Selective erythrocyte potassium efflux following pulse treatment with tellurite.

Human erythrocytes exposed to 0.1 mM tellurite (K2TeO3) in an isotonic buffered choline chloride medium for 15 min at 37 degrees C, washed, and incubated further in the absence of the chemical in the buffer, exhibited selective leakiness for potassium within minutes. The potassium efflux curve was sigmoidal, with an initially slow leakage followed by a sharp rise (first-order kinetics) and a plateau by 60 min. After 15 min, 30-50% of the total potassium concentration had leaked from the cells, although less than 1% lysis had occurred. The control cells incubated in buffer with no K2TeO3 exhibited no potassium leakage. The mean volume of the K2TeO3-treated erythrocytes increased and their median density decreased, indicating changes in the colloid osmotic state and physical characteristics of the cells. However, cells pretreated with K2TeO3 exhibited no significant change in glutathione (GSH) concentration and no membrane lipid peroxidation, unlike cells pretreated with t-butylhydroperoxide (Deuticke et al., Biochim. Bio phys. Acta, 899, 125-128, 1987). The enhanced potassium permeability of the K2TeO3-treated erythrocytes preceded the increase in cell volume, intracellular hydration, and a decrease in median density. We suggest that perturbation of the lipid-protein interaction in the membrane by the oxidant alters the potassium permeability and results in the selective leakage with eventual hemolysis.     

Inhibition of acid production in Streptococcus mutans R9: Inhibition constants and reversibility

Abstract The pac gene encoding the penicillin G acylase (PGA) of Bacillus megaterium ATCC 14945 has been cloned in Escherichia coli HB101 ( proA, leuB ) using a selective minimal medium containing phenylacetyl-L-leucine instead of L-leucine. The nucleotide sequence of this gene has been determined and contains an open reading frame of 2406 nucleotides. The deduced amino acid sequence shows significant similarity with other β-lactam acylases. Although the PGA of B. megaterium is extracellular, the enzyme produced in E. coli appears to have a cytoplasmic localization.     

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Reorganization of the Endoplasmic Reticulum during Meiotic Maturation of the Mouse Oocyte

The endoplasmic reticulum (ER) of live metaphase II mouse eggs and prophase I-arrested oocytes was compared using the fluorescent, lipophilic dicarbocyanine dye, DiI. DiI, dissolved in soybean oil, was microinjected into oocytes and eggs; the dye diffused throughout the cytoplasm to label the ER, which was imaged by confocal microscopy. The mature egg had a fine reticular network of ER throughout the cell and numerous dense accumulations of membrane in the cortex. These ER accumulations, 1-2 μm in diameter, were generally absent deeper in the cytoplasm. A similar staining pattern was observed when the eggs were fixed within 1 min of injection, providing evidence that the cortical accumulations of membrane are part of a continuous ER membrane system, since membrane trafficking could not occur in a fixed egg. Cortical ER accumulations were localized to the same region of the egg as the cortical granules and were not observed in the cortical granule-free region adjacent to the meiotic spindle. In contrast, ER accumulations were rarely found in the cortex of the immature, prophase I-arrested oocyte, but larger and less well-defined membrane clusters were found throughout the deeper cytoplasm of the oocyte. The appearance of ER clusters in the egg cortex following oocyte maturation correlates with an increased ability of the mature egg to release calcium at fertilization. Since the ER is a calcium store, structural reorganization of the ER may be necessary to permit the large release of calcium and resulting cortical granule exocytosis at fertilization.