Uptake and accumulation of Mn2+ and Sr2+ in Saccharomyces cerevisiae

Initial uptake of Mn2+ and Sr2+ in the yeast Saccharomyces cerevisiae was studied in order to investigate the selectivity of the divalent cation uptake system and the possible involvement of the plasma-membrane ATPase in this uptake. The initial uptake rates of the two ions were not significantly different. This ruled out a direct role of the plasma-membrane ATPase, since this ATPase is specific for Mn2+ compared to Sr2+. After 1 h uptake, Mn2+ had accumulated 10-times more than Sr2+. Influx of Mn2+ and Sr2+ remained unchanged during that time, however. The differences in accumulation level found for Mn2+ and Sr2+ could be ascribed to a greater efflux of Sr2+ as compared with Mn2+. Probably this greater efflux of Sr2+ was only apparent, since differential extraction of the yeast cells revealed that Mn2+ is more compartmentalised than Sr2+, giving rise to a lower relative cytoplasmic Mn2+ concentration.     

Mutation analysis of patients with Hermansky-Pudlak syndrome: a frameshift hot spot in the HPS gene and apparent locus heterogeneity.

Hermansky-Pudlak syndrome (HPS) is a rare, autosomal recessive disorder in which oculocutaneous albinism, bleeding, and lysosomal ceroid storage result from defects of multiple cytoplasmic organelles-melanosomes, platelet-dense granules, and lysosomes. As reported elsewhere, we mapped the human HPS gene to chromosome segment 10q23, positionally cloned the gene, and identified three pathologic mutations of the gene, in patients from Puerto Rico, Japan, and Europe. Here, we describe mutation analysis of 44 unrelated Puerto Rican and 24 unrelated non-Puerto Rican HPS patients. A 16-bp frameshift duplication, the result of an apparent founder effect, is nearly ubiquitous among Puerto Rican patients. A frameshift at codon 322 may be the most frequent HPS mutation in Europeans. We also describe six novel HPS mutations: a 5' splice-junction mutation of IVS5, three frameshifts, a nonsense mutation, and a one-codon in-frame deletion. These mutations define an apparent frameshift hot spot at codons 321-322. Overall, however, we detected mutations in the HPS gene in only about half of non-Puerto Rican patients, and we present evidence that suggests locus heterogeneity for HPS.     

Isolation and characterization of an inhibitory subunit of the Mg2+--Ca2+-ATPase of Escherichia coli.

1. Stimulation of the Escherichia coli ATPase activity by urea and trypsin shows that the ATPase activity both in the membrane-bound and the solubilized form is partly masked. 2. A protein, inhibiting the ATPase activity of Escherichia coli, can be isolated by sodium dodecyl sulphate polyacrylamide gel electrophoresis of purified ATPase. The inhibitor was identified with the smallest of the subunits of E. coli ATPase. 3. The molecular weight of the ATPase inhibitor is about 10,000, as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and deduced from the amino acid composition. 4. The inhibitory action is independent of pH, ionic strength or the presence of Mg2+ or ATP. 5. The ATPase inhibitor is heat-stable, insensitive to urea but very sensitive to trypsin degradation. 6. The Escherichia coli ATPase inhibitor does not inhibit the mitochondrial or the chloroplast ATPase.     

The prevalence of the Staphylococcus aureus tst gene among community- and hospital-acquired strains and isolates from Wegener's Granulomatosis patients

To allow rapid identification of toxic shock syndrome toxin-1 (TSST-1)-producing Staphylococcus aureus strains, a real-time PCR assay for the detection of the tst gene, which encodes TSST-1, was developed. The assay was applied to S. aureus isolates from patients with Wegener's Granulomatosis (WG), as well as isolates that were classified as either community- (CA) or hospital-acquired (HA). No significant difference in the percentage of tst-positive strains was observed between isolates from WG patients and CA isolates (24% and 25%, respectively). In contrast, only 14% of the HA isolates were tst-positive (p<0.05). Investigation of the clonal relationship between tst-positive CA and HA strains could indicated the recent emergence of a virulent S. aureus clone in the community.     

Formaldehyde fixation and microwave irradiation

Summary Formaldehyde is the most commonly used fixative in pathology laboratories. However, due to time pressures, this fixative is often not optimally exploited. the majority of biopsies are only partly fixed when histoprocessing is started, with adverse effects. This paper reports how formaldehyde fixation is improved, by using 1.5 min of microwave irradiation of tissue previously soaked for four hours in the fixation solution. It is argued that this beneficial effect of microwave irradiation can be attributed to the acceleration of the reaction of formaldehyde to the tissue. Formation of free formaldehyde, by the dehydration of methylene glycol present in the tissue when the irradiation starts, is also enhanced. Five different formaldehyde-containing fixatives were evaluated, using five different working protocols. Spleen was taken as a suitable tissue for these tests. The technique described leads to uniform microscopical results. It is a simple method and is suitable for use in routine laboratories.