Enhanced cell survival and resistance to apoptosis during thermotolerance correlates with an increased expression of heat shock proteins (Hsps). Here we present additional evidence in support of the hypothesis that the induction of Hsp27 and Hsp72 during acquired thermotolerance in Jurkat T-lymphocytes prevents apoptosis. In thermotolerant cells, Hsp27 was shown to associate with the mitochondrial fraction, and inhibition of Hsp27 induction during thermotolerance in cells transfected with hsp27 antisense potentiated mitochondrial cytochrome c release after exposure to various apoptotic stimuli, despite the presence of elevated levels of Hsp72. Caspase activation and apoptosis were inhibited under these conditions. In vitro studies revealed that recombinant Hsp72 more efficiently blocked cytochrome c-mediated caspase activation than did recombinant Hsp27. A model is presented for the inhibition of apoptosis during thermotolerance in which Hsp27 preferentially blocks mitochondrial cytochrome c release, whereas Hsp72 interferes with apoptosomal caspase activation.

Water compartments, permeability, and the possible active translocation of various substances in rat liver microsomes were studied by using radioactive compounds and ultracentrifugation. The total water of the microsomal pellet, 3.4 #l/rag dry weight, is the sum of water in the extramicrosomal and intramicrosomal spaces, or 56 and 44%, respectively. Sucrose space accounts for 77% of the intramicrosomal water and the hydration water ~ 14 %, leaving almost no sucrose-impermeable space when using the ultracentrifugation approach. With increasing sucrose concentration, microsomes do not show an osmotic response. The intramicrosomal water decreases greatly-in the presence of Cs + and Mg ++ in rough but not in smooth microsomes. Uncharged substances of molecular weight of up to at least 600 freely penetrate microsomal membranes, which already become impermeable to charged substances at a molecular weight of 90. These substances also induce an osmotic response. The vesicles can be made permeable to charged substances after water treatment and cooling, which, however, does not increase glucose-6-phosphatase and inosine diphosphatase (IDPase) activities, and these enzymes can still be activated by deoxycholate. IDPase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation reactions, performed in vitro, also disproved the hypothesis of an accumulation of charged substances inside of vesicles of being a major pathway. The products of the enzymic reactions as well as the glucuronidated form of a hydroxylated product can be recovered on the cytoplasmic side of membranes, and little accumulation occurs in the intravesicular compartment.

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The capacity of isolated membrane fractions to catalyse transfer of sugars from sugar nucleotides to alpha-saturated and non-saturated forms of phosphorylated C85 and C55 polyprenols and retinyl phosphate was examined. The amount of endogenous lipid acceptor present for various sugars was also measured. It appears that the types and amounts of polyprenyl phosphates present in rough- and smooth-microsomal fractions and Golgi membranes are different and the individual polyprenyl phosphates exhibit specificity as sugar acceptors.

Mannosylation of the proteins of microsomal and Golgi membranes was investigated both after incubation in vitro of the isolated subfractions with GDP‐[14C]mannose and after injection of [3H]mannose into rats followed by separation of these subfractions. Mannosylation of endogenous and added exogenous dolichol phosphate and also of dolichol pyrophosphate‐oligosaccharide occurs in all three fractions. It was essential to inhibit antagonistic enzymes during incubation and to centrifuge after incubation. The presence of detergent in the incubation mixture influences the incorporation pattern of the different fractions in very different ways. In a system in vitro predominantly membrane proteins and not secretory proteins are mannosylated. Trypsin treatment of intact vesicles removes components from the outer surface only; such treatment liberates about one third of the radioactive mannose associated with lipid, releases radioactivity from the protein acceptor to the same extent and causes some inactivation of the transferase activities. It appears that a part of the mannosyl transferase system in rough and smooth endoplasmic reticulum and in Golgi membranes is localized at the cytoplasmic side of these membranes. This activity is probably involved in the glycosylation of proteins localized at the cytoplasmic surface of the endoplasmic reticulum.

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Normal rabbit alveolar macrophages were infected in vitro with Candida albicans. Early after infection, germ tube formation by phagocytized C. albicans was inhibited in contrast to extracellular (nonphagocytized) C. albicans. Over an 8-h period, plate counts of C. albicans incubated with alveolar macrophages revealed a decrease in colony-forming units in contrast to C. albicans alone. In addition, an assay was developed which specifically measured C. albicans [3H]leucine incorporation in the presence of alveolar macrophages. Using this assay, we observed a 71 to 93% inhibition of macromolecular synthesis in C. albicans when incubated with alveolar macrophages. Autoradiographic studies showed that the inhibition of leucine incorporation was restricted to the ingested Candida.

Lysosomal-rich fractions, obtained from normal rabbit alveolar macrophages, were extracted and tested for their effects on Candida albicans. The uptake and incorporation of various compounds (amino acids, uridine, 2-deoxy-D-glucose, and Rb+) by C. albicans were measured in the presence and absence of extract. These studies demonstrated that the extract had a specific effect on the uptake of certain amino acids by C. albicans. Of the amino acids tested, the uptake of methionine, valine, lysine, phenylalanine, and leucine was drastically reduced in the presence of extract, whereas proline and glutamic acid uptake was unaffected. Those amino acids whose uptake was inhibited have been shown to be transported in other yeasts by a general amino acid permease. The existence of a general amino acid permease in C. albicans is compatible with our data. Additionally, the extract 506 on July 16, 2020 by guest http://iai.asm.org/ Downloaded from