Medium For Periods Of Time Research Articles

Cisplatin-induced lipid peroxidation and decrease of gluconeogenesis in rat kidney cortex: Different effects of antioxidants and radical scavengers

The present in vitro study was performed to investigate the effect of the nephrotoxic anticancer agent cisplatin (CP) on lipid peroxidation, on pyruvate-stimulated gluconeogenesis and on p-aminohippurate (PAH) accumulation in rat renal cortical slices. In addition, the inhibitory effects of the antioxidants and radical scavengers N,N′-diphenyl-p-phenylenediamine (DPPD), (+)-cyanidanol-3 or α-tocopherol on CP-induced lipid peroxidation and CP-induced decrease of gluconeogenesis and the inhibitory effect of DPPD on CP-induced decrease of PAH accumulation were evaluated. Slices were incubated in a CP-containing medium for different periods of time (7.5–300 min) and at different concentrations (0.025–1.5 mg/ml). Lipid peroxidation was monitored by measuring the production of malondialdehyde (MDA). Accumulation of PAH was expressed as slice to medium concentration ratio. Pyruvate-stimulated gluconeogenesis, measured as glucose production, was determined after a subsequent 60- or 15-min incubation in a pyruvate-containing, CP-free medium.CP led to a time- and concentration-dependent increase in MDA production, a time- and concentration-dependent decrease of pyruvate-stimulated gluconeogenesis and a time-dependent decrease of PAH accumulation in renal cortical slices. Decrease of gluconeogenesis preceded MDA production and decrease of PAH accumulation. Antioxidants reduced CP-induced MDA production and CP-induced decrease of accumulation of PAH, but did not reverse CP-induced decrease of gluconeogenesis. This might indicate, that the generation of free radicals and subsequent lipid peroxidation may play a role, at least in part, in inducing CP nephrotoxicity. There could be more than one mechanism of CP-induced nephrotoxicity, since decrease of gluconeogenesis preceded MDA production and decrease of PAH accumulation and could not be inhibited by antioxidants and radical scavengers.

What is schizophrenia?

The psychotic syndrome at the core of schizophrenia appears to be invariable across cultures. The risk of morbidity also seems to vary very little from country to country and over medium periods of time. Moreover, apart from gender differences in first onset, the cumulative lifetime risk is the same in females and males. A similar epidemiological pattern is only found in pathological conditions that are characterized by a precisely defined section of a psychopathological dimension with a continuous distribution in the population, e.g. severe mental retardation being the extreme section of normally distributed IQ values. The interpretation of schizophrenic psychosis as the extreme section of a psychopathological dimension or disposition that is almost evenly distributed in all populations is supported by the fact that milder psychiatric disorders occur more frequently before the onset of the psychosis and in close relatives of schizophrenic patients. The psychopathological heterogeneity of these disorders argues against the assumption of a manifest psychopathological dimension with a continuous transition from the schizophrenic psychosis to the "normal" schizothymic personality. More probable is a continuously distributed latent vulnerability to schizophrenia--with or without a threshold effect--which in severe degrees disposes to the uniform reaction pattern of the schizophrenia syndrome. Smaller degrees of vulnerability are associated with an increased risk for milder patterns of disturbances, which are also more strongly determined by environment and personality and therefore are rather heterogeneous. These assumptions lead to other epidemiological and genetic models than Kraepelin's early concept of a disease entity does.

The effect of protein supplementation on single-cell mouse embryos in vitro

For determination of the effect of protein supplementation on single-cell mouse embryos in vitro, 842 single-cell embryos were cultured initially in protein-free medium for variable periods of time and then transferred to bovine serum albumin (BSA)-supplemented medium. The embryos cultured for 24 hours or longer in protein-free medium demonstrated significantly reduced growth rates. An additional 770 embryos were cultured in BSA-supplemented medium initially and then transferred to human fetal cord serum (HCS)-supplemented medium. Significantly reduced growth rates were observed when transfer from BSA to HCS occurred at 48 hours or earlier, and an increased hatching rate was seen if culture in BSA-supplemented medium continued for up to 57 hours. Another 492 embryos were cultured in HCS-supplemented medium and then transferred to BSA. Significantly reduced growth rates were observed when the transfer occurred at 24 hours or later. HCS was then separated by ultrafiltration (10,000 mol wt cutoff), and 1109 embryos were cultured in the media with BSA, whole HCS, small molecular weight fraction (SMCS), or large molecular weight fraction (LMCS) of HCS or BSA and SMCS. The embryos cultured with SMCS demonstrated a significantly reduced growth rate even if BSA was added in the medium. In summary, it appears that the type and timing of protein supplementation of the culture medium is important to embryo growth. Moreover, it seems that serum contains properties that may even be toxic to embryo survival in vitro.

Histones and the first cell cycle in maize germination

The timing of the onset of cell division during seed germination in maize and the role of histones for this process have been studied. Embryonic axes of maize seeds (Zea mays L. hybrid H‐30) were incubated in a sterile nutrient medium for different periods of time. For some experiments putrescine was also added. Mesocotyl, root tip and scutellar node were dissected at specific periods after incubation and the mitotic indices were determined in these tissues. Embryonic axes were incubated in the same medium either with [14C]‐lysine or [32P]‐phosphate. The incorporation of either 14C or 32P into histones was followed, both in postribosomal supernatant and in nuclei. It was found that during germination, there is specific timing for meristematic cells entering into cell division. Among the tissues tested, the mesocotyl meristem was the first to initiate this process. De novo synthesis of histones was detected as early as after 6 h of imbibition and the rate increased up to 12 h. Putrescine stimulated cell division and phosphorylation of the histones. The implications of these findings are discussed.

An established avian fibroblast cell line without mitochondrial DNA.

An established avian fibroblast cell line (LSCC-H32) has been found to be inherently resistant to the growth-inhibitory effect of ethidium bromide, when supplied with exogenous uridine. After long-term exposure to ethidium bromide (90 days), the cell population has been transferred to drug-free medium for 60 days, and then seeded at low cell density. Three clones have been isolated and propagated in drug-free medium for 5, 6, and more than 12 months, respectively. It was found that none of these cell lines had detectable cytochrome c oxidase activity and that they were virtually devoid of cytochromes aa3 and b. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 300 copies of mitochondrial DNA per cell was found in LSCC-H32 cells. Analysis of DNA extracted from cell populations exposed to ethidium bromide for 90 days and then transferred to drug-free medium for long periods of time revealed no mitochondrial DNA molecules by reassociation kinetics or by Southern blot hybridization of HindIII-or AvaI-digested total cellular DNA.

Membrane potential changes induced by the ouabain-like compound extracted from mammalian brain.

The electrical membrane potential (delta psi) of chicken embryo fibroblasts in tissue culture was determined to be -30.5 +/- 2.9 mV as measured by distribution of the lipophilic [3H]tetraphenylphosphonium cation (Ph4P+). Stimulation of the electrogenic activity of the Na+,K+-ATPase by the ionophore monensin induces a hyperpolarization of approximately 47 mV and a new delta psi of -77.3 +/- 5.7 mV. The effects of the cardiac glycoside ouabain and an "ouabain-like compound" (OLC), which was extracted and partially purified from sheep brain, were contrasted using both the resting and hyperpolarized fibroblasts. Addition of OLC or ouabain to the incubation medium for short periods of time does not alter the cells' resting delta psi. However, OLC and ouabain block monensin-induced hyperpolarization. The inhibitory effects of OLC, like ouabain, are dose dependent, with half-maximal inhibition occurring at an amount of OLC equivalent to that found in 1.6 g of brain (wet weight) per ml and at 0.85 microM ouabain. In addition, the maximal actions of ouabain and OLC are not additive. These results show that the endogenous OLC specifically affects the delta psi of intact cells by a mechanism analogous to that of ouabain--i.e., inhibition of the Na+,K+-ATPase.

DNA heterogeneity and genetic control of tumorigenesis in nicotiana tumorous and nontumorous genotypes

AbstractThe possible relevance of changes in amounts of highly repetitive DNA sequences for plant differentiation and dedifferentiation processes has been suggested in several cases. Data are lacking however on (1) the genetic control of these phenomena and (2) cause‐effect relationships between DNA amplification and specific ontogenetic patterns.The present study was carried out on a Nicotiana genetic system consisting of the tumorous amphidiploid N glauca X N langsdorffii, a nontumorous mutant of it, their F1, and a backcross to the tumorous parent. Backcross segregation ratios were shown to be compatible with a “single gene” hypothesis, the F1 plant being nontumorous but showing a low percentage of tumors induced by wounds, 6‐azauracil or X‐rays.In vitro studies of excised pith tissue grown on Linsmaier and Skoog medium for different periods of time showed the presence, confirmed by cytological analyses, of amplification of highly repetitive sequences only in the nontumorous stock, as judged by reassociation experiments in the first 24–96 hours of culture. CsCl analytical ultracentrifugation of those sequences showed the appearance in the same stock of a heavy DNA satellite (density = 1.721 gm/ml), whose presence was also confirmed by derivative melting curves.Amplification seemed to be essential for the initiation of cell division, which was completely inhibited in the nontumorous genotype and partially influenced in the F1 by incorporation during the critical period (24–96 hours of the primary explant) of 5‐bromo‐2′‐deoxy‐uridine.The results are discussed in terms of an hypothesis of an integrated gene‐controlled, hormone‐mediated regulatory system of cell proliferation involving changes in target repetitive DNA sequences.

Alteration of Acrylonitrile-Methylacrylate-Butadiene Terpolymer by Nocardia rhodochrous and Penicillium notatum

[C]Barex-210, a terpolymer of acrylonitrile, methylacrylate, and butadiene, was tested for bioconversion. Powdered samples of polymer, each specifically C labeled at different carbon atoms of the polymer, were incubated with either Nocardia rhodochrous or Penicillium notatum in an enriched growth medium for various periods of time. After 6 months of incubation, the C-labeled polymer was transformed from a high-molecular-weight material completely soluble in dimethyl formamide (DMF) into both a lower-molecular-weight form still soluble in DMF and a second form that was no longer soluble in DMF. The amount of C-labeled carbon atoms converted into DMF-insoluble material was 8% of the backbone carbon-carbon atoms and 12% of the side-chain nitrile and acrylate atoms from the acrylonitrile-methylacrylate copolymer and 60% of the elastomer (acrylonitrile-butadiene copolymer) atoms. Metabolism of the polymer was not established from measurements of metabolic CO(2). Evolution of CO(2) amounted to only 0.3, 0.6, 1.8, and 3.3% of these four fractions, respectively. Although the transformation of high-molecular-weight polymer into DMF-insoluble material was rapid in the early stages of microbial growth, the accompanying CO(2) evolution was much slower. Further evidence of polymer alteration was indicated by the infrared spectrum of the insoluble material, which showed a disappearance of the nitrile and methylacrylate peaks.

Foetal-associated Material: Its Expression in Long-term Cultured Human Skin

Using immunofluorescence and electron microscopy, we have demonstrated the presence of "foetal substances" (antigens) in skin kept in tissue culture medium for periods of time ranging from 9 days-18 weeks. This is a specific reaction demonstrated with an anti-human foetal serum raised in New Zealand white rabbits. It is suggested that the expression of foetal substances by cells in the skin under these abnormal conditions might lead to the masking or modification of the normal histo-compatible antigens, enabling such skins to survive transplantation without rejection.

Morphological studies of cultured swine aorta media explants.

Expiants obtained from the media of swine abdominal aortas, free of intimai cells and of vasa vasis, were cultivated in a semisynthetic medium for various periods of time, up to 50 days. Under these conditions it was observed that some smooth muscle cells, apparently independently from their location, undergo dedifferentiation, become rich in organelles, migrate and possibly multiply. These cells, found outside the original expiant, are capable of synthesis of collagen and elastic substance. Other cells seem unable to become activated and after some time die. It is suggested that cells capable of dedifferentiation “in vitro” may be the same that “in vivo” under adequate stimulation are responsible for proliferative atherosclerotic lesions.

Megalodiscus temperatus: Comparative radioautography of glucose- 3H and galactose- 3H incorporation

Whole worms and slices of the trematode, Megalodiscus temperatus, were incubated for varying periods of time in media containing either galactose- 3H or glucose- 3H. Using radioautographic techniques for light and electron microscopy, the distribution of the label of galactose- 3H was compared with that of glucose- 3H. Three morphological areas were used in the comparison, the subtegumental cells, the subtegumental musculature, and the tegument with glycocalyx. The glucose label was incorporated only into the subtegumental musculature, the galactose label was incorporated primarily into the subtegumental cells and the tegument with glycocalyx. Galactose label was located first over the Golgi area and later over the rod-shaped vesicles in the tegument and glycocalyx. Whole worms incubated in the various media did not display label even after 5 hr of incubation. It is suggested that in the natural state, the only mechanism by which precursors of glycogen and other complex carbohydrates can be introduced into the worm is by way of ingested food, and the tegument and glycocalyx of Megalodiscus temperatus are probably protective in function. It is further hypothesized that the galactose is incorporated into the neutral mucopolysaccharide comprising the glycocalyx, and this incorporation occurs initially in the Golgi region of the subtegumental cells.

Cytogenetic analysis of pig oocytes matured in vitro.

AbstractPrimary oocytes from the ovaries of prepubertal pigs were stimulated to undergo maturation by removing them from small follicles and incubating them in a defined culture medium for various periods of time. The cultured oocytes were air‐dried and their chromosomes examined for the stage of meiosis and for the presence of abnormalities. Of 107 oocytes recovered after 26–27.5 hours of culture 41% were at diakinesis‐metaphase I and 2% were at telophase I. Of 96 oocytes recovered after 36–37 hours of culture 46% were at diakinesis‐metaphase I while only 14% were at metaphase II. These observations suggest that the oocytes were delayed at diakinesis‐metaphase I, since it has been shown previously that at 36–37.25 hours after HCG injection only 23% of oocytes are at metaphase I and 45% are at metaphase II in vivo. When recovered after 42.5–55 hours of culture 128 of 232 oocytes (55%) had reached metaphase II.Of 56 cultured oocytes whose chromosomes were analysed at metaphase I, only one exhibited a chromosomal aberration (19 bivalents plus three fragments) and one oocyte contained 18 bivalents plus two monovalents.Of 123 oocytes analysed at anaphase I, telophase I and metaphase II, 33 (27%) exhibited such chromosomal abnormalities as heteroploidy, fragments, chromosomal bridges, tripolar spindles and lagging chromosomes. In addition 31% of 128 oocytes at metaphase II displayed degenerate chromosomes. Therefore, of 232 immature oocytes cultured for 42.5–55 hours only about 20% could be regarded as chromosomally normal, mature secondary oocytes.The delay at diakinesis‐metaphase I may be related to a failure of the supporting granulosa cells to supply some substance, possibly an energy source, required by the oocytes. It is suggested that during a delay at diakinesis‐metaphase I, the chromosomes of pig oocytes which failed to divide, may have become “sticky,” leading to the chromosomal aberrations observed at subsequent stages of meiosis.

Effect of peptides on animal cells in culture: I. Maintenance of beating heart cells

Chick embryo heart cells grown as organ cultures or monolayer cultures maintained beating for varying periods of time in media supplemented with Bacto-Peptone or Proteose Peptone but in the absence of serum. Electron micrographs of these cultures suggested a “normal ultrastructure”, and initial fractionation studies indicated that the factor(s) in either Bacto-Peptone or Proteose Peptone which contributed to the maintenance of beating could be partially purified by gel filtration. Serum, routinely used in tissue culture studies, is not only complex but the response of cells to different lots of serum has been reported to vary significantly [4, 8, 21, 23, 24]. These problems might be resolved by identifying the active component(s) of serum or by substituting less complex materials for the serum component. The latter has been reported by Amborski & Moskowitz [1], and by Amborski et al. [2]. These results suggested that peptides might be used in the place of serum in studies on the growth and development of primary cultures. This report describes the effects of similar peptides on primary cell cultures derived from embryonic chick hearts cultivated under different conditions; in organ culture, in aggregate culture and in monolayer culture.