OPTIMAL CONDITIONS FOR CELL SUSPENSION CULTURES OF THLASPI CAERULESCENS AND BRASSICA NAPUS

Thlaspi caerulescens (Brassicaceae), known as a Zn hyper accumulator, is able to accumulate and tolerate Zn, Ni, Cu, and Cd at high concentrations in its biomass. We are examining the feasibility of using cell suspensions of T. caerulescens and B. napus to study the effect of selected heavy metals on growth and nutrient uptake. Callus was initiated by culturing seedlings on basal medium containing MS salts supplemented with MS or B5 vitamins, 1, 2, 5, or 10 mg 2,4-D/liter, and 0.7% Phytagar. Cell suspensions were initiated by transferring calli to liquid basal medium containing MS or B5 vitamins, and 1 or 2 mg 2,4-D/liter, and were incubated on a gyratory shaker at 120 rpm. Growth of suspensions inoculated at 0.2, 0.4, or 0.6 g/25 ml was monitored for 13 days. Optimal conditions required to initiate and maintain suspension cultures of T. caerulescens and B. napus include MS medium supplemented with B5 vitamins and 1 mg 2,4-D/liter, an inoculation density of 0.4 g/25 ml, and a 2-week subculture schedule.

The biotransformation of tranylcypromine by Cunninghamella echinulata

When incubated alone for 7 days with the fungus Cunninghamella echinulata, tranylcypromine was extensively metabolized. As observed in mammalian systems, N-acetyltranylcypromine was the major metabolite recovered along with lesser amounts of 4-hydroxytranylcypromine, as its N,O-diacetyl derivative. The rate and extent of tranylcypromine biotransformation was affected by whether incubation was on either 30° or flat brackets with a gyratory shaker. There is a strong association between the rate of biotransformation and the utilization of glucose, formation of ammonia, and pH. The slowest rates of biotransformation and metabolic response were observed with the large fungal pellets formed during incubation on flat brackets. These findings raise the possibility that, as in mammalian systems, fungal metabolism of xenobiotics can be affected by nutrient and environmental conditions. Key words: Cunninghamella echinulata, tranylcypromine, biotransformation, toxicity model.

On the reduced intercellular adhesiveness of virally transformed BHK21 cells

Baby hamster kidney fibroblasts (BHK21 cells) transformed by polyoma or Rous sarcoma viruses aggregate less than the untransformed parental cells when incubated in growth medium in a gyratory shaker for 18–24 h. This difference can be measured by electronic particle counting, or by filtering aggregated suspensions of 32P-labelled cells through bolting fabric. The aggregation of transformed derivatives is not enhanced by the presence, during aggregation of epsilon-amino caproic acid, an inhibitor of plasmin activation. Some lines of transformed BHK21 cells do not appear less adhesive than untransformed cells in a short-term aggregation assay, and none adheres markedly less well when seeded onto homotypic cell sheets. The decreased aggregation of transformed cells is consistent with suggestions that LETS protein is involved in intercellular adhesion of fibroblasts as well as in attachment of cells to non-cellular substrates. If so, the short-term aggregation of freshly trypsinized cells may depend on secretion of LETS from an intracellular pool.

The effects of tumour cells on embryonic cell aggregation, adhesion and detachment

The presence of small numbers of tumour cells inhibits the aggregation of embryonic chicken neural retina cells grown in gyratory shaker culture. The aggregation of neural retina cells was also inhibited by ascites cell medium. We investigated whether the inhibitory effect of the tumour cells on aggregate size is effected by inhibition of the initial adhesion or by enhancement of their separation. The number of neural retina cells adherent to microtest plate surfaces was significantly reduced after incubation with either Ehrlich ascites cells or cell-free, conditioned medium, while the percentage of cells removed from glass by shearing was unchanged under those conditions. These results suggest that the reduction in neural retina cell aggregate size produced by Ehrlich ascites cells and their products is due to partial inhibition of neural retina cell adhesion processes, as distinct from enhancement of separation.

Immunological procedure for the rapid and specific identification of Coccidioides immitis cultures

An immunological procedure for the rapid and specific identification of Coccidioides immitis isolates has been developed. The specificity of the procedure is based on the fact that C. immitis produces antigens that are not produced by morphologically similar fungi. The procedure involved the transfer of heavy mold-form inocula to flasks that contained small volumes of brain heart infusion broth. Shake cultures were grown at 25 degrees C on a gyratory shaker at 150 rpm. The concentrated supernatant antigens so obtained from broth cultures were tested in parallel with coccidioidin by a microimmunodiffusion technique against C. immitis antiserum. The ability of the immunological procedure to identify C. immitis cultures was evaluated by testing the supernatant antigens of 166 unknown isolates, many of which, by gross or microscopic examination or both, resembled C. immitis or belonged to the family Gymnoascaceae. Each culture was also identified by conventional laboratory procedures. Comparative evaluation showed that the immunological test for C. immitis was 100% sensitive and specific. The diagnostic precipitin bands that were produced by the interaction of the C. immitis supernatants and antisera were shown to consist of (i) a heat-stable antigen comparable to that active in the tube precipitin test and (ii) two heat-labile antigens, one of which was associated with complement fixation reactivity. With pure cultures, this immunological procedure permitted the identification of C. immitis isolates within 5 days.

Reconstruction of Mammalian Heart Tissue from Isolated Embryonic Heart Cells

Although monolayer cultures are useful in various cell studies, they are not always adequate for examining the nature of cellular interrelationships and interactions in the formation, differentiation, and function of tissues. The procedures of aggregation in vitro of dissociated cells (Moscona & Moscona, 1966) enable one to study in detail the formation of cell contacts, the assembly of cells into multicellular systems, and cell cooperation in forming organized and differentiating tissues. We adapted the techniques of cell aggregation by rotation to studies on embryonic mammalian heart cells. Cell suspensions from the 18-day-old embryonic rat were prepared by dissociation with 0. 5% trypsin. The cells were dispersed in a culture medium which consisted of Eagle's basal medium with 10% fetal bovine serum, 1% glutamine solution, and 1% penicillin-streptomycin mixture. Cultured in 25ml Erlenmeyer flasks on a gyratory shaker at 70 rpm at 37°C were 3ml aliquots of the cell suspensions.

Histotypic Cell Aggregation in the Presence of Cytochalasin B

A study was made of the effects of cytochalasin B on (a) specific sorting of reaggregating cells; (b) redistribution of cell types after treatment of preformed aggregates; and (c) the ability of aggregates of one cell type to incorporate and sort cells of another type. Freshly disaggregated neural retina and heart cells were cultured on a gyratory shaker at 70 rev/min and the aggregates formed analysed for sorting of cell types. Cytochalasin B disrupted the sorting of forming aggregates at concentrations of 1 µg/ml and greater. The distribution of cell types in aggregates that were treated with cytochalasin after 24 h of culture was more random than the control. Untreated cultures of retinal aggregates and heart cell suspension resulted in pure retinal and pure heart aggregates, but with more than 50 % mixed and sorted aggregates. Cytochalasin B treatment resulted in fewer mixed aggregates and a higher proportion of pure retina and pure heart aggregates.

On the Mechanism of the Increased Aggregation by Neuraminidase of 16 C Malignant Rat Dermal Fibroblasts in Vitro

The aggregation of briefly trypsinized 16 C malignant rat dermal fibroblasts as measured by gyratory shaker/electronic particle counting technique is increased by neuraminidase. Enzyme treatment also exposes soybean and Ricinus communis agglutinin-binding sites as judged by cell agglutination. Desialysed bovine submaxillary mucin and desialysed, agalacto fetuin reversed the neuraminidase-stimulated increase in aggregation, whereas desialysed fetuin and ovalbumin did not. The effective glycoproteins also acted as acceptors in cellular sialyl- and galactosyl-transferase reactions, and these activities are detected in plasma membrane fractions. The possibility that neuraminidase increases cellular aggregation by generating acceptor sites for interaction with cell surface glycosyltransferases, is discussed.

The analysis of spatial distributions in mixed cell populations: a statistical method for detecting sorting out

1. This work presents a quantitative measure, α, of the degree of segregation of two cell types in sections of aggregates, and some results obtained with the measure relating to ‘sorting out’. The method is designed particularly for the case where labelling of one type of cell is incomplete, and the importance of this effect is assessed. Possible problems in formulating such a model are discussed. The measure α is compared with methods used in investigations of segregation in plant communities. 2. Segregation of chick heart and limb-bud cells in mixed aggregates has been analysed using α. In control aggregates of mixtures of labelled and unlabelled cells of one type, α is near to its random value of 1, and we suggest that the departure from random can be adequately accounted for by cell division. In mixed aggregates, significant segregation is consistently found, even in aggregates formed after 2 and 4 h. Both disaggregation procedures (EDTA, trypsin or trypsin + EDTA) and reaggregation methods (reciprocating or gyratory shaker) are found to have an effect on the degree of segregation. Possible reasons for these findings are discussed. 3. Positioning of the cells relative to the outside of aggregates is also investigated for some of the aggregates.