Hormone and methylxanthine action on breast epithelial cells

Human mammary carcinoma cells and normal mouse breast epithelial cells desensitized as the result of treatment with beta-adrenergic agonists. Accumulation of cAMP in the same cells was affected only slightly by caffeine and there was no detectable desensitization or hypersensitization as a result of that treatment. However, as in many other cell types, caffeine was an effective inhibitor of adenosine action. These observations do not support the hypothesis made by Minton and his co-workers (1), that treatment of breast epithelial cells with agents that increase cAMP accumulation leads to hypersensitization rather than desensitization.     

Inhibition of protein carboxyl methylation by S-adenosyl-L-homocysteine in intact erythrocytes. Physiological consequences

S-Adenosyl-L-homocysteine was used to inhibit the methylation of carboxylic acid residues of membrane proteins in intact human erythrocytes. Incubation of erythrocytes for 24 h with 5 mM each of adenosine and L-homocysteine resulted in the intracellular accumulation of S-adenosyl-L-homocysteine and substantially inhibited membrane protein carboxyl methylation. From the degree of inhibition and from the observed turnover of methylated proteins, we estimate that the number of protein methyl esters in cells incubated with adenosine and L-homocysteine for 20 h is less than 20% that of cells incubated without these inhibitors. No significant differences in the physical deformability properties of the membrane of these hypomethylated cells were detected. However, there was a small but significant (p less than 0.001) increase in the amount of membrane protein D-aspartyl residues in these cells compared to control cells. These observations are consistent with the hypothesis that methylation of membrane proteins at D-aspartyl residues may result in the selective removal or repair of these uncommon residues.     

Kinetic and Physical Studies of Cell Death Induced By Chemotherapeutic Agents Or Hyperthermia

The kinetics of three physical parameters: cell density, relative cytoplasmic viscosity and DNA stability to denaturation have been measured during the period preceding cell death induced by hyperthermia, methylprednisolone and a series of cancer chemotherapeutic agents. This series of measurements employed cultured human lymphoblastoid cells as an experimental system to establish the changes that can be observed in the early stages of cell death, prior to applying such measurements to tissue biopsies from solid human tumours. Cell death, induced by hyperthermia up to 43°C, methylprednisolone, vincristine, 5-fluorouracil, BCNU and melphalan, showed essentially identical and reproducible changes corresponding to those which characterize programmed cell death (apoptosis). Such changes could also be observed following hyperthermia above 43°C, but reproducibility was poor and increasing damage to the cell membranes was evident. In cells treated with adriamycin or methotrexate, cell sub-populations showing an increase in cell density were not detected. Measurements of DNA stability were readily performed by flow cytofluorometry thus allowing rapid quantitation of the fraction of cells in the early stages of cell death. Modified flow cytometric instrumentation would further allow measurement of cytoplastic viscosity as an additional parameter to indicate entry into programmed cell death. This suggests that these measurements could readily be applied to cell suspensions derived from tumour tissue biopsies for a more accurate assessment of tumour growth rate, and to allow monitoring of response to therapy in sequential tumour biopsies.     

Studies of the mobility of maleimide spin labels within the erythrocyte membrane

We have confirmed a method yielding reproducible and reliable spectrometric parameters derived from spin-labeled erythrocyte ghosts using nitroxide derivatives of maleimide compounds. The disorder parameter, W/S, was shown to vary with changes in the structure of the label, the conditions utilized for labeling such as ionic strength and erythrocyte age and the presence of drugs such as alcohol and acetaminophen. The nitroxide spectrum was also found to change with increasing and decreasing temperature in an irreversible manner. These findings should permit increased reliance to be placed on the spin-labeling technique when used to monitor changes in membrane lipid or protein assembly.     

Development of the cytosolic defence system against microsomal lipid peroxidation in rat liver

Ascorbate-induced lipid peroxidation in rat liver microsomes reaches the adult level in 2-3 days. NADPH-induced peroxidation develops more gradually, in parallel with the activity of NADPH-cytochrome P-450 reductase, attaining adult levels by 10-12 days. The glutathione-dependent cytosolic enzyme activity which inhibits peroxidation is inhibited by bromosulphophthalein. The development of this system lags behind the development of microsomal lipid peroxidation between the ages of 2 and 20 days, allowing peroxidation to proceed.     

Lipid enrichment of thylakoid membranes. I. Using soybean phospholipids

(1) Using asolectin (mixed soybean phospholipids) liposomes, extra lipid, with or without additional plastoquinone, has been introduced into isolated thylakoid membranes of pea chloroplasts. (2) Evidence for this lipid enrichment was obtained from freeze-fracture which indicated that a decrease in the numbers of EF and PF particles per unit area of membrane occurred with increasing lipid incorporation. The decrease was not due to loss of integral membrane polypeptides as judged by assay of cytochrome present or SDS-polyacrylamide gel electrophoresis of lipid-enriched membrane fractions. Moreover, the enrichment procedure did not lead to extraction of low molecular weight lipophilic membrane components or of thylakoid membrane lipids. (3) The introduction of phospholipids into the membrane affected steady-state electron transport. Inhibition of electron transport was observed when either water (Photosystem (PS) II + PS I) or duroquinol (PS I) was used as electron donor with methyl viologen as electron acceptor, and the degree of inhibition increased with higher enrichment levels. Introduction of exogenous plastoquinone with the additional lipid had little effect on whole-chain electron transport, but caused an increase in the 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)-sensitive rate of PS I electron transport. The inhibition was also detected by flash-induced oxidation-reduction changes of cytochrome f.     

The optical properties of CuA in bovine cytochrome c oxidase determined by low-temperature magnetic-circular-dichroism spectroscopy.

The visible-near-i.r.-region m.c.d. (magnetic-circular-dichroism) spectrum recorded at low temperature in the range 450-900 nm is reported for oxidized resting mammalian cytochrome c oxidase. M.c.d. magnetization curves determined at different wavelengths reveal the presence of two paramagnetic species. Curves at 576, 613 and 640 nm fit well to those expected for an x,y-polarized haem transition with g values of 3.03, 2.21 and 1.45, i.e. cytochrome a3+. The m.c.d. features at 515, 785 and 817 nm magnetize as a S = 1/2 paramagnet with average g values close to 2, and simulated m.c.d. magnetization curves obtained by using the observed g values of CuA2+, i.e. 2.18, 2.03 and 1.99, fit well to the experimental observations. The form of the m.c.d. magnetization curve at 466 nm is curious, but it can be explained if CuA2+ and cytochrome a3+ contribute with oppositely signed bands at this wavelength. By comparing the m.c.d. spectrum of the enzyme with that of extracted haem a-bisimidazole complex it has been possible to deconvolute the m.c.d. spectrum of CuA2+, which shows transitions throughout the spectral region from 450 to 950 nm. The m.c.d.-spectral properties of CuA2+ were compared with those of a well-defined type I blue copper centre in azurin isolated from Pseudomonas aeruginosa. The absolute intensities of the m.c.d. signals at equal fields and temperatures for CuA2+ are 10-20-fold greater than those for azurin. The optical spectrum of CuA2+ strongly suggests an assignment as a d9 ion rather than Cu(I) bound to a thiyl radical.     

An estimation of tissue damage and thermal history in the cryolesion

The cooling and thawing rates at fixed sites within an expanding ice ball have been recorded using a temperature probe containing six integral thermocouples. The cell changes relevant to these sites have been followed through the 48 hr postfreeze-thaw period by light and electron microscopy. The results suggest that cooling rates that are compatible with cell survival in cell suspension are associated with uniform tissue cellular death in an organized tissue.