Differences in reactivity of confluent and nonconfluent cultures of human endothelial cells toward thrombin-stimulated platelets or heparinized salt solution

Summary This study examined whether nonconfluent endothelial cell cultures reacted differently than confluent ones toward thrombin-stimulated platelets or a heparinized salt solution. The adherence to the endothelial cell cultures of⁵¹Cr-labeled human platelets stimulated at different thrombin concentrations was studied. There was significantly higher adherence of stimulated platelets to nonconfluent cultures compared with confluent ones. This was confirmed by scanning electron microscopy, which also revealed a tendency for the platelets to adhere at the cell periphery. Electron microscopy also showed that thrombin-stimulated platelets induced endothelial cell contraction. Part of the peripheral endothelial cell surface toward the bottom of the culture dish was inverted, facing the lumen of the dish. This phenomenon was particularly seen in nonconfluent cultures. When⁵¹Cr-labeled endothelial cultures were incubated with a mildly injurious fluid as heparinized sodium acetate and 20% serum, at 20° C for 30 min, the nonconfluent cultures showed significantly more cell detachment and release of⁵¹Cr than the confluent ones. We conclude that under the conditions of the present experiments there are differences in the reactivity of confluent and nonconfluent endothelial cell cultures. These differences probably reflect biological dissimilarities. In experiments where properties of cultured endothelium are studied, care should be taken that the degree of confluency is standardized.     

Antigenic profiles of human,bovine and canine articular chondrocytes

Summary Human, bovine and canine articular chondrocytes have been shown to bear cartilage matrix, chondrocyte-specific and histocompatibility antigens. These cell-surface antigens of chondrocytes were demonstrated both simultaneously and separately either by complement-mediated cytotoxicity or by immunohistochemical reactions. The chondrocyte-specific antigens involve subsets of species-common and species-specific determinants, which are also present on the surfaces of rib and laryngeal chondrocytes. In addition to these antigens, human and calf articular chondrocytes also express unique cell-surface components that are capable of producing a blastogenic stimulation of autologous T-lymphocytes in vitro. These putative autoantigens segregated from lymphocytes in vivo could be released in trauma and in inflammatory joint diseases triggering the immune system of the host.     

Cell envelope phospholipid changes in a moderate halophile during phenotypic adaptation to altered salinity and osmotic stress

Abstract The increased content of negatively-charged phospholipids in membranes of Vibrio costicola grown at high salinities is mediated by increased phospholipid synthesis of phosphatidylglycerol relative to phosphatidylethanolamine. This phenomenon provides a system for investigating the factors involved in triggering and controlling haloadaptation in this moderately halophilic bacterium. We review recent experiments, which show that when subjected to sudden increases in external salinity, V. costicola senses both the absolute NaCl concentration and the magnitude of the salt shift. We show that the latter is sensed at least in part via osmotic pressure effects, since shift-up into sucrose-containing media triggers comparable changes in growth and in phospholipid composition and synthesis.     

An enzymatic assay for acetate in spent bacterial culture supernatants

A method is presented for the rapid enzymatic determination of acetate in spent bacterial culture supernatants. The assay is based on a previously published assay for acetate kinase [Bergmeyer et al. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. V., ed.), Vol. 1, pp. 425-426, Verlag Chemie-Academic Press, New York/London], and is sufficiently sensitive to detect acetate levels of 50 microM. The assay is cheaper than commercially available assays and is particularly useful for occasional use by laboratories not equipped for routine acetate analysis using gas chromatography. The application of the assay to the measurement of acetate in bacterial cultures is described, though it should also be applicable to other biological fluids and foodstuffs.     

On the Phospholipid Metabolism of Glial Cell Primary Cultures.

Abstract: Primary cultures were prepared from newborn rat brain. After 16-18 days, they consisted mainly of mature and immature astrocytes and oligodendrocytes, as judged by immunohistochemistry. To study the metabolism of ethanolamine glycerophospholipids, the cells were incubated with 1-[1-³H]alkyl- sn -glycero-3-phosphoethanolamine (1-alkyl-GPE), for 1–20 h. Five main products were formed: 1-alkyl-2-acyl-GPE; 1-alkyl-2-acyksn-glycero-3-phosphocholine (1-alkyl-2-acyl-GPC); 1-alkenyl-2-acyl-GPE (ethanolamine plasmalogen); 1-alkenyl-2-acyl-GPC (choline plasmalogen); and 1-alkyl-glycerol. Acylation of the substrate was the main reaction during the first 3 h of incubation, whereas desaturation to plasmaiogen reached a maximum after 12 h. Greater amounts of radioactivity were observed in the phosphatidylcholine fraction after longer incubation times. Only small amounts of choline plasmalogen were observed. The phosphatidylethanolamine fraction consisted of 26.5% diacyl-, 27.5% alkyl-acyl-, and 46.0% alkenyl-acyl- compounds, whereas the corresponding data for the phosphatidylcholine fraction were 78.5, 16.4, and 5.1%, respectively, after 20 h of incubation. Hydrolysis of the substrate to 1-alkyl-glycerol was a minor reaction.     

The relationship between serum and cell type on the development of rat and pig cultured preadipocytes

Stromal-vascular cells from rats and pigs were isolated from adipose tissue and used to measure preadipocyte proliferation and differentiation. Cells from rats and pigs were grown in either 2.5% pig serum or 2.5% rat serum. Cells were either supplemented or unsupplemented with insulin after five days of growth in culture. In these cultures, pig fat cells developed as discrete clusters while rat fat cells developed as loose clusters or as individual cells. Rat cells had greater levels of sn-glycerol phosphate dehydrogenase activity compared to pig cells. Rat serum increased soluble protein in plated cells when compared to cells grown in pig serum. Pig serum increased glycerol phosphate dehydrogenase specific activity when compared to rat serum. In this system, there was no response to insulin. The cells grown in rat serum did not resemble adipocytes in regard to the presence of large lipid droplets (oil red 0 staining). These results demonstrate that rat and pig stromal-vascular cells in culture are morphologically different. Cells from both species, however, responded similarly to sera from either species showing that cells from rats and pigs responded to the growth and differentiation factors present in these sera.Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Comparison of α1-Adrenergic Receptor-Stimulated Inositol Phosphate Formation in Primary Neuronal and Glial Cultures

alpha 1-Adrenergic receptor binding sites and norepinephrine-stimulated 3H-inositol phosphate (3H-InsP) accumulation were measured in primary cultures of neurons and glia from 1-day-old rat brains. The density of alpha 1-adrenergic receptor binding sites was approximately three times higher in membranes from neurons compared to glia. Although norepinephrine was slightly more potent in stimulating 3H-InsP formation in neurons than in glia, the maximal response was greater in glial cells. Norepinephrine-stimulated 3H-InsP formation remained constant for [3H]inositol prelabelling periods of 1-14 days in neurons, whereas the response increased with time in glia and was maximal after 7-10 days of prelabelling. Both the incorporation of [3H]inositol into lipid and basal levels of 3H-InsPs were lower in glial cells than in neurons, which accounted for the greater percent stimulation in glia. Pretreatment with phenoxybenzamine decreased norepinephrine-stimulated 3H-InsP formation in a dose-dependent manner in both neurons and glia by decreasing the maximal response without altering potency. HPLC separation showed that similar types of 3H-InsPs were accumulated in neurons and glial cells. These results demonstrate that alpha 1-adrenergic receptors exist on both neurons and glial cells and activate 3H-InsP accumulation in both cell types. Although receptor density is higher in neurons than in glia, the 3H-InsP response is higher in glia. This difference does not appear to be due to different receptor reserves, but may be due to differential coupling mechanisms in the two cell types.     

Effects of three herbicides on soil microbial biomass and activity

Three post-emergence herbicides (2,4-D, picloram and glyphosate) were applied to samples of an Alberta agricultural soil at concentrations of 0, 2, 20, and 200 μg g⁻¹. The effects of these chemicals on certain microbial variables was monitored over 27 days. All herbicides caused enhancement of basal respiration but only for 9 days following application, and only for concentrations of 200 μg g⁻¹. Substrate-induced respiration was temporarily depressed by 200 μg g⁻¹ picloram and 2,4-D, and briefly enhanced by 200 μg g⁻¹ glyphosate. It is concluded that because changes in microbial variables only occurred at herbicide concentrations of much higher than that which occurs following field application, the side-effects of these chemicals is probably of little ecological significance.     

Actions of Excitatory Amino Acids on Somatostatin Release from Cortical Neurons in Primary Cultures

L-Glutamate, N-methyl-D-aspartic acid (NMDA), quisqualate, and kainate were found to increase endogenous somatostatin release from primary cultures of rat cortical neurons in a dose-dependent manner. The rank order of potency calculated from the dose-response curves was quisqualate greater than glutamate = NMDA greater than kainate, with EC50 values of 0.4, 20, and 40 microM, respectively. Alanine, glutamine, and glycine did not modify the release of somatostatin. The stimulation of somatostatin release elicited by L-glutamate was Ca2+ dependent, was decreased by Mg2+, and was blocked by DL-amino-5-phosphonovaleric acid (APV) and thienylphencyclidine (TCP), two specific antagonists of NMDA receptors. The NMDA stimulatory effect was strongly inhibited by APV in a competitive manner (IC50 = 50 microM) and by TCP in a noncompetitive manner (IC50 = 90 nM). The release of somatostatin induced by the excitatory amino acid agonists was not blocked by tetrodotoxin (1 microM), a result suggesting that tetrodotoxin-sensitive, sodium-dependent action potentials are not involved in the effect. Somatostatin release in response to NMDA was potentiated by glycine, but the inhibitory strychnine-sensitive glycine receptor did not appear to be involved. Our data suggest that glutamate exerts its stimulatory action on somatostatin release essentially through an NMDA receptor subtype.