Transmission electron microscopic studies on the mitotic cycle of nucleolar proteins impregnated with silver

Hela cells were impregnated with silver according to Paweletz et al. (1967). In cells in mitosis not only the nucleolar organizer regions (NORs) are strongly impregnated but also part of the nucleolar material, which accumulates in and around the chromosomes. The treatment with adenosine, which in interphase cells spreads the nucleolar material within the nucleus, also distributes the argentophilic material in and around the chromosomes. During the reconstruction phase this material reassembles around the NORs to form parts of the new nucleolus. The silver impregnation technique clearly demonstrates that two main components are responsible for the argentophily of the nucleolus. This is in agreement with the results obtained by Lischwe et al. (1979).     

Familial breast/ovarian cancer and BRCA1/2 genetic screening: the role of immunohistochemistry as an additional method in the selection of patients.

Only 20-25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene.     

Slow heat rate increases yeast thermotolerance by maintaining plasma membrane integrity.

Thermal resistance of Saccharomyces cerevisiae was found to be drastically dependent on the kinetics of heat perturbation. Yeasts were found to be more resistant to a plateau of 1 h at 50 degrees C after a slope of temperature increase (slow and linear temperature increments) than after a shock (sudden temperature change). Thermotolerance was mainly acquired between 40-50 degrees C during a heat slope, i.e., above the maximal temperature of growth. The death of the yeasts subjected to a heat shock might be related to the loss of membrane integrity: intracellular contents extrusion, i.e., membrane permeabilization, was found to precede cell death. However, the permeabilization did not precede cell death during a heat slope and, therefore, membrane permeabilization was a consequence rather than a cause of cell death. During a slow temperature increase, yeasts which remain viable may have time to adapt their plasma membrane and thus maintain membrane integrity.     

Receptor-binding and down-regulatory properties of 22000-Mr human growth hormone and its natural 20000-Mr variant on IM-9 human lymphocytes.

Our earlier binding studies of the 22000- and 20000-Mr variants of human growth hormone (somatotropin) to pregnant-rabbit liver and mammary receptors [Closset, Smal, Gomez & Hennen (1983) Biochem. J. 214, 885-892] suggested that the 20000-Mr variant was a lower-affinity analogue of the 22000-Mr molecule. Since the receptor population in these tissues is not fully characterized, we have now investigated the binding of both variants to the well-characterized and highly specific human-growth-hormone receptor of the human lymphocyte IM-9 cell line. The maximum bindability of radioiodinated 22000- and 22000-Mr to IM-9 cells was 60 and 45% respectively. Both hormone variants have essentially the same binding characteristics: slow association (equilibrium reached in 8-10h at 30 degrees C), poor reversibility ('tight binding'), linear Scatchard plot, same specificity as shown by lack of competition by bovine, porcine or equine growth hormones or human growth hormone-(32-46)-(missing in the 20000-Mr variant),-(1-134)- and -(141-191)-peptides. Both unlabelled hormones inhibit binding of both tracers completely, with the 20000-Mr variant being only half as potent as the 22000-Mr one. The apparent affinity is 2.8 X 10(9)M-1 for the 22000-Mr variant and 1.6 X 10(9)M-1 for the 20000-Mr variant. This decreased affinity of the 20000-Mr variant appears to be due to a lower association rate constant. Concentrations (5 ng/ml) of the two variants that occupy about 15% of the total sites induce a marked down-regulation of the receptors after 18h incubation, but the 20000-Mr variant (50% decrease) has a smaller effect than the 22000-Mr variant (75% decrease). Thus the only consequence of the residues-32-46 deletion in the 20000-Mr variant is a lower association rate and affinity for the IM-9 lymphocyte human-growth-hormone receptor. The close binding characteristics of the two forms suggest that the known differences in their insulin-like effects cannot be explained by differences in the nature of their interaction with the human-growth-hormone receptor.     

A new species of bound ubisemiquinone anion in QH2: cytochrome c oxidoreductase

Using a combination of EPR and low temperature diffuse reflectance spectroscopy, a new species of semiquinone anion has been detected in QH2:cytochrome c oxidoreductase in submitochondrial particles under conditions of oxidant-induced extra reduction of cytochrome b. In contrast to the previously detected semiquinone anion, this new species is insensitive to antimycin but sensitive to treatment with 2,3-dimercaptopropanol and O2. The two species can easily be distinguished on the basis of their respective EPR properties since they differ in g-value, line width, and microwave power saturation behavior. It is concluded that the two species of semiquinone anion are bound to different domains on QH2:cytochrome c oxidoreductase. The existence of two different semiquinone anions in the enzyme strongly supports a mechanism of electron flow as proposed in the Q-cycle.