Evaluation of different staining procedures for the quantification of fibroblasts cultured in 96-well plates.

Comparison of published methods for the quantification of adherent cell numbers by the measurement of absorbance of bound stain indicates a wide variation in their sensitivity. This study aimed at comparing the sensitivities of five different staining procedures (Coomassie brilliant blue G in perchloric acid, Coomassie brilliant blue G in phosphoric acid, methylene blue, crystal violet, and toluidine blue) applied to three separate types of cultured fibroblasts (3T3 cells, Vero cells, and human gingival fibroblasts) at concentrations from 0.125 x 10(4) to 10 x 10(4) per well in 96-well microplates. Absorbance values of Coomassie blue-stained cells were measured in situ. Those of the remaining cells were measured after solubilization of the dye with 1% sodium dodecyl sulfate. All absorbance values were measured using an Elisa reader at 620 or 570 nm for crystal violet. The relationship between cell number and absorbance over the entire cell concentration range was best fitted with quadratic regression analysis, in contrast with the linear relationship described elsewhere. The order of sensitivity of the staining procedures was the same for each cell type: Coomassie blue in perchloric acid less than Coomassie blue in phosphoric acid less than methylene blue less than crystal violet less than toluidine blue. With the latter two stains absorbance values began to plateau at approximately 8 x 10(4) cells per well. However, staining with Coomassie blue in perchloric acid and methylene blue resulted in an almost linear relationship between cell number and absorbance over the entire concentration range tested.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and ultrastructure of adrenocorticotropic hormone cells in goats in anoestrus, gestation and milk production.

The structural and ultrastructural characteristics of adrenocorticotropic hormone cells in adult female goats in anoestrus, gestation and milk production were studied with an immunohistochemical method (peroxidase-antiperoxidase). Only one cellular type has been identified and is characterized by numerous secretory granules of different electron density and an average diameter of 275 nm. During pregnancy these cells increase in number and size, and there is a frequent presence of vacuoles. During lactation the number of size of the cells decreases but without reaching the state observed in anoestrus and the involution of the cytoplasmic vacuolizations which appear in pregnancy.     

Effect of proteases on the activity of enolase from muscle of carp (Cyprinus carpio) and pig.

The treatment of enolase from pig and carp (Cyprinus carpio) with proteases resulted in a decrease of enzymatic activity, which depended on the kind of protease used. The most active were trypsin and subtilisin. Substrate and magnesium ions protected enolase against inactivation. The enolase from pig muscle was much more resistant to protease action than this enzyme from carp muscle. Some differences in the structure between the two enolases are suggested.     

Involvement of superoxide dismutase in heat-induced stimulation of photosystem I-mediated oxygen uptake

We have investigated the effect of heat-treatment of chloroplast thylakoid membranes on photosystem I-mediated electron transport. Spectroscopic techniques, oxidation of dichlorophenolindophenol (donor side) and reduction of NADP or methyl purple (acceptor side), showed no indication of an increased activity of photosystem I electron transport. Enhancement of oxygen uptake in the heat-treated (40 degrees C-48 degrees C) samples could largely be accounted for by decline in the activity of superoxide dismutase.     

Tau-related protein present in paired helical filaments has a decreased tubulin binding capacity as compared with microtubule-associated protein tau

We have isolated, after exhaustive detergent treatments, a 33 kDa tau-related protein isolated from paired helical filaments from Alzheimer's disease patient brains. The N-terminal sequence of the 33 kDa protein begins at residue 71 of the sequence described for human fetal tau protein. This truncated form of tau is not the consequence of the translation of a tau RNA lacking a region at its 5' end, as measured by primer extension analyses, suggesting that the 33 kDa protein must be generated by proteolysis of previously synthesized tau. This tau-related protein has only one blocked cysteine residue and also has a decreased tubulin binding capacity as compared with that of tau protein.     

Lipid-alginate interactions render changes in phospholipid bilayer permeability

Lipid vesicles, e.g. liposomes, generally release their contents in a continuous manner. However, when these vesicles are entrapped in Ca-alginate and coated with poly(L-lysine), they release their contents in an unusual fashion, in 'bursts'. Molecular-level studies indicated that lipid-alginate interactions are responsible for changes in the barrier properties of lipid vesicles. Differential scanning calorimetry revealed that exposure of liposomes to alginate resulted in a 4-fold reduction in the phase transition enthalpy, with no change in the melting temperature. Size-exclusion chromatography of liposomes-in-alginate gave an additional liposomal peak with a smaller elution volume. These studies suggested that alginate is inserted into the lipid bilayer of vesicles. Lipid-alginate interactions were highly dependent on phospholipid head group charge and the phase transition temperature of the phospholipid. Based on these interactions, a mechanism to explain the 'burst' from these entrapped liposomes is suggested.     

Cytochrome c oxidase subunit II mRNA levels during T-lymphocyte proliferation and liver regeneration.

We studied here the variations in the mRNA levels of the mitochondrially-encoded subunit II of cytochrome c oxidase (COII) during the proliferation of thymocytes, splenic T-cells and hepatocytes. The COII mRNA levels increased during thymocyte proliferation and decreased when they were growth arrested. However, its levels remained nearly constant during splenic T-cell proliferation and liver regeneration after partial hepatectomy. The different pattern of COII gene expression in the cellular systems analyzed suggests that an increment in the oxidative metabolism could not always be necessary during cell proliferation.     

The characteristics of macrophage-like cell lines derived from normal sheep spleens

Macrophage-like cell lines were derived from sheep spleens using conditioned medium from L-929 mouse cells as a source of colony stimulating factor. In seven out of ten attempts colonies of macrophage-like cells appeared after 2-3 weeks of culture. The cells were established in culture as cell lines, and survived 120 passages. They were strongly (+ +) positive for non-specific esterase but negative for peroxidase and produced detectable but small amounts of lysozyme (0.21-1.76 micrograms/10(6) cells). Latex particles were actively phagocytosed. Bacteria (Staphylococcus albus, Staphylococcus aureus) attached to the cell surface and were internalized in the presence of specific antibody. Expression of receptors for immunoglobulin and complement varied somewhat between the different cell lines: the proportion of receptor-bearing cells ranged between 9 and 26% FC-receptors, and 10 and 38% for C-receptors. The cell lines displayed a peculiar karyotype as well as protein profile that were different from normal sheep but similar between the different cell lines. Culture supernatants of the cell lines contained a colony stimulating activity which was used to establish further cell lines. They also spontaneously produced an interleukin-1-like activity that had no effect on baseline proliferation of sheep lymphocytes but enhanced their response to PHA (1.7-fold) particularly in conjunction with sheep IL-2 (4-fold). Prostaglandin E2 was produced in a growth-cycle dependent manner: the peak production occurred on the second day (77-140 pg/ml) at 2 x 10(5) cells and declined to 33-50 pg/ml on the eighth day when cell numbers had increased to 2-3 x 10(6). These easily cultured cell lines derived from normal tissue without the introduction of viral DNA should provide a useful source of material for studies of macrophage function in sheep.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Expression of anchorin CII mRNA by cultured chondrocytes

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during “in vitro” chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.The anchorin mRNA level was low in dedifferentiated chondrocytes, progressively increased after the cell transfer into suspension (a condition promoting differentiation), reached its maximal value after 4 weeks and decreased after 5 weeks.Therefore anchorin CII mRNA reaches its maximum level in hypertrophic stage II chondrocytes.