The morphological and spectral phenotype of apoptosis in HeLa cells varies following exposure to UV-C and the addition of inhibitors of ICE and CPP32

Numerous extra- and intracellular factors, including UV radiation, can initiate a programme of cell death by apoptosis. While apoptosis is commonly defined morphologically, the relationships between morphology and molecular events are not well established. To investigate these relationships in HeLa cells, eight morphometric criteria for cell proliferation and damage and 10 criteria for apoptotic phenotype were examined using light microscopy, and corroborated by ultrastructure and spectral imaging. They were identified (1) during a time course after irradiation with 0, 10 or 30 J/m² UV-C; (2) after separation of apoptotic from normal cells on a Percoll gradient; and (3) after irradiation with UV-C plus perturbation of the apoptotic pathway by treatment with inhibitors of two caspases, ICE and CPP32. The number of cells in apoptosis increased in a dose-dependent manner after UV-C treatment. Centrifugation of irradiated cells on a Percoll gradient increased the collection of apoptotic cells tenfold. The stereotypical apoptotic phenotype, in which cells have deep cytoplasmic blebbing and highly condensed DNA, comprised only a few percent of all apoptoses, and was rarely seen in groups receiving caspase inhibitors. The most common apoptotic phenotype was a rounded cell with large spherical nucleolus and associated DNA. After treatment with UV-C plus inhibitors the apoptotic index was decreased by about 30% compared to UV-C radiation alone. These apoptotic cells had dark spherical cytoplasm with small blebs, greatly increased numbers of cytoplasmic ribosomes, abundant nucleolar material with a large separate granular component, and chromatin condensed at the nuclear membrane. Using the technique of spectral imaging, it was found that the spectrum obtained from the granular component of the nucleolus, which was elevated in apoptotic cells treated with UV-C plus inhibitors, was similar to the dense accumulation of ribosomes in the apoptotic cytoplasm. The data indicate that spectral imaging may be a useful tool for identifying and characterizing variations in the apoptotic process, and that the caspase inhibitors used here do not completely abolish UV-C induced apoptosis, but rather alter its incidence and progression.     

Influence of species, sex and drug pretreatment on the metabolism of metyrapone

The metabolism of metyrapone was investigated in three mammalian and four non-mammalian species, and keto reduction was found to be the major metabolic route (except in the cat). Toad, lizard and tortoise did not form metyrapone N-oxides. Rat and cat formed both isomeric N-oxides of metyrapone, whereas rabbit and pigeon have a limited capacity to form only the N-oxide II and N-oxide I, respectively. There were marked sex differences in both keto reduction and N-oxidation in the rat. Anthracene did not affect metyrapone N-oxides formation in the male rat; however phenobarbitone and pregnenolone significantly induced N-oxide II formation, whereas ethanol induced both isomeric N-oxides formation. Cimetidine, a known cytochrome P-450 inhibitor, inhibited the N-oxide II formation but with an enhanced N-oxide I formation.     

Membrane electrical parameters in turtle bladder measured using impedance-analysis techniques

Summary Equivalent-circuit impedance analysis experiments were performed on the urinary bladders of freshwater turtles in order to quantify membrane ionic conductances and areas, and to investigate how changes in these parameters are associated with changes in the rate of proton secretion in this tissue. In all experiments, sodium reabsorption was inhibited thereby unmasking the electrogenic proton secretion process. We report the following: (1) transepithelial impedance is represented exceptionally well by a simple equivalent-circuit model, which results in estimates of the apical and basolateral membrane ionic conductances and capacitances; (2) when sodium transport is inhibited with mucosal amiloride and serosal ouabain, the apical and basolateral membrane conductances and capacitances exhibit a continual decline with time; (3) this decline in the membrane parameters is most likely caused by subtle time-dependent changes in cell volume, resulting in changes in the areas of the apical and basolateral membranes; (4) stable membrane parameters are obtained if the tissue is not treated with ouabain, and if the oncotic pressure of the serosal solution is increased by the addition of 2% albumin; (5) inhibition of proton secretion using acetazolamide in CO₂ and HCO ₃ ^– -free bathing solutions results in a decrease in the area of the apical membrane, with no significant change in its specific conductance; (6) stimulation of proton transport with CO₂ and HCO ₃ ^– -containing serosal solution results in an increase in the apical membrane area and specific conductance. These results show that our methods can be used to measure changes in the membrane electrophysiological parameters that are related to changes in the rate of proton transport. Notably, they can be used to quantify in the live tissue, changes in membrane area resulting from changes in the net rates of endocytosis and exocytosis which are postulated to be intimately involved in the regulation of proton transport.     

Evalution of Home Nurse Attachment in Bristol

By the end of June 1969 home nurses in Bristol were attached to 18 general practices caring for about 137,000 patients, or about one-third of the city''s population. Attachment was associated with an increase by about one-third in the number of patients referred by general practitioners for home nursing. Additional benefits derived from attachment during the nine months from January to September 1969 were 2,047 items of service performed by nurses in general practitioners'' surgeries, 65 home visits to patients who were not receiving domiciliary nursing care, improved communications between general practitioners and nurses, and opportunities for both doctors and nurses to widen their fields of work. The travelling expenses paid to Bristol''s nurses increased by 9·5%.It is suggested that the benefits to patients, doctors, and nurses of attachment far outweigh the costs and that there is scope for extending the role of the attached nurse in the surgery and in home visiting.