In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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1-Benzyl-1,2,3,4-tetrahydroisoquinoline (1 BnTIQ) was detected as a novel endogenous amine in mouse brain and parkinsonian CSF by using the gas chromatography-selected ion-monitoring method . The level of 1 BnTIQ was very high in CSF of some parkinsonian patients compared with that of controls with other neurological diseases, the mean value being three times higher (parkinsonians : 1 .17 ± 0 .35 ng/ml of CSF, n = 18 ; vs . controls : 0.40 -0 .10 ng/ml of CSF, n = 11 ; mean !-SEM, not significantly different) . The pole test, a toxicological examination to evaluate behavior abnormalities related to Parkinson's disease, was used to examine the pharmacological effect of 1 BnTIQ in mice . Repeated administration of 1 BnTIQ induced behavior abnormalities, which pretreatment with 1-methyl-1,2,3,4-tetrahydroisoquinoline could prevent. We suggest that 1 BnTIQ may be related to the idiopathic Parkinson's disease.

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Tributyltin chloride (TBT), an endocrine-disrupting chemical, has been used as a heat stabilizer, agricultural pesticide, and component of antifouling paints. In this study, we investigated the concentration dependence of the mechanisms of tributyltin cytotoxicity in PC12 cells. Exposure of PC12 cells to both 500 nM and 2 microM tributyltin increased the number of cells showing nuclear fragmentation, a typical apoptotic feature, and activated caspase-3. The peak Ca(2+) concentration in 2 microM tributyltin-treated cells was higher than that in 500 nM tributyltin-treated cells. The intracellular Ca(2+) increase induced by 2 microM tributyltin was mediated by Ca(2+) release from both inositol 1,4,5-trisphosphate receptor and ryanodine receptor, while the Ca(2+) increase induced by 500 nM tributyltin was mediated through the voltage-dependent calcium channel (VDCC). Next, we investigated whether the mechanisms leading to cell death after Ca(2+) increase were different. Reactive oxygen species (ROS) were involved only in 2 microM tributyltin-induced cell death, while c-jun N-terminal kinase (JNK) mediated only 500 nM tributyltin-induced toxicity. Thus, caspase-dependent apoptosis caused by 2 microM tributyltin was mediated by a large Ca(2+) increase via inositol 1,4,5-trisphosphate receptor and ryanodine receptor, followed by generation of ROS. Apoptosis caused by 500 nM tributyltin was mediated by a moderate Ca(2+) increase through the VDCC, followed by phosphorylation of JNK. These results suggest that apoptosis by TBT is induced via distinct pathways depending on the TBT concentration, and we showed a rare example that upstream mechanisms of apoptosis are distinct depending on strength of toxic insult.

ABSTRACT:Accurate prediction of pharmacokinetics (PK) parameters in humans from animal data is difficult for various reasons, including species differences. However, chimeric mice with humanized liver (PXB mice; urokinase-type plasminogen activator/severe combined immunodeficiency mice repopulated with approximately 80% human hepatocytes) have been developed. The expression levels and metabolic activities of cytochrome P450 (P450) and non-P450 enzymes in the livers of PXB mice are similar to those in humans. In this study, we examined the predictability for human PK parameters from data obtained in PXB mice. Elimination of selected drugs involves multiple metabolic pathways mediated not only by P450 but also by non-P450 enzymes, such as UDP-glucuronosyltransferase, sulfotransferase, and aldehyde oxidase in liver. ). Elimination half-life (t 1/2 ) after intravenous administration also showed a good correlation (r 2 ‫؍‬ 0.886, p ‫؍‬ 1.506 ؋ 10 ؊4) between humans and PXB mice. The rank order of CL and t 1/2 in human could be predicted at least, although it may not be possible to predict absolute values due to rather large prediction errors. Our results indicate that in vitro and in vivo experiments with PXB mice should be useful at least for semiquantitative prediction of the PK characteristics of candidate drugs in humans.