha mRNA, suggesting that estrogen acts on early events of osteoclast differentiation. Finally, 10-8 M 17beta-estradiol has no effect on the release of IL-6 and IL-6-sr into the medium of marrow mononuclear cells cultured for 5 or 15 days. Osteoclast apoptosis was not affected by estradiol or Raloxifene after 15 days of culture under our conditions. In conclusion, we have shown that both estradiol and Raloxifene inhibit osteoclast differentiation in human bone marrow mononuclear cultures. The biological effect that can mimic in vivo differentiation could be mediated through ER-alpha.     

Methylcellulose effect on cell proliferation and glucose utilization in chemically defined medium in large stationary cultures

Three different established strains of mammalian cells were grown in chemically defined medium in large cultures. The degree of proliferation of cells of an established strain from human skin in large stationary cultures was significantly greater in the presence of methylcellulose (medium NCTC 135M) than in its absence (medium NCTC 135). The relatively fragile cells of a derivative of monkey kidney LLCMK2 strain were carried in large stationary cultures through 11 transfer generations during 152 days. The presence of methylcellulose was associated with higher cell population levels, proliferation rates, and cell viability. Cells of this strain utilized glucose at an extremely high rate; during two representative periods the rate averaged 1.2 mg/10⁶ cells/day in cultures on medium 135M and 1.9 mg in medium 135. In a 53-day experiment with mouse fibroblast 2071-L cells, the cells in suspension culture during the first 28 days went through the normal lag, logarithmic plateau, and initial decline phases in medium 135M, and then were transferred to large stationary cultures, where they proliferated for 7 days at uniformly high rates in both medium 135 and medium 135M. It appeared that cells of strain 2071-L in such stationary cultures had no need for Methocel as a protective agent. Glucose utilization rates while these cells were carried in large stationary cultures averaged 2–4 times the rates while they were in suspension cultures: about 0.8 and 0.2 mg/10⁶ cells/day, repectively.