Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages

Resident peritoneal macrophages of the mouse, cultivated for 3 d, have been studied by quantitative subcellular fractionation using differential centrifugation and density equilibration in linear gradients of sucrose. Density equilibration experiments were carried out on untreated cytoplasmic extracts, on cytoplasmic extracts treated with digitonin or sodium pyrophosphate, and on cytoplasmic extracts derived from cells cultivated for 24 h in the presence of Triton WR-1339. The enzyme distributions obtained distinguished six typical behaviors characteristic of distinct subcellular entities. Acid α-galactosidase and other acid hydrolases displayed the highest average velocity of sedimentation and equilibrium density. Culturing in a medium that contained Triton WR-1339 markedly decreased their density, most likely as a result of Triton WR-1339 accumulation within lysosomes. Cytochrome c oxidase and the sedimentable activity of malate dehydrogenase showed a narrow density distribution centered around 1.17, very similar under all the experimental situations; their rate of sedimentation fell within the range expected for mitochondria. Catalase was particle-bound and exhibited structure-linked latency (80 percent); it was released in soluble and fully active form by digitonin, but this required a much higher concentration than in the case of lysosomal enzymes. Differences relative to all the other enzymes studied suggest the existence of a particular species of organelles, distinctly smaller than mitochondria, and possibly related to peroxisomes. Many enzymes were microsomal in the sense that the specific activities, but not the yields, were greater in microsomes than in other fractions obtained by differential centrifugation. These enzymes were distinguished in three groups by their properties in density equilibration experiments. NAD glycohydrolase, alkaline phosphodiesterase I, and 5’-nucleotidase had low equilibrium densities but became noticeably more dense after addition of digitonin. The other microsomal enzymes were not shifted by digitonin, in particular N-acetylglucosaminyltransferase and galactosyltransferase, which otherwise equilibrated at the same position in the gradient. We assign the digitonin-sensitive enzymes to plasma membranes and possibly to related endomembranes of the cells, and the two glycosyltransferases to elements derived from the Golgi apparatus. Finally, α-glucosidase, sulphatase C, NADH cytochrome c reductase, NADPH cytochrome c reductase, and mannosyltransferase, equilibrated at a relatively high density but were shifted to lower density values after addition of sodium pyrophosphate. These properties support their association with elements derived from the endoplasmic reticulum

Guidelines for peer support in high‐risk organizations: An international consensus study using the delphi method

Despite widespread adoption of peer‐support programs in organizations around the world whose employees are at high risk of exposure to potentially traumatic incidents, little consensus exists regarding even the most basic concepts and procedures for these programs. In this article, consensus refers to a group decision‐making process that seeks not only agreement from most participants, but also resolution of minority objections. The aim of the current study was to develop evidence‐informed peer‐support guidelines for use in high‐risk organizations, designed to enhance consistency around goals and procedures and provide the foundation for a systematic approach to evaluation. From 17 countries, 92 clinicians, researchers, and peer‐support practitioners took part in a 3‐round web‐based Delphi process rating the importance of statements generated from the existing literature. Consensus was achieved for 62 of 77 (81%) statements. Based upon these, 8 key recommendations were developed covering the following areas: (a) goals of peer support, (b) selection of peer supporters, (c) training and accreditation, (d) role of mental health professionals, (e) role of peer supporters, (f) access to peer supporters, (g) looking after peer supporters, and (h) program evaluation. This international consensus may be used as a starting point for the design and implementation of future peer‐support programs in high‐risk organizations