Fluoreszenzmikroskopische Untersuchungen über das Haftprotein bei Stärkekörnern

Ohne Zusammenfassung     

De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy

ABSTRACT

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using ¹³C-labeled choline and ¹³C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues.     

Effects of intracranial implants of dihydrotestosterone on the reproductive physiology and behavior of male guinea pigs

An experiment was conducted to determine whether the intracranial application of dihydrotestosterone (DHT), a nonaromatizable androgen, would stimulate male guinea pig mating. Of three castrate groups studied, one was a control group in which subjects were implanted both in the medial preoptic area (MPOA) and under the skin with cannulae containing cholesterol (NoDHT). Males in one of the experimental groups received implants of cholesterol in the MPOA plus subcutaneous implants containing DHT (ScDHT). Members of the other experimental group were subcutaneously implanted with cholesterol and simultaneously given intracranial implants of DHT (IcDHT). Compared to either the NoDHT control group of the ScDHT experimental group, greater numbers of males in the IcDHT group displayed mounts (P less than 0.01), intromissions (P less than 0.01), and ejaculations (P less than 0.001). Additionally, the hypothalamic implants of DHT had no significant effects on peripheral target tissues. These data indicate that androgenic stimulation of the guinea pig brain is sufficient to activate masculine sexual behavior in this species.