Characterization of epithelial cell islets in primary monolayer cultures of human breast carcinomas by the tetrazolium reaction for glucose 6-phosphate dehydrogenase

Epithelial cell islets in primary monolayer cultures of human breast biopsies were characterized by combined immuno-, enzyme- and DNA cytochemistry as well as by analysis of attachment-, spread- and growth patterns. For cultivation we used explants from reduction mammoplasties, benign lesions, primary carcinomas and metastases. Milk fat globule membrane antigen (MFGM-A) was detected with a monoclonal antibody, and the tetrazolium reaction for glucose 6-phosphate dehydrogenase (G6PDH) as well as DNA content of the cultured cells were quantified. Spreading and growth of individual islets were studied by image analysis. Fibroblast-like cells did not express MFGM-A, and whereas epithelial (MFGM-A positive) cell islets of normal and benign origin showed cells with no or low G6PDH reaction, respectively, the majority of epithelial cell islets from 11 out of 21 carcinomas showed strong reaction. Cell islets with strong G6PDH reaction were sometimes hyperdiploid. Moreover, whereas cell islets with no or low reaction from both benign lesions and carcinomas readily attached and spread in a serum-free medium and showed population doubling times of 30 to 110 h, cell islets with strong reaction from carcinomas and metastatic lesions required serum for attachment and their growth rate was too low to be determined.     

Epithelial differentiation in Drosophila pupae

The construction of cell hairs on the wings in developing pupae of Drosophila provides a unique system for studies of the regulation of differentiation in the absence of cell division. Early steps in hair construction are the extrusion of cell hairs and the deposition of the external impervious layer called "cuticulin." Some properties of six of the most abundant proteins that are present during the early stages of hair construction are described. These proteins make up about 40% of the total protein of the preparation.     

Human T cell responses to the Epstein-Barr nuclear antigen-1 (EBNA-1) as evaluated by synthetic peptides

A panel of synthetic peptides derived from Epstein-Barr virus (EBV) nuclear antigen 1 (EB-NA-1) was used to examine human T cell responses to this antigen. In six of seven normal persons with past EBV infection, T cell precursors specific for five peptides (P27, amino acid residues 83-101;P62, 148-166;E31, 353-367;E41, 368-381; and E11, 461-474) were detectable. The precursor frequencies were in the range of 1:20,000 to less than 1:100,000 peripheral blood mononuclear cells as determined by limiting dilution analyses. Only two of these peptides were predicted as alpha-helices; all peptides were glycine-rich. Four other peptides were not reactive in the seven individuals tested. T cell responses were not detectable in donors without prior EBV infection. Infectious mononucleosis patients investigated 4-6 weeks after diagnosis had likewise no detectable peptide-specific T cell precursors. Thus, it appears that T cells recognizing peptides from EBNA-1 arise and persist in people with past EBV infection.     

Intracellular ADP activates K+ channels that are inhibited by ATP in an insulin-secreting cell line

The effect of ADP on ATP-sensitive K+ channels in the insulin-secreting RINm5F cell line has been investigated with the help of single-channel current recording from saponin-permeabilized cells. ADP (100-500 microM) markedly activates K+ channels when added to the bath solution in contact with the membrane inside. ADP-beta-S cannot mimick this effect. During sustained ATP (500 microM)-evoked inhibition of K+ channel opening, 500 microM ADP markedly and reversibly activates the channels. Conversely ATP markedly reduces the opening probability of ADP-activated channels. It is suggested that the physiological control of K+ channel opening in the insulin-secreting cells is mediated by changes in ATP/ADP ratio rather than being solely determined by the ATP concentration.     

Accuracy and reliability of flow cytometric DNA analysis using a simple, one-step ethidium bromide staining protocol

Sources of variation and error were investigated for a simple flow cytometric analysis of DNA content of detergent-isolated nuclei stained with ethidium bromide. Using the ploidy classes of mouse liver nuclei, deviations from linearity were assessed for three different instruments. In more extreme settings, the maximum deviations for a FACS instrument were up to 6 to 9%, but in general deviations were around 1% or lower for all instruments. As biological DNA standards, human peripheral lymphocytes and trout erythrocytes appeared to be suitable and easy to store frozen. The erythrocytes had dye-binding characteristics similar to those of human lymphocytes and a 20% lower fluorescence, thus being well suited as an internal standard, as was demonstrated in tumor ploidy analyses performed with varied tissue concentration. Staining homogeneity was improved when staining time was extended to 24 h, at which time male and female lymphocytes were completely separated with an average difference in DNA content of 1.9%. A small difference in fluorescence between mitogen-stimulated and unstimulated lymphocytes was reduced to less than 1% after 24 h of staining. In general, the manipulations of the conditions for the analysis resulted in maximum variations of around 1%, indicating the robustness and reliability of the technique.     

Short and reversible uncoupling evokes little change in the gap junctions of pancreatic acinar cells

Three different preparations of mouse pancreatic fragments where all the cells tested electrophysiologically showed (a) complete electrical coupling (control), (b) complete uncoupling (after 1-to 2-min exposure to 100% CO2), or (c) complete recoupling (1-2 min after removal of 100% CO2) were fixed, with the electrodes in situ, with 0.2% glutaraldehyde and freeze-fractured for quantitative analysis of acinar cell gap junctions. No obvious difference was observed between gap junctions of coupled and uncoupled acinar cells. However, quantitation revealed a small (2.3-5.6%) increase in particle diameter and spacing within junctions of uncoupled cells. Such increase was rapidly reversed upon cell recoupling. In all preparations, most of the gap junctions were made up of disordered arrays of particles but a few of them showed a more tight packing of their particles of which most had lost the usual globular appearance. These "amorphous" gap junctions had larger particle diameter but smaller particle spacing than the other gap junctions and these parameters were not modified during cell uncoupling. However, "amorphous" gap junctions were more frequent in the latter condition.