Graphical abstractHighlightsThe occurrence of PCR artifacts depends on template, non-template and primer concentrations in the reaction.Reliability and reproducibility of qPCR experiments requires standardized technical aspects and short on-bench times.Measurement of artifact-associated fluorescence can be minimized by primer design and qPCR protocol.
In the larval fat body of Calliphora erythrocephala, protein accumulation and autophagic activity occur prior to the onset of puparium formation. The involvement of the lysosomal system in the degradation of sequestered protein and cell organelles can be demonstrated by the electron-microscopical cytochemical localization of the lysosomal marker enzyme acid phosphatase in so-called protein granules. These granules contain not only newly synthetized or absorbed protein but also remnants of cell organelles such as mitochondria and endoplasmic reticulum. Ligation of the larvae behind the brain-ring gland complex prevents the appearance of these acid phosphatase-positive granules. They can be induced in ligated larvae by the injection of 20-hydroxyecdysone into the abdomen. These findings are briefly discussed in relation to the role of moulting hormones in normal development, especially with regard to the induction of autophagic activity.
Arteriosclerosis in the Wistar rat carotid artery was induced by air drying of the endothelium, a procedure that caused media necrosis. We describe a number of technical modifications that facilitate the procedure and minimize damage to the vessel wall (i.e., media necrosis). A morphometric study of vessel wall changes induced by endothelial denudation at various air-flow rates showed that necrosis of the inner medial layer and lesion size and position were constant at all flow rates used (=s 28 ml/min). The extent of necrosis of the outer medial layers, the endothelial repair, and the shape of the lesions varied with the air-flow rate used. Since at an air-flow rate of 28 ml/min medial necrosis was minimal and myointimal lesion development was as extensive as at higher flow rates, we consider this air-flow rate to be optimal for this model of experimental arteriosclerosis. We hypothesize that the shape of the myointimal lesion is determined by the timespan of endothelial denudation and by the availability of smooth muscle cells in necrotic and normal media. Furthermore, the proliferation of the smooth muscle cells and their migration into the intima is probably dominant over the repopulation of the media by these cells. (Arteriosclerosis 3:441-451, September/October 1983) T he formation of arteriosclerotic, fibromuscularelastic intimal thickening following experimental endothelial denudation has been well documented in a number of animal models. 1 " 4 In rat arteries mechanical injury, 5 air-drying, 6 and balloon catheterization 78 have been shown to remove the endothelium and to cause myointimal thickening. Although these experiments were aimed at specificially causing endothelial injury, the underlying media was often affected as well.8 " 10 Fishman et al. 6 and Clowes et al.
11used the air-drying technique in the rat and claimed that injury, at least at the time of denudation, was confined to the endothelium. This apparent selectivity led us to choose the air-drying model for a study of the effect of experimental conditions on lesion development.In our study however, we found that this injury was not as selective as in other studies; 6 media necrosis (disappearance of smooth muscle cells) occurred in certain areas of the vessel wall. Furthermore, a different intimal thickening pattern was observed. Preliminary studies indicated that the velocity of infused air was important. To find the optimal conditions, we performed the air-drying method by lowering the airflow rate in steps of approximately 5 ml/min down to the lower limit of lesion induction. The air-drying technique of Fishman et al. 6 was modified, thereby facilitating surgery, avoiding mechanical injury at air inflow and outflow holes, and minimizing media necrosis.In this paper we shall present a univariate quantitative analysis of the changes in vessel wall morphology that we found when applying diminished air-flow rates to denude the artery. Special attention will be paid to parameters describing the resulting myointimal lesions, endothelial repai...
Acid phosphatase activity was demonstrated by EM-cytochemistry in 4 day old third instar larvae of the fly Calliphora erythrocephala Meigen, but not in younger stages. During larval development, the activity increased, reaching a maximum at the onset of pupariation. The reaction product was localized in Golgi vesicles and sacculi, in vacuoles and in protein granules of varying size and composition, confirming the autophagic character of the protein granules. Throughout larval development, the reaction product was restricted to membrane-bound structures and no indications of "free" cytoplasmic activity that might be related to cytolysis were found. Enzyme activity could be evoked by transplanting inactive fat body lobes into host larvae of a later developmental stage. High enzyme activity was induced in these transplants within 18 h. The sites of activity were roughly the same, but a portion of the activity in the transplants was found in the vacuoles. The induction could be inhibited by cyclohexamide.
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