Detection of secretion from pancreatic islets using chemically modified electrodes

Secretion of insulin from pancreatic islets was monitored indirectly by detecting zinc. Anodic stripping voltammetric measurements of zinc were done on a bismuth-modified electrode. Comparison of the performance of bismuth-modified electrodes and mercury film electrodes showed that bismuth is an appropriate alternative for Zn detection. The bismuth-coated electrode was used to detect zinc in insulin samples and insulin secreted from pancreatic islets upon stimulation with high concentrations of K(+). Detection of zinc released from pancreatic islets was done in the culture medium without any further cleanup. This detection method can be used to monitor secretion from pancreatic islets in their native environment.     

Zinc effects on LDH, MDH and alkaline phosphatase from cultures of fathead minnow cells.

1. Three purported zinc metalloenzymes have been investigated from cell cultures of the fathead minnow (Pimephales promelas). 2. With the addition of increasingly higher concentrations of zinc to the tissue culture medium, the specific activity of LDH increased. 3. The results with MDH were equivocal. 4. The specific activity of alkaline phosphatase decreased in the presence of increasing amounts of zinc in the growth medium. 5. Zinc exogenously added to the LDH enzyme assay did not alter the LDH enzyme activity of cells grown without zinc.     

Growth-modulating tripeptide (glycylhistidyllysine): association with copper and iron in plasma, and stimulation of adhesiveness and growth of hepatoma cells in culture by tripeptide-metal ion complexes

The tripeptide H-Gly-His-Lys-OH (GHL) is a human plasma constituent which has been previously shown to modulate the growth and viability of a variety of cell types and organisms. Experimental observations presented herein indicate that GHL is complexed with the transition metal ions Cu++ and Fe++ in vivo and may exert its biological effects as a peptide-metal chelate. At physiological pH in vitro, GHL associates with ionic copper, cobalt, iron, molybdenum, manganese, nickel, and zinc, but has no affinity for calcium, manganese, potassium, and sodium. GHL acts synergistically with copper, iron, cobalt, and zinc to alter patterns of cell growth in monolayer cultures of a tumorigenic hepatoma cell line (HTC4). These transition metals induce cellular flattening and adhesion to support surfaces, and inhibit DNA synthesis and lactic acid production when growth is limited by reduction of serum concentrations in medium. These inhibitory effects are neutralized, and intercellular adhesion and growth are stimulated by GHL in medium at nanomolar concentrations. Cu and Fe are the most active metals when combined with GHL. The results suggest that the inability of HTC4 cultures to replicate without adequate concentrations of serum in medium may reflect deficiency of GHL and transition metals, which appear to form complexes prior to interaction with cells. Chelation of transition metals with GHL and, potentially, with other growth-modulating peptide factors in plasma or medium, may provide a mechanism for expression and regulation of biological activities influenced by transition metals and polypeptide growth factors. The observed effects of GHL-metal complexes, including stimulation of cellular adhesiveness to substratum (flattening) and intercellular attachment (monolayer formation), appear to satisfy requirements for growth of hepatoma cells in monolayer culture.     

A simple and effective method for the removal of trace metal cations from a mammalian culture medium supplemented with 10% fetal calf serum

Direct batch addition of sterile Chelex ion-exchange resin to Dubecco's modified Eagle's medium supplemented with 10% fetal calf serum with gentle stirring removed a very wide variety of trace metal ions from the medium to varying extents dependent upon Chelex content (between 0.01 and 4% w/v), exposure time (between 5 min and 10 days) and temperature 4, 25 and 37 °C). Prolonged treatment (10 days) with 4% w/v Chelex at 4°C reduced the concentration of zinc, strontium, aluminum, copper, manganese, nickel and chromium from 100 to 2.7, 12.1, 7.7, 22.6, 13.0, 14.7 and 53.3% of their original concentrations, respectively. Re-supplementation of the metal depleted medium with a defined cocktail of metals restored the growth potential of the medium which was then capable of supporting growth over at least three subcultures without a decrease in fibroblast cell yield, demonstrating its suitability in cell culture studies on trace metal ions.     

Amino acid abundance and composition in cell culture medium affects trace metal tolerance and cholesterol synthesis

Amino acid compositions of cell culture media are empirically designed to enhance cell growth and productivity and vary both across media formulations and over the course of culture due to imbalance in supply and consumption. The interconnected nature of the amino acid transporters and metabolism suggests that changes in amino acid composition can affect cell physiology. In this study, we explore the effect of a step change in amino acid composition from a DMEM: F12-based medium to a formulation varying in relative abundances of all amino acids, evaluated at two amino acid concentrations (lean LAA vs. rich HAA). Cell growth was inhibited in LAA but not HAA. In addition to the expected effects on expression of the cell cycle, amino acid response and mTOR pathway genes in LAA, we observed an unanticipated effect on zinc uptake and efflux genes. This was accompanied by a lower tolerance to zinc supplementation in LAA but not in the other formulations. Histidine was sufficient but not necessary to prevent such zinc toxicity. Additionally, an unanticipated downregulation of genes in the cholesterol synthesis pathway was observed in HAA, accompanied by an increase in cellular cholesterol content, which may depend on the relative abundances of glutamine and other amino acids. This study shows that changes in the amino acid composition without any evident effect on growth may have profound effects on metabolism. Such analyses can help rationalize the designing of medium and feed formulations for bioprocess applications beyond replenishment of consumed components.     

Antibacterial testing of metal ions using a chemically defined medium

Data from in-vitro tests on potential germicides can be greatly influenced by the culture medium. The bioavailability and biochemical reactivity of the biocides can be influenced by chemical interference with media components (Spooner & Sykes 1972). Bird et al . (1985) showed that metal ions are particularly prone to chemical interferences. A chemically defined solid medium has been developed to monitor the antibacterial activity of metal ions. The minimum inhibitory concentrations of zinc and silver have been determined against a range of bacteria using this medium.     

Selection and characterization of cadmium tolerant cells in tomato

Tomato (Lycopersicon esculentum cv. VFNT-Cherry)cell lines tolerant of to 5 mM cadmium (Cd) were selected by progressively elevating the level of CdCl₂ in the culture medium (the lethal concentration of Cd for unselected tomato cells is 400 μM). Cd tolerance was not lost during long-term culture (up to 12 months) in the absence of Cd stress. In all the cell lines examined, Cd uptake was rapid and Cd concentration within the cells exceeded that in the culture medium by several fold. While Cd included the synthesis and accumulation of phytochelatins (PCs) [poly[γ-glutamyl-cysteiny)glycine], little change has been observed in protein synthesis during short term Cd stress. PCs formed complexes with Cd. However, uptake and accumulation of Cd was not affected if PC synthesis was inhibited by treatment with buthionine sulfoximine. Selected and unselected cells were compared for their growth characteristics in the presence of various other metal ions. Cd tolerant cells showed a slightly higher tolerance of copper but not of mercury, zinc, lead or silver.     

Labile intracellular zinc is associated with 3T3 cell growth

Increasing evidence shows that labile intracellular zinc is metabolically important. Depletion of labile intracellular zinc using chelators suppresses DNA synthesis. In this study, we tested the hypothesis that labile intracellular zinc could be modulated via varying zinc nutrition. This could result in an altered availability of labile intracellular zinc, which, in turn, could influence zinc-dependent cellular events involved in cell proliferation and ultimately suppress growth. Labile intracellular zinc was detected by using N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), a membrane-permeable fluorescence probe. After 48 h culture in a zinc-depleted medium, labile intracellular zinc in 3T3 cells was diminished along with a suppressed DNA synthesis and cell proliferation. In contrast, supplementation of zinc to the zinc-depleted medium increased the labile intracellular zinc and promoted DNA synthesis and cell proliferation. Furthermore, growth factor-dependent stimulation of DNA synthesis and cell proliferation was also accompanied by increased labile intracellular zinc. Together, our data showed an association between the labile intracellular zinc, detected using TSQ, and 3T3 cell growth, suggesting that labile intracellular zinc could be an important cellular link between zinc nutrition and growth.     

Effect of nanopreparations on development of the populations of <Emphasis Type="Italic">Saccharomyces</Emphasis> brewer’s yeasts

Effect of nanoparticles of different nature on development of the populations of top and bottom fermentation brewers’ yeasts (Saccharomyces cerevisiae) on various media and on ethanol accumulation during wort fermentation was studied. In some cases the presence of nanoparticles in the medium was shown to affect the monitored parameters, primarily the yeast cell titer. Direction and intensity of this effect depend on a number of factors: nanopreparation kind and concentration, medium composition, and cultivation conditions. Inhibition of development of yeast populations was most pronounced in the presence of the silver nanopreparation, while zinc nanoparticles and zinc oxide nanopreparation had the least effect. At the same time, specific concentrations of some nanoparticles stimulated growth of the population and yeast metabolism.     

Effects of Trace Metals on the Production of Aflatoxins by Aspergillus parasiticus

Certain metals added as salts to a defined basal culture medium influenced the level of aflatoxin production by Aspergillus parasiticus in the low micrograms-per-milliliter range of the added metal. In many cases no change or a relatively small change in mat weight and final pH of the medium accompanied this effect. With zinc at added levels of 0 to 10 mug/ml in the medium, aflatoxin increased 30-to 1,000-fold with increasing of zinc, whereas mat weight increased less than threefold. At 25 mug of added zinc per ml, aflatoxin decreased, but mat weight did not. At an added level of 25 mug or less of the metal per ml, salts of iron, manganese, cooper, cadmium, trivalent chromium, silver, and mercury partly or completelyinhibited aflatoxin production, without influencing mat weight.     

Zinc levels in zinc-stabilized insulin are inhibitory to the growth of cells in vitro

Adult human prostatic epithelium was cultured in a defined medium consisting of RPMI 1640 supplemented with transferrin, insulin, epidermal growth factor, dexamethasone, and vitamin A. In the presence of insulin, stabilized with zinc, maximum epithelial multiplication was obtained at an insulin concentration of 0.03 to 0.1 U/ml, corresponding to a zinc concentration of 1.4 X 10(-7) M. At higher insulin concentrations, growth stimulation declined. Zinc-free insulin, on the other hand, stimulated cell multiplication with an optimum concentration of 0.3 to 1.0 U/ml. At this concentration, the maximum growth was twice that obtained with zinc-stabilized insulin. Results demonstrate that growth inhibition caused by zinc limits the concentration of zinc-stabilized insulin, which can be used in serum-free, defined culture media.     

Hybridoma cell growth and anti-neuroblastoma monoclonal antibody production in spinner flasks using a protein-free medium with microcarriers

The main disadvantages of foetal calf serum as the world-wide common serum supplement for cell growth are its content of various proteins of variable concentrations between batches as well as its high cost. The use of serum-free and protein-free media is gradually becoming one of the goals of cell culture especially for standardizing culture conditions or for simple purification of cell products like monoclonal antibodies. The mouse hybridoma cells 14/2/1 were cultivated either in protein-free UltraDOMA medium or in serum-containing RPMI medium with and without microcarriers to generate high quantities of monoclonal antibodies against neuroblastoma tumour cells. Cell growth rate, IgG production, viability, glucose and lactate concentrations, attachment rate and doubling time have been used as investigation criteria. Modifications of culture procedures (static or stirred), inoculum density, and microcarrier concentration caused an improvement of monoclonal antibody production. The kinetics of antibody synthesis was best in spinner culture with 2 ml of microcarriers in protein-free medium. These results of short-term microcarrier culture in stirred spinner flasks indicate that IgG yields in protein-free medium 2.5-fold higher to those in serum-supplemented medium can be achieved.     

Copper accumulation and metabolism in primary monolayer cultures of rat liver parenchymal cells

The characteristics of hepatic copper accumulation and metabolism were studied using primary monolayer cultures of adult rat liver parenchymal cells. Accumulation of copper from serum-free medium was temperature dependent and strongly inhibited by cyanide and N-ethylmaleimide. Addition of various concentrations of zinc to the medium did not alter copper accumulation by the cells. Furthermore, it was found that supplementation of the cell cultures with dexamethasone significantly stimulated zinc accumulation without affecting the accumulation of copper. Cycloheximide substantially stimulated accumulation of copper from the culture medium, whereas actinomycin D had no effect. Efflux experiments showed that copper is rapidly sequestered by intracellular components and becomes unavailable for exchange soon after it is transported into the cells. Gel chromatography of liver cytosol demonstrated that most of the cooper that is initially accumulated is bound to the low molecular weight cytoplasmic protein metallothionein.     

Penicillium expansum, a resident fungal strain of the orbital complex Mir, producing xanthocillin X and questiomycin A

It was demonstrated that the fungus Penicillium expansum 2-7, a resident strain of the orbital complex Mir, which became dominating at the end of a long-term space flight, formed biologically active secondary metabolites (antibiotics). Using physicochemical methods, these metabolites were identified as xanthocyllin X and questiomycin A. Time courses of their biosyntheses during growth and development of the producer culture were studied. Addition of zinc to the culture medium affected both the growth of the culture and the biosyntheses of the antibiotics. The concentrations of zinc in the medium, optimum for xanthocyllin X and questiomycin A production, amount to 0.3 and 3.0 mg/l, respectively.     

Variation in the growth medium during the culture cycle of Tetrahymena: with special reference to ammonia (NH3), ammonium (NH4+), and pH1

The ciliate Tetrahymena pyriformis was grown in a peptone medium without added glucose. The interrelationship between increasing cell density and pH of the growth medium was studied from mid-log to the stationary phase, i.e. from 50,000 to 1,000,000 cells/ml, by continuous registration of the pH of the growth medium. The present findings correlate with the known physiological, biochemical, and structural changes occurring in Tetrahymena as it passes through the culture cycle. The ammonia production of the cells and the buffer capacity of the growth medium were determined throughout the growth cycle. The results revealed that the ammonia excreted by the cells can explain the increase in pH of the medium from 6.8 to about 8.3 normally seen during the culture cycle. Moreover, neither the increased pH nor the raised level of ammonia were found to be the responsible factor for cessation of cell proliferation in the stationary growth phase although these factors may affect cell proliferation in concentrations well beyond the range found in normal cultures.     

Cell Wall Composition of Neurospora crassa Under Conditions of Copper Toxicity

The mycelia of Neurospora crassa grown in the presence of high concentrations of copper were blue in color, but only on a medium containing inorganic nitrate and phosphate as the nitrogen and phosphate sources, respectively. The cell wall isolate of the blue mycelia contained large amounts (12%) of copper and higher amounts of chitosan, phosphate, and amino groups, with a 42% decrease in the chitin content. Although all the glucosamine of the cell wall of control cultures could be released within 6 h of hydrolysis with acid, that of the blue mycelium required prolonged hydrolysis for 24 h. On removal of copper, the cell wall of the blue mycelium could quantitatively bind again to copper as well as to zinc. Although zinc binding was fivefold greater, copper alone was preferentially bound from a mixture of the two metal ions. Supplementation of iron along with copper in the culture medium resulted in the disappearance of the blue color of the mycelium and restoration of normal growth and composition of the cell wall, probably by limiting the uptake of copper from the medium. The possibility of the cell wall being a specific site of lesion in copper toxicity in the mold is discussed.     

Short- and long-term effects of silver nanoparticles on human microvascular endothelial cells

AIM: To study the response to silver nanoparticles (Ag NP) of human microvascular endothelial cells, protagonists of angiogenesis.METHODS: We cultured human microvascular endothelial cells and endothelial colony-forming cells in their corresponding growth medium. Stock solutions of Ag NP were prepared in culture medium and sonicated before use. They were added at different concentrations and for different times to culture media. The toxicity of Ag NP was investigated by measuring the reduction of yellow tetrazolium salt to dark purple formazan (MTT assay) at 575 nm. After staining with trypan blue, cell proliferation was assessed by counting viable cells. The lactate dehydrogenase leakage assay was performed on culture media by following the oxidation of NADH to NAD+ and monitoring the reaction kinetically at 340 nm. Reactive oxygen species production was quantified using 2’-7’-dichlorofluorescein diacetate. The alkaline comet assay was performed after mixing the cells with low melting-point agarose. Electrophoresis was then conducted and the samples were stained with ethidium bromide and analyzed with a fluorescence microscope.RESULTS: Ag NP are cytotoxic in a dose and time dependent fashion for HMEC. At high concentrations, Ag NP determine loss of membrane integrity as demonstrated by the increased activity of lactate dehydrogenase in the culture medium. Ag NP rapidly stimulate the formation of free radicals. However, pre-incubation with Trolox, apocynin, or N-acetyl-L-cysteine, antioxidants which have different structure and act through different mechanisms, is not sufficient to prevent cytotoxicity. Ag NP also induce DNA damage dose-dependently, as shown by comet assay. When exposed to sublethal concentrations of Ag NP for long times, the cells remain viable but are growth retarded. Interestingly, removal of Ag NP partially rescues cell growth. Also genotoxicity is reversible upon removal of Ag NP from culture medium, suggesting that no permanent modifications occur. It is noteworthy that Ag NP are cytotoxic and genotoxic also for endothelial progenitors, in particular for endothelial colony-forming cells, which participate to angiogenesis.CONCLUSION: Silver nanoparticles are cytotoxic and genotoxic for human microvascular endothelial cells and might become a useful tool to control excessive angiogenesis.     

Regulation of growth and differentiation of a rat hepatoma cell line by the synergistic interactions of hormones and collagenous substrata

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine. To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic. Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.(ABSTRACT TRUNCATED AT 400 WORDS)