Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

The covalent marking of proteins by methyl group addition to arginine residues can promote their recognition by binding partners or can modulate their biological activity. A small family of gene products that catalyze such methylation reactions in eukaryotes (PRMTs) work in conjunction with a changing cast of associated subunits to recognize distinct cellular substrates. These reactions display many of the attributes of reversible covalent modifications such as protein phosphorylation or protein lysine methylation; however, it is unclear to what extent protein arginine demethylation occurs. Physiological roles for protein arginine methylation have been established in signal transduction, mRNA splicing, transcriptional control, DNA repair, and protein translocation.

Methylation of arginine (Arg) and lysine residues in histones has been correlated with epigenetic forms of gene regulation. Although histone methyltransferases are known, enzymes that demethylate histones have not been identified. Here, we demonstrate that human peptidylarginine deiminase 4 (PAD4) regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. PAD4 targets multiple sites in histones H3 and H4, including those sites methylated by coactivators CARM1 (H3 Arg17) and PRMT1 (H4 Arg3). A decrease of histone Arg methylation, with a concomitant increase of citrullination, requires PAD4 activity in human HL-60 granulocytes. Moreover, PAD4 activity is linked with the transcriptional regulation of estrogen-responsive genes in MCF-7 cells. These data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones.

Reinvestigation of the chemical structure of (-amyloid nonpathological by-product of cellular metabolism, whereas A3-(1-42) may have the more important role in the formation of neuritic plaques.With this in mind, we reexamined amyloid from the cerebrovasculature of AD brains and found that A,B-(1-42) is also the major form in these deposits. Interestingly, the amount of racemization and isomerization at aspartyl residues is much less than in neuritic plaque Af-(1-42). The localization of these undegradable aggregates suggests that their deposition might be linked to a compromised blood-brain barrier. MATERIALS AND METHODSHuman brains obtained at autopsy met the diagnostic criteria for AD established by the National Institutes of Health Neuropathology Panel (10) and by the Consortium to Establish a Registry for AD (CERAD) (11). Morphometric analyses identified brains that contained large amounts of AP in compact cores and in the blood vessels. Left hemispheres were analyzed histopathologically, while right hemispheres were stored at -70°C for subsequent A,3 isolation.Purification of A,B from Leptomeningeal Blood Vessels. The leptomeninges were gently pulled from the surface of 1-cmthick coronal sections with the aid of a dissecting microscope and immersed in 0.1 M Tris HCl (pH 8.0; TB) at 4°C. Blood vessels larger than 1 mm in diameter were discarded, and the remaining tissue was cut with scissors into 1-to 2-mm pieces. The tissue was washed eight times with 1 liter of TB at 4°C with continuous stirring for 10 min and was collected by filtration (45-,um mesh). After resuspension of this material in 20 vol of 2 mM CaCl2 in TB, 0.3 mg of collagenase (Worthington) and 10 p.g of DNase I (Worthington) were added per ml, and the suspension was shaken for 18 hr at 37°C. Large debris was removed by filtration (350-,gm mesh), and the smaller insoluble material was recovered by centrifugation at 6000 x g for 15 min. The resulting pellet was resuspended in 100 vol of 2% SDS in TB and incubated for 2 hr at room temperature, after which the insoluble material was again recovered by centrifugation (see above) and washed twice with distilled water. The pellet was then dissolved with 8 vol of 98% (vol/vol) glass-distilled formic acid (15 min at room temperature) and centrifuged at 430,000x g for 15 min in Polyallomer tubes in a TLA 100.2 rotor (Beckman). Pure A8 was isolated from the clear supernatant by size-exclusion chromatography with a Superose 12 column (10 x 300 mm) on a Pharmacia-LKB fast protein liquid chromatography (FPLC) system with a running buffer of 75%

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The TIS21 immediate-early gene and leukemia-associated BTG1 gene encode proteins with similar sequences. Two-hybrid analysis identified a protein that interacts with TIS21 and BTG1. Sequence motifs associated with S-adenosyl-L-methionine binding suggested this protein might have methyltransferase activity. A glutathione Stransferase (GST) fusion of the putative methyltransferase modifies arginine residues, in appropriate protein substrates, to form N G -monomethyl and N G ,N Gdimethylarginine (asymmetric). We term the proteinarginine N-methyltransferase (EC 2.1.1.23) gene "PRMT1," for protein-arginine methyltransferase 1. GST-TIS21 and GST-BTG1 fusion proteins qualitatively and quantitatively modulate endogenous PRMT1 activity, using control and hypomethylated RAT1 cell extracts as methyl-accepting substrates. PRMT1 message appears ubiquitous, and is constitutive in mitogen-stimulated cells. Modulation of PRMT1 activity by transiently expressed regulatory subunits may be an additional mode of signal transduction following ligand stimulation.The protein products of the immediate-early/primary response genes are thought to act as "third messengers," mediating phenotypic alterations in cells in response to ligands such as growth factors, hormones, neurotransmitters, cytokines, and neurotrophins. Many immediate-early genes encode transcription factors (e.g. Fos, Jun, Egr-1) that initiate transcriptional cascades required for proliferation or differentiation . Other ligand-induced immediate-early genes encode paracrine mediators of cellular communication whose products (e.g. prostaglandin synthase-2, inducible nitricoxide synthase, and cytokines such as MCP-1) modulate the behavior of neighboring cells (Smith and Herschman, 1995).Because immediate-early/primary response genes have been cloned on the basis of their induction characteristics, rather than the functions of their protein products, a number of these genes encode proteins whose biological roles have not yet been determined. One such immediate-early gene is TIS21. The TIS21 cDNA was cloned by differential screening, both from a cDNA library prepared from mitogen-treated, quiescent murine Swiss 3T3 cells (Fletcher et al., 1991) and from a cDNA library prepared from nerve growth factor-treated rat PC12 pheochromocytoma cells (Bradbury et al., 1991). The predicted rat and mouse TIS21 proteins differ at only four out of 158 amino acid residues. We demonstrated, by metabolic labeling followed by immunoprecipitation, that maximal TIS21 protein synthesis occurs within the first hour after exposure to ligand, both in mitogen-stimulated Swiss 3T3 cells and in nerve growth factor-stimulated PC12 cells (Varnum et al., 1994). Moreover, the half-life of both mitogen-and nerve growth factor-induced TIS21 protein is less than 15 min (Varnum et al., 1994). Despite substantial investigation into both the structure of the TIS21 gene and the induced expression of the TIS21 message and protein, no function has been identified for this protein.The human BTG1 gene was cloned and ...

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