Accumulation of cadmium and copper by the terrestrial snail Arianta arbustorum L.: kinetics and budgets

Summary Specimens of the terrestrial gastropod Arianta arbustorum were fed on cadmium- or copper-enriched agar plates with the aim of performing an input/output analysis and of studying the distribution of these metals in several organs of the snails. After a feeding period of 20 days about 45% of cadmium were lost. 36% accumulated in the hepatopancreas, where a cadmium concentration of more than 500 g/g was measured. The efficiency of cadmium assimilation decreased from about 90% at the beginning to about 55% after 20 days. Copper was distributed more evenly than cadmium, but the main site of copper storage seemed to be the foot/mantle tissues, where 49% of the ingested copper were found. The efficiency of copper assimilation always exceeded 95%. The patterns of distribution and assimilation of copper and cadmium are discussed in relation to differences in the cytological and biochemical detoxification mechanisms which exist for these metals in molluscs.     

Effects of auxin and cytokinin on induction of sister chromatid exchanges in cultured cells of wheat (Triticum aestivum L.)

Summary In order to know the mutagenic effects of synthetic auxins (NAA, 2,4-D, and 2,4,5-T) and a cytokinin (kinetin) in vitro, sister chromatid exchanges (SCEs) were analyzed in cultured cells of a hexaploid wheat (Triticum aestivum L.). In the MS medium supplemented with 2.0 mg/l 2,4-D, the mean number of SCEs per cell was 15.2, and per pg of DNA, 0.42. No significant effect was found in the treatments of NAA or 2,4-D at concentrations of 0.5–10.0 mg/l, whereas more than 2.0 mg/l of 2,4,5-T induced dramatic increases of SCEs. Kinetin itself had no significant effect on SCE induction, but there was a tendency that SCEs induced by 2,4,5-T were suppressed by kinetin.     

Glomerular podocytes in cultured rat kidney slices

Summary The ultrastructure of rat glomerular epithelial cells (podocytes) in kidney slices in vitro was examined using qualitative and quantitative electron microscopy. The kidney slices were cultured in Medium 199 with Hanks' salts in a 5% CO₂/95% O₂ environment for up to 14 days. Few changes in podocyte ultrastructure occurred in the first 12 h of culture, but by 24 h cell bodies were rounded, microvilli were present on all podocyte surfaces, and some foot processes had been replaced by flattened expanses of cytoplasm. These changes were more pronounced by 3 days, when some podocytes had developed pseudopodal extensions and appeared to be migrating from glomeruli onto the slice surface. Podocytes could still be identified after 8, 10 and 14 days of culture, although relatively few glomeruli remained at 14 days. Morphometric methods were used to analyse podocyte shape, volume and surface area during the first 4 days of culture. The most significant change involved loss of foot processes: the number of filtration slits per 100 m of basement membrane decreased from 211.8 ± 15.0 (mean ± SD) at the commencement of culture, to 55.3 ± 22.6 after 2 days (P < 0.001). These data provide baseline information for in vitro studies on the effects of nephrotoxins on podocytes.     

An in vitro demonstration of peroxisome proliferation and increase in peroxisomal β-oxidation system mRNAs in cultured rat hepatocytes treated with ciprofibrate

Using the normal adult rat hepatocytes, plated on rat tail collagen-coated dishes and fed a chemically defined medium, we demonstrate here that ciprofibrate at 0.1 mM concentration, increases significantly the mRNA levels of fatty acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and thiolase (the three enzymes of the β-oxidation system), and causes peroxisome proliferation. Increase in mRNA levels of these genes was evident within 1 h and was maximal 24 h after the addition of ciprofibrate. In hepatocytes cultured in the absence of ciprofibrate, the basal levels of these enzymes were low and further declined with time. Concomitant treatment of hepatocytes with cycloheximide did not inhibit or superinduce the mRNA levels, indicating that this induction may represent a primary (direct) effect of this compound on the expression of these genes and does not apparently involve short-lived repressor protein(s).     

Extracellular matrix influences hormone and protein production by human chorionic villi

Summary Increasing evidence confirms that the extracellular matrix greatly influences cell behaviour and function. Collagen and fibrin are in contact with trophoblast throughout pregnancy. To investigate whether these two matrices influence hormon production by the trophoblast, explants from first-trimester chorionic villi were cultured for up to 30 days either a) in medium with agitation, b) embedded in type-I collagen (three-dimensional gels), or c) embedded in fibrin (three-dimensional gels). The supernatant culture medium was changed every 48 h and tested by radioimmunoassay for hCG, progesterone and pregnancy-associated plasma protein A. In addition, after 3, 7, 15, and 30 days of culture villi were fixed and studied by light and electron microscopy. Embedding in the extracellular matrix showed higher and longer-lasting production rates of all measured products and superior structural preservation as compared to cultures with agitation. Collagen matrix proved to be superior to fibrin. As established by several tests, this difference was neither due to thrombin used to polymerize fibrinogen, nor to differences in the diffusion rates through the two different matrices used. We conclude that extracellular matrix, particularly collagen, influences the synthesis of trophoblastic products. Embedding of the villous explants in three-dimensional gels constitutes a new method for long-term cultures of chorionic villi.This study was presented at the workshop Placental-and decidual-specific protein synthesis and secretion: regulation, role and interaction, Zemun, Belgrade, Yugoslavia, 19–20 May, 1988 (Bischof and Castellucci 1988; see also J. Aplin 1989), and at the 11th Rochester Trophoblast Conference, Rochester, N.Y. USA, 9–12 October 1988 (Castellucci et al. 1988)     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.     

红豆草组织培养物的超低温保存及其超微结构的观察

红豆草(Onobrychis viciaefolia Scop.)组织培养物在5%DMSO+10%甘油+8%蔗糖的冰冻保护剂及以1℃/分钟的速度降温到-35—-40℃,停留2小时后,投入液氮,40℃水浴快速化冻等条件下,存活率达60—70%,并保持了高的分化能力。电子显微镜的观察结果表明,快速冰冻和1℃/分钟慢速冰冻至-35℃—40℃不停留,对细胞结构造成严重的致死性破坏;-35℃停留30分钟对细胞结构的损伤是可逆性的;停留2小时的其超微结构基本上与对照材料无明显差别。     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.     

Sex determination in the dioecious Melandrium

Summary Melandrium album (2n=24), a dioecious species with heteromorphic sex chromosomes (XY, males and XX, females), has a strong genetic commitment for sex determination. We report here a procedure for obtaining haploid plants from cultured anthers and show that genotype, pollen stage, cold treatment and certain culture media components are essential for a reproducible yield of embryos. Our procedure increased the number of responsive anthers and not the number of responsive microspores per anther. Most likely, our experimental system allows the recovery of competent microspores, and this on a medium containing either an auxin or a cytokinin. All of the 36 anther-derived plants tested expressed a female phenotypic sex instead of the theoretical one male one female ratio. When analysed cytologically, the plants exhibited the corresponding female genetic sex (one or two X chromosomes).     

High density microcarrier culture with a new device which allows oxygenation and perfusion of microcarrier cultures

A novel system useful for aeration and cell retention in continuous perfused microcarrier cultures is described. The system is based on a vibrating cage that separates cells and microcarriers from the oxygenation chamber and allows gas bubble free oxygen transfer. In the cultivation of monkey kidney cells (VERO) on gelatin coated microcarriers, using different concentrations (5, 10 and 15 g Cytodex 3/liter) cell densities up to 10⁷ cells per ml were obtained. The described system is scaleable.