Calcium was localized in the red cell membrane by light microscopy using chlorotetracycline hydrochloride (CTC) as chelate probe. Treating human erythrocytes with CTC dissolved in saline free of divalent cations, leads to a 530 nm fluorescence emission in the cell border and to characteristic cell shape changes which were evaluated to assess intramembrane calcium distribution. CTC prevented and reverted erythrocyte crenation induced either by washing or superfusing the cells with saline. The ionophore A23187, EGTA and glucose depletion depressed the shape modifying effect of CTC. Thus, CTC appears to act on red cell shape by complex formation with membrane associated calcium. This is further confirmed by the failure of degraded CTC, devoid of metal binding capacity, to modify the crenated shape. The CTC effect can be reverted by superfusing the erythrocytes with CTC-free medium. Thus, calcium binds more tightly to the membrane than to CTC and is not displaced by the antibiotic. If the bilayer couple hypothesis [Sheetz, M.P., Singer, S.J., Proc. Natl. Acad. Sci. USA 71, 4457-4461 (1974)]applies, crenation is reverted by expansion of the inner membrane half relative to the outer membrane half. Expansion of the inner membrane half results from intercalation of CTC which binds to calcium. Thus, calcium in the red cell membrane preferentially occupies the inner leaflet of the bilayer.

A simple and versatile tool facilitating micropuncture of small cells is described which utilizes a commercial piezoelectric element made from a stacked column of monomorph ceramic discs. The device is able to advance complete input stage-electrode-assemblies with high speed and can be used in combination with conventional micromanipulators. Advancing characteristics as recorded optically at high magnification demonstrated less axial vibration, although faster action, than two other modern micropositioners driven by step motors. In biological experiments on selected tissues (Necturus gallbladder epithelium, Amphiuma renal distal tubule cells, rabbit and human corneal endothelium) the combined use of micromanipulator and piezo-stepper was, in all cases, superior to the use of a micromanipulator alone: the percentage of successful cell penetrations increased, cell potentials were stable for a longer time, and the durability of electrode-tips improved.

During consideration of three film mammographic systems (see article I), the concept of signal/noise ratio is developed as a quantitative measure of film quality. The ability to recognise detail related to detail size, film blackening and exposure geometry was studied for various systems, and the quality profiles are discussed. There is a considerable difference in quality between industrial films without screens and film-screen combinations; however, exposure geometry during mammography has a considerable effect which tends to reduce the difference. Consequently, detail sizes of 200 mu to 1,000 mu (including the majority of mammographic micro-calcifications) are shown about equally well. Contrast for the lo-dose system (D = 1.1) is somewhat less than for adequately exposed industrial film (D = 2.0) or for Cronex 75 m with lo-dose screen (D = 1.5 to 2.0). The lo-dose system (D = 1.1) is 8.6 times more sensitive than Cronex 75 m (D = 2.0), and the system Cronex 75 m with lo-dose screens is 11.6 times more sensitive. Over-exposure with the lo-dose system, contrary to industrial film, rapidly leads to unsatisfactory results (loss of contrast and increased granularity for D greater than 1.5). On the other hand it is often not possible to obtain an adequate exposure (D = 2.0) when using industrial film. For these reasons it is often an advantage to examine large breasts (with considerable geometric unsharpness) and the dense breasts of young women with a film-screen combination (eg. lo-dose system) which requires approximately one eighth of the dose necessary for industrial film. For small or easily compressable breasts (little geometric unsharpness) best results are obtained, using an adequate exposure by employing industrial film; radiation dose it then acceptable. Detailed analysis of the systems has shown that the recording system unsharpness of film-screen combinations in mammography should be reduced.

Kidneys of male Sprague Dawley rats have been isolated and perfused in vitro in order to study the metabolism of corticosterone (B). B is the main endogenous corticosteroid in this species. Using 3H-B and HPLC for the separation of steroid metabolites it has been possible to detect radioactive derivatives of B which have been denoted as met I, II and III. These substances were purified and compared with authentic reference hormones under different isocratic and gradient elution techniques. We observed chromatographic identity of met I with 11-dehydro-20-dihydro-B, of met II with 20-dihydro-B and of met III with 5 alpha-H-4,5-dihydro-B. From the fact that conversion of B can be observed with normal (50 g X l-1 albumin in perfusate) and elevated (75 g X l-1) colloid osmotic pressure of the recirculating perfusate it can be concluded that B gets access to the metabolic site in renal tissue not solely by glomerular filtration and tubular reabsorption. The metabolites identified presently are excreted in the urine. Metopirone increased the concentration of met I and decreased the concentration of met II. This is compatible with the concept of a stimulatory effect of metopirone on a C-20-hydroxysteroid oxidoreductase and a C-11-hydroxysteroid dehydrogenase.

The physical parameters of three radiographic filming systems used for mammography were analysed (industrial film without screens, lo dose system (Dupont), industrial film with lo dose screens). Their effect on image quality was studied and the influence of the exposure geometry on sharpness and detail was investigated experimentally. Geometrical unsharpness so reduces the difference in definition between screenless industrial film and a film-screen combination that the total unsharpness at the centre of the breast parenchyma of an average breast becomes approximately the same. In particular, recognition of fine detail (microcalcification) is reduced by geometric unsharpness. Final grading of the three film systems is only possible after development of a concept of film quality which takes account of the parameters of the filming system and of exposlre geometry, together with physiological and practical requirements (paper II).