Membrane resting potentials in cultured mouse neuroblastoma cells

Summary Membrane resting potentials (MRP) were measured systematically in cultured mouse N₂A neuroblastoma cells: 1) in the logarithmic growth phase; 2) in subconfluent cultures; 3) in confluent cultures; 4) after dBcAMP had induced morphological differentiation. Neurite extension was accompanied by a significant increase in MRP as compared to the appropriate controls. No significant differences in MRP were observed with regard to the different growth phases.     

Multiple flavonoid-binding sites within multidrug resistance protein MRP1

Recombinant nucleotide-binding domains (NBDs) from human multidrug resistance protein MRP1 were overexpressed in bacteria and purified to measure their direct interaction with high-affinity flavonoids, and to evaluate a potential correlation with inhibition of MRP1-mediated transport activity and reversion of cellular multidrug resistance. Among different classes of flavonoids, dehydrosilybin exhibited the highest affinity for both NBDs, the binding to N-terminal NBD1 being prevented by ATP. Dehydrosilybin increased vanadate-induced 8-N₃-[-³²P]ADP trapping, indicating stimulation of ATPase activity. In contrast, dehydrosilybin strongly inhibited leukotriene C₄ (LTC₄) transport by membrane vesicles from MRP1-transfected cells, independently of reduced glutathione, and chemosensitized cell growth to vincristine. Hydrophobic C-isoprenylation of dehydrosilybin increased the binding affinity for NBD1, but outsite the ATP site, lowered the increase in vanadate-induced 8-N₃-[-³²P]ADP trapping, weakened inhibition of LTC₄ transport which became glutathione dependent, and induced some cross-resistance. The overall results indicate multiple binding sites for dehydrosilybin and its derivatives, on both cytosolic and transmembrane domains of MRP1.Received 1 May 2003; received after revision 18 June 2003; accepted 24 June 2003     

Effects of prolactin and growth hormone on DNA synthesis of rat mammary carcinomas in vitro

Summary Explants derived from mammary carcinomas of DMBA-treated female Sprague-Dawley rats were cultured for 5 days in Medium 199 containing insulin and corticosterone. The addition of ovine prolactin to the culture media resulted in a consistent significant increase in H³-thymidine incorporation into DNA. DNA synthesis of explants treated with either ovine or human growth hormone was intermediary to prolactin-treated cultures and control cultures. A combination of prolactin and human growth hormone often increased DNA synthesis above either hormone alone, suggesting a possible growth synergism between these peptides.Supported by NIH research grant No. CA-13777 and American Cancer Society research grant No. ET-59.NIH Research Career Development Awardee No. CA-35027.     

Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease

RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7–10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.     

Clonogenic growth of acute non-lymphocytic leukemia cells in serum-free medium

We devised a serum-free medium for growth of leukemic colony-forming units (CFU-L), enriched with albumin, transferrin, lipids, insulin, hydrocortisone and oligoelements. Blast cells from 15 patients affected by acute non-lymphocytic leukemia were grown in this medium in the presence of human placental conditioned medium obtained under serum-free conditions (sfHPCM). Their clonogenic growth was comparable with that obtained in a serum-containing system. Furthermore, when serum-free cultures were carried out in absence of sfHPCM, either CFU-L growth was prevented or, if clones were obtained, the cultures showed a marked decrease in clonogenicity, indicating their strict dependence on growth factors.     

Baker's yeast,an attractant for baiting traps for Chagas' disease vectors

We tested the attraction of volatile compounds, produced by the aerobic growth ofSaccharomyces cerevisiae on saccharose forTriatoma infestans. For these tests, we exploited the behavioural characteristic of these haematophagous insects of dropping when searching for food. In olfactometer assays, yeast cultures activated and attracted bugs as effectively as a mouse. The attraction of the cultures was significantly reduced when the carbon dioxide released was partially eliminated using potassium hydroxide. Yeast cultures were also tested as lures in a novel trap device. A baited device for trapping Chagas' disease vectors using the behavioural peculiarities ofT. infestans and this simple attractant is described.     

Metionina,etionina, colina nel metabolismo intermedio dei fibroblasti coltivatiin vitro

Summary The administration of ethionin to tissue cultures of fibroblastsin vitro causes a rapid and total inhibition of the growth of the transplanted tissues.It was observed that equimolar quantities of methionin are, on the contrary, well tolerated. The inhibition is therefore seen to be due to the dismetabolic activity of the antagonist.The contemporary administration of cholin is not able to restore the growth capacity of ethionin inhibited tissue cultures.In conclusion, ethionin produces a direct action on fibroblastsin vitro, probably intervening in the process of protein synthesis of the transplanted elements.     

Clonogenic growth of acute non-lymphocytic leukemia cells in serum-free medium

Summary We devised a serum-free medium for growth of leukemic colony-forming units (CFU-L), enriched with albumin, transferrin, lipids, insulin, hydrocortisone and oligoelements. Blast cells from 15 patients affected by acute non-lymphocytic leukemia were grown in this medium in the presence of human placental conditioned medium obtained under serum-free conditions (sfHPCM). Their clonogenic growth was comparable with that obtained in a serum-containing system. Furthermore, when serum-free cultures were carried out in absence of sfHPCM, either CFU-L growth was prevented or, if clones were obtained, the cultures showed a marked decrease in clonogenicity, indicating their strict dependence on growth factors.     

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: effect of nafenopin and electroporation

Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive beta-oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes. In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. 'Whorl formation' by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.     

Effet de divers bois sur certains micro-organismesin vitro

Summary Mycobacterium tuberculosis was brought into contact with stoved beech-tree wood in physiological saline solution. After a contact of 6, 12, and 24 h, respectively, between the beech-wood and the suspension of microorganisms in saline, this latter was sowed on Loewenstein culture medium. The suspensions which were in contact with the beech-wood for 6 h, gave cultures not different from the controls without wood. With the 12 h ones, the growth of mycobacterium tuberculosis was greatly delayed, whereas with the 24 h ones the cultures remained sterile.     

Chemical control of eucaryotic and blue-green algae in anaerobic photoreactors culturing Rhodospirillaceae

Summary To control the growth of eurocaryotic and blue-green algae in anaerobic reactors of photosynthetically grown Rhodospirillaceae, the effect of algae inhibitors with different modes of action was examined. Tests were performed with mixed populations of green algae and blue-green algae, besides strains of the purple nonsulphur bacteriaRhodopseudomonas capsulata, Rhodospirillum rubrum andRhodomicrobium vannielii. Chloroxuron, a urea-derivative, was found to inhibit completely growth of green and blue-green algae at 5 ppm. When it was applied to the Rhodospirillaceae cultures, growth was not reduced and nitrogenase activity was not inhibited.     

The use of primary cultures of adult rat hepatocytes to study induction of enzymes and DNA synthesis: Effect of nafenopin and electroporation

Summary Primary cultures of adult rat hepatocytes maintained in a well-differentiated state, in a chemically defined medium containing 2% DMSO, have been utilized to study the effect of non-mutagenic hepatocarcinogens such as the peroxisome proliferator nafenopin. The parameters chosen in this in vitro system were those that paralleled the major in vivo effects of nafenopin on the liver, mainly: the proliferation of the endoplasmic reticulum and induction of cytochrome P-452, the proliferation of the peroxisome compartment and the induction of cyanide-insensitive -oxidation of fatty acids and the stimulation of liver growth as measured by the DNA synthetic activity of the hepatocytes.In this review, we also describe the morphology of hepatocyte cultures prepared from previously electroporated hepatocytes and the potential for the use of electroporation to introduce growth related genes into hepatocyte cells to study the mechanisms of hepatocyte growth at the molecular level. In addition we describe the formation of endoplasmic reticulum whorls in these cultures as a consequence of nafenopin treatment. Whorl formation by hepatotrophic chemicals has been previously shown to occur in vivo; in this report, it is described for the first time in vitro.     

Effects of carmine and carminic acid on embryonic tissue cell cultures

Summary The biological reaction to carmine and carminic acid at cellular level on in vitro cultures was tested and significant variables were controlled. Results suggested that proliferation and metabolism of these cultures were not affected by the 2 stains.     

Adenosine triphosphatase ofAspergillus nidulans: Stimulation by aminoacids

Summary Ca²⁺-dependent ATPase ofAspergillus nidulans was found to be stimulated by aminoacids in vitro. Both histidine and arginine stimulated the enzyme more effectively than the aromatic aminoacids. Supplementation of the growth medium with basic or aromatic aminoacids increased the enzyme activity in vivo 2–6-fold. The enhanced activity observed in these cultures in vivo was not mediated through the synthesis of new isoenzyme, as observed in proteinenriched cultures, but appeared to be through the activation of enzyme activity.     

Kinetic analysis,size profiling,and bioenergetic association of DNA released by selected cell lines in vitro

Although circulating DNA (cirDNA) analysis shows great promise as a screening tool for a wide range of pathologies, numerous stumbling blocks hinder the rapid translation of research to clinical practice. This is related directly to the inherent complexity of the in vivo setting, wherein the influence of complex systems of interconnected cellular responses and putative DNA sources creates a seemingly arbitrary representation of the quantitative and qualitative properties of the cirDNA in the blood of any individual. Therefore, to evaluate the potential of in vitro cell cultures to circumvent the difficulties encountered in in vivo investigations, the purpose of this work was to elucidate the characteristics of the DNA released [cell-free DNA (cfDNA)] by eight different cell lines. This revealed three different forms of cfDNA release patterns and the presence of nucleosomal fragments as well as actively released forms of DNA, which are not only consistently observed in every tested cell line, but also in plasma samples. Correlations between cfDNA release and cellular origin, growth rate, and cancer status were also investigated by screening and comparing bioenergetics flux parameters. These results show statistically significant correlations between cfDNA levels and glycolysis, while no correlations between cfDNA levels and oxidative phosphorylation were observed. Furthermore, several correlations between growth rate, cancer status, and dependency on aerobic glycolysis were observed. Cell cultures can, therefore, successfully serve as closed-circuit models to either replace or be used in conjunction with biofluid samples, which will enable sharper focus on specific cell types or DNA origins.     

Influence of progesterone on protein and RNA synthesis in cultured chick embryo liver cells

Summary Chick embryo liver primary cultures, when added with progesterone, exhibit, in comparison with the controls, a normal growth, a decline of both³H-uridine uptake and incorporation into total RNA, a decline of³H-leucine and¹⁴C serine incorporation into the proteins. Progesterone is not able to stimulate phosvitin synthesis induction.These studies were supported in part by the Italian CNR grants.     

Effects of carmine and carminic acid on embryonic tissue cell cultures

The biological reaction to carmine and carminic acid at cellular level on 'in vitro' cultures was tested and significant variables were controlled. Results suggested that proliferation and metabolism of these cultures were not affected by the 2 stains.     

Nicotinamide desamidase in the culture medium of neuroblastoma cells]

Important amounts of nicotinic acid appear in the growth medium of neuroblastoma cell cultures. It is shown that an enzyme released by the cells into the growth medium deamidates the nicotinamide supplied by the growth medium to nicotinic acid.     

Bovine microvascular endothelial cells of separate morphology differ in growth and response to the action of interferon-γ

Five cell types recently isolated from the bovine corpus luteum differed in their epithelioid morphology and their cytoskeleton, but shared common criteria of microvascular endothelial cells^1,2. To give strong evidence for the separate entity, the growth rate of the 5 phenotypically different cells was studied. They were seeded at low density on day 0. Most of these cells were treated with 200 to 1000 U recombinant bovine interferon- (IFN-) for 3 days. The untreated remainder served as controls. Cell counts were made for all cultures on days 4, 7, 10 and 13. morphology: 13 d after treatment with IFN- senescent cells as well as intact cells occurred in cultures of cell types 1 to 4. Cultures of cell type 5 were apparently unchanged and resembled their untreated counterparts. Desminpositive cells in cultures of cell type 2 developed cell processes. Growth rate: In the absence of IFN-, the growth rate was high for cell types 3 and 4, moderate for cell type 1, and low for cell types 2 and 5. The presence of IFN- caused anti-proliferative effects. These were higher for cell types 3 and 4 than for cell types 1 and 2. IFN- could be cytotoxic on cell type 3. In contrast, the cytokine tended to support the cell growth of cell type 5. These findings substantiate the postulate that endothelial cells exhibiting separate morphology in culture also function differently.     

Use of aggregating cell cultures for toxicological studies

Relatively simple techniques are now available which allow the preparation of large quantities of highly reproducible aggregate cultures from fetal rat brain or liver cells, and to grow them in a chemically defined medium. Since these cultures exhibit extensive histotypic cellular reorganization and maturation, they offer unique possibilities for developmental studies. Therefore, the purpose of the present study was to investigate the usefulness of these cultures in developmental toxicology. Aggregating brain cell cultures were exposed at different developmental stages to model drugs (i.e., antimitotic, neurotoxic, and teratogenic agents) and assayed for their responsiveness by measuring a set of biochemical parameters (i.e., total protein and DNA content, cell type-specific enzyme activities) which permit a monitoring of cellular growth and maturation. It was found that each test compound elicited a distinct, dose-dependent response pattern, which may ultimately serve to screen and classify toxic drugs by using mechanistic criteria. In addition, it could be shown that aggregating liver cell cultures are capable of toxic drug activation, and that they can be used in co-culture with brain cell aggregates, providing a potential model for complementary toxicological and metabolic studies.