Cell-cell interaction can influence drug-induced differentiation of murine embryonal carcinoma cells

When cultured in the presence of either retinoic acid (RA) or dimethyl sulfoxide (DMSO), aggregates of the P19 line of mouse embryonal carcinoma (EC) cells differentiate and the spectrum of cell types formed depends on the drug dose. It is shown here the EC cells rapidly lose their colony-forming ability when cultured as aggregates in the presence of DMSO. This loss of plating efficiency (PE) also occurs rapidly following RA treatment. Loss of PE has been used as a quantitative procedure for assessing the rate of drug-induced differentiation. The relationship between drug dose and loss of PE is much steeper for DMSO than for RA, suggesting that these two drugs affect different stages of the differentiation decision-making apparatus. Mutant EC cell lines (D3 and RAC65) do not differentiate in the presence of drug-inducers (DMSO and RA, respectively). Neither differentiation-deficient mutant has an altered ability to form gap junctions. When D3 and P19 cells were mixed within the same DMSO-treated aggregates, the D3 cells remained undifferentiated and the P19 cells differentiated much less efficiently than if they were cultured in the absence of the D3 cells. When RAC65 and P19 cells were mixed in RA-treated aggregates, each cell responded to the drug as though the other were absent. Thus RA behaves as a cell-autonomous inducer of differentiation, whereas DMSO-induced differentiation seems to be mediated by interactions between neighboring cells.     

Studies of heterogeneous mitochondrial populations in a mouse cell line: the effects of selection for or against mitochondrial genomes that confer chloramphenicol resistance

A single nucleotide substitution in a highly conserved region of the mitochondrial genome of a mouse cell line confers both chloramphenicol resistance and an alteration to the recognition site for the endonuclease Eco RV. This has enabled a detailed study on the effects of selection on a mitochondrial population comprising initially both chloramphenicol-resistant and chloramphenicol-sensitive mitochondrial genomes. The mutation confers advantage to cells grown in the presence of chloramphenicol, but is apparently deleterious in its absence. Selection at the cellular level is sufficient to explain the results observed. Fixation, which results in cells having mitochondria of only a single type, is slow. It is probable, therefore, that mammalian oocyte mitochondria are derived from only a small number of progenitors. This would allow fixation of new mutations and explain the observed uniformity in mitochondrial genomes of the individual in the presence of extensive variation between different members of the population.     

Redistribution and characterization of (H+ + K+)-ATPase membranes from resting and stimulated gastric parietal cells.

When isolated from resting parietal cells, the majority of the (H+ + K+)-ATPase activity was recovered in the microsomal fraction. These microsomal vesicles demonstrated a low K+ permeability, such that the addition of valinomycin resulted in marked stimulation of (H+ + K+)-ATPase activity, and proton accumulation. When isolated from stimulated parietal cells, the (H+ + K+)-ATPase was redistributed to larger, denser vesicles: stimulation-associated (s.a.) vesicles. S.a. vesicles showed an increased K+ permeability, such that maximal (H+ + K+)-ATPase and proton accumulation activities were observed in low K+ concentrations and no enhancement of activities occurred on the addition of valinomycin. The change in subcellular distribution of (H+ + K+)-ATPase correlated with morphological changes observed with stimulation of parietal cells, the microsomes and s.a. vesicles derived from the intracellular tubulovesicles and the apical plasma membrane, respectively. Total (H+ + K+)-ATPase activity recoverable from stimulated gastric mucosa was 64% of that from resting tissue. Therefore, we tested for latent activity in s.a. vesicles. Permeabilization of s.a. vesicles with octyl glucoside increased (H+ + K+)-ATPase activity by greater than 2-fold. Latent (H+ + K+)-ATPase activity was resistant to highly tryptic conditions (which inactivated all activity in gastric microsomes). About 20% of the non-latent (H+ + K+)-ATPase activity was also resistant to trypsin digestion. We interpret these results as indicating that, of the s.a. vesicles, approx. 55% have a right-side-out orientation and are impermeable to ATP, 10% right-side-out and permeable to ATP, and 35% have an inside-out orientation.     

Enzyme kinetics of a highly purified mitochondrial creatine kinase in comparison with cytosolic forms

Summary Mitochondrial creatine kinase (CK) purified from canine myocardium showed a single protein band on SDS-PAGE and was free of MMCK. Its amino acid composition was different than MMCK or BBCK and did not react to antiserum to MMCK or BBCK. Using purified mitochondrial, MM and BBCK, the velocity of reaction (V) was estimated for creatine phosphate (CP), creatine (C), adenosine triphosphate (ATP) and adenosine diphosphate (ADP) over a wide range of concentrations including those at V_max. The values for K_m (mM/L) derived from Lineweaver-Burke plots are shown: The affinity of mitochondrial CK for C is much greater than MMCK which is compatible with the energy shuttle hypothesis, namely ATP is converted by mitochondrial CK to CP, and then diffuses to the myofibril for conversion to ATP for utilization.     

The effect of alterations in haematocrit on tumour sensitivity to X-rays

Hypoxic cells in human tumours probably contribute to the failure of radiotherapy in some sites. Changes in the oxygen carrying capacity of the blood, such as in anaemia, have been shown to influence tumour response. The effect of acute and chronic changes in haematocrit on the radiosensitivity of three mouse tumours (EMT6, KHT and RIF-1) were studied. Alterations in haematocrit were achieved by bleeding followed by retransfusion. When radiation was preceded immediately by an acute reduction in haematocrit (anaemia), radiosensitivity was markedly reduced in each tumour. An acute rise in haematocrit (polycythaemia) increased or decreased X-ray sensitivity depending on its severity. The optimum haematocrit for maximum sensitivity was always found to be at a level 5-10 per cent above normal. When the time between induction of anaemia and irradiation was increased, simulating a progressively longer duration of anaemia, marked changes in radiosensitivity of all the tumours were observed. A short duration of anaemia resulted in a resistant tumour with each cell line, but the resistance was gradually lost as the anaemia was prolonged, even though no recovery in haematocrit occurred. The rate of recovery to normal radiosensitivity varied from 24 to 72 hours in the different tumours. Therefore, only haematocrit changes which occurred within 1-3 days of a dose of radiation affect the radiosensitivity of these tumours.     

Cellular location of heat-labile enterotoxin in Escherichia coli.

We demonstrated that both the A and B subunits of heat-labile enterotoxin from Escherichia coli are located in the periplasm. The toxin was shown to form aggregates in Tris-EDTA buffers which are routinely used for isolating membranes. The aggregates pellet upon centrifugation, and this may explain why several previous investigators have concluded that enterotoxin is associated with membranes.     

Growth and mortality of zebra fish, Brachydanio rerio (Hamilton Buchanan), maintained at two temperatures and on two diets

One hundred zebra fish per tank were maintained for 112 days at 24°C or 28°C in glass aquaria and fed a diet of flaked food made without cellulose (13.45 kJ g^?1, metabolizable energy, Type A) or with cellulose (8.71 kJ g^?1, metabolizable energy, Type B). Each experimental condition was repeated in triplicate (12 tanks). The weight of food given daily to the fish was based on daily records of survivors (from which mortality rates were calculated) and wet wt of fish (measured every 14 days) in each tank. All fish were fed with the same weight of food per day and the quantity of energy in the food in excess of standard metabolism (as a proportion of SM) was approximately 0–5 for fish maintained at 28°C and fed food B, 1–0 for fish maintained at 24°C and fed food B, 1–5 for fish maintained at 28°C and fed food A, and 2.2 for fish maintained at 24°C and fed food A. Non-ionized ammonia, nitrite and nitrate nitrogen in the tanks did not reach toxic levels although there was an increase in total ammonium nitrogen in one tank and a subsequent heavy mortality. It was assumed that this was caused by the build up of pathogenic bacteria. Apart from this tank, mortality was highest in tanks at 28°C with fish fed food A and second highest in tanks at 24°C with fish fed on the same diet. Growth was measured in units of length, wet and dry weights, carbon and energy. There was a good correlation (P < 0.001) between carbon (mgC mg^?1) and calorific (J mg^?1) values and a conversion factor of 46.2 J (mgC)^?1 was derived. Fish maintained at 24°C and fed food A had the highest rates of growth both in weight and in energy value per unit weight. Fish fed the same diet but kept at 28°C had the lowest growth rates. Both these groups of fish had the highest coefficients of variation in wet weights which increased during the experiment, indicating an increase in interaction within the tanks. There was agreement between the energy value of fish sampled for growth and a condition factor based on the length-weight relationships of fish remaining in the tanks. A correlation (P < 0.05) was found between instantaneous mortality and growth rates for fish between tanks when those maintained at 28°C and fed on food A were ignored.     

IL 1 requirement for B cell activation revealed by use of adult serum

Fetal calf serum is an essential component of the culture medium developed by Mishell and Dutton for the immunization of murine spleen cells in vitro. Serum from adult donors (mouse, human, rabbit) does not support antibody synthesis in this system. This "deficiency" of adult serum can be overcome with IL 1. Adult serum in the presence of IL 1 is as effective in stimulating a B cell response against xenogeneic red cells as fetal calf serum. We attribute the capacity of fetal calf serum to support an immune response in the absence of exogenous IL 1 to serum factors that cause macrophages to release IL 1 endogenously. Our findings strengthen the notion that IL 1 plays an essential role in the process of B cell activation and suggests that the use of fetal calf serum should be avoided in studies concerned with the function of interleukin 1.