A Simple Cargo Sequestration Assay for Quantitative Measurement of Nonselective Autophagy in Cultured Cells.

Autophagy (self-eating) is a common term for various processes by which cellular components are transferred to lysosomes for degradation. In macroautophagy, intracellular membrane structures termed "phagophores" expand to encapsulate autophagic cargo into sealed, double-membrane vacuoles termed "autophagosomes," which subsequently may fuse with endosomes to form intermediary vacuoles called "amphisomes," and finally with lysosomes to have their contents degraded and recycled. Autophagy is frequently analyzed by monitoring phagophore- and autophagosome-associated markers such as LC3. Although useful, it is becoming increasingly clear that very few, if any, of these marker proteins are entirely specific to the autophagic process. Moreover, phagophore/autophagosome markers cannot be used to measure autophagic activity since they are part of the autophagic machinery, or "cart," rather than autophagic cargo. Thus, there is a great need for functional assays in autophagy research. Here, we describe a method that quantitatively measures the nonselective autophagic sequestration of endogenous cytosolic cargo. The method is based on a crude separation of sedimentable cellular material from cytosol and a subsequent measurement of the fraction of a cytosolic enzyme activity transferred to the sedimentable fraction by autophagic sequestration. The original assay was first developed in 1990, but during the last few years we have systematically downscaled and simplified the method into the time- and cost-efficient procedure presented here, which can be performed with standard laboratory equipment and is suitable for any cell type.

UAP1 is overexpressed in prostate cancer and is protective against inhibitors of N-linked glycosylation.

Prostate cancer is the second most common cause of cancer-associated deaths in men, and signaling via a transcription factor called androgen receptor (AR) is an important driver of the disease. Consequently, AR target genes are prominent candidates to be specific for prostate cancer and also important for the survival of the cancer cells. Here we assess the levels of all hexosamine biosynthetic pathway (HBP) enzymes in 15 separate clinical gene expression data sets and identify the last enzyme in the pathway, UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), to be highly overexpressed in prostate cancer. We analyzed 3261 prostate cancers on a tissue microarray and found that UAP1 staining correlates negatively with Gleason score (P=0.0039) and positively with high AR expression (P<0.0001). Cells with high UAP1 expression have 10-fold increased levels of the HBP end-product, UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is essential for N-linked glycosylation occurring in the endoplasmic reticulum (ER) and high UAP1 expression associates with resistance against inhibitors of N-linked glycosylation (tunicamycin and 2-deoxyglucose) but not with a general ER stress-inducing agent, the calcium ionophore A23187. Knockdown of UAP1 expression re-sensitized cells towards inhibitors of N-linked glycosylation, as measured by proliferation and activation of ER stress markers. Taken together, we have identified an enzyme, UAP1, which is highly overexpressed in prostate cancer and protects cancer cells from ER stress conferring a growth advantage.

[Stages of grief in coping with diabetes?]. / Trauerphasen in der Diabetesbewältigung?

Forms of coping with diabetes were determined on the basis of self-ratings of emotions (depressive, sad, angry, anxious) and responses on a 30-item "stages of grief" questionnaire by 52 inpatients aged between 25 and 50 (diabetes duration 1 to 35 years) at the beginning of a 12 day diabetes education program. Psychometric qualities of the instruments were evaluated. To test empirically the notion of grief stages in coping with diabetes, the frequency of emphasis on one or more emotional areas or stages of grief was determined and tested to ascertain whether various conditions occurred more frequently than was expected by chance. Results indicated that shortly after diabetes manifestation scores on protest and depression with respect to diabetes were elevated. The majority of cases could not be assigned to a single emotion or stage of grief but displayed negative emotional reactions in different content areas simultaneously. Consequently the concept of grief stages for coping with diabetes as well as recommendations based on this concept are questioned.