Transfection of Mv1Lu mink lung type II alveolar cells with ␤1-6-N-acetylglucosaminyl transferase V is associated with the expression of large lysosomal vacuoles, which are immunofluorescently labeled for the lysosomal glycoprotein lysosomal-associated membrane protein-2 and the ␤1-6-branched N-glycan-specific lectin phaseolis vulgaris leucoagglutinin. By electron microscopy, the vacuoles present the morphology of multilamellar bodies (MLBs). Treatment of the cells with the lysosomal protease inhibitor leupeptin results in the progressive transformation of the MLBs into electron-dense autophagic vacuoles and eventual disappearance of MLBs after 4 d of treatment. Heterologous structures containing both membrane lamellae and peripheral electron-dense regions appear 15 h after leupeptin addition and are indicative of ongoing lysosome-MLB fusion. Leupeptin washout is associated with the formation after 24 and 48 h of single or multiple foci of lamellae within the autophagic vacuoles, which give rise to MLBs after 72 h. Treatment with 3-methyladenine, an inhibitor of autophagic sequestration, results in the significantly reduced expression of multilamellar bodies and the accumulation of inclusion bodies resembling nascent or immature autophagic vacuoles. Scrape-loaded cytoplasmic FITC-dextran is incorporated into lysosomal-associated membrane protein-2-positive MLBs, and this process is inhibited by 3-methyladenine, demonstrating that active autophagy is involved in MLB formation. Our results indicate that selective resistance to lysosomal degradation within the autophagic vacuole results in the formation of a microenvironment propicious for the formation of membrane lamella. INTRODUCTIONMultilamellar bodies (MLBs) are membrane-bound cellular organelles, which vary in size from 100-2400 nm, are composed of concentric membrane layers, and frequently exhibit an electron-dense core. MLBs are found in numerous cell types where they function in lipid storage and secretion (Schmitz and Mü ller, 1991). In lung type II alveolar cells, MLBs function as secretory granules whose exocytosis results in the deposition of the tubular myelin forms of surfactant on the surface of the alveolae (Hatasa and Nakamura, 1965;Ryan et al., 1975;Williams, 1977). The surfactant film over the alveolar epithelium regulates the surface tension at the air-cell interface and protects the alveola from collapse during respiration (Haagman and van Golde, 1991).Although the secretory function of MLBs in type II alveolar cells is well established, the precise mechanism of MLB biogenesis remains unclear. Autoradiographic studies of murine type II alveolar cells of mouse lungs showed that although phospholipids labeled with [ 3 H]choline are delivered directly from the Golgi to the MLB, proteins metabolically labeled with [ 3 H]leucine are visualized within multivesicular bodies before delivery to MLBs (Chevalier and Collet, 1972). Surfactant proteins A, B, and C are delivered via multivesicular bodies to MLBs, and multivesicular bodies are proposed as th...

Nonenterotoxigenic porcine Escherichia coli strains belonging to the serogroup O45 have been associated with postweaning diarrhea in swine and adhere to intestinal epithelial cells in a characteristic attaching and effacing (A/E) pattern. O45 porcine enteropathogenic E. coli (PEPEC) strain 86-1390 induces typical A/E lesions in a pig ileal explant model. Using TnphoA transposon insertion mutagenesis on strain 86-1390, we found a mutant that did not induce A/E lesions. The insertion was identified in a gene designated paa (porcine A/E-associated gene). Sequence analysis of paa revealed an open reading frame of 753 bp encoding a 27.6-kDa protein which displayed 100, 51.8, and 49% homology with Paa of enterohemorrhagic E. coli O157:H7 strains (EDL933 and Sakai), PEB3 of Campylobacter jejuni, and AcfC of Vibrio cholerae, respectively. Chromosomal localization studies indicated that the region containing paa was inserted between the yciD and yciE genes at about 28.3 min of the E. coli K-12 chromosome. The presence of paa and eae sequences in the porcine O45 strains is highly correlated with the A/E phenotype. However, the observation that three eae-positive but paa-negative PEPEC O45 strains were A/E negative provides further evidence for the importance of the paa gene in the A/E activity of O45 strains. As well, the complementation of the paa mutant restored the A/E activity of the 86-1390 strain, showing the involvement of Paa in PEPEC pathogenicity. These observations suggest that Paa contributes to the early stages of A/E E. coli virulence.Attaching and effacing (A/E) Escherichia coli (AEEC) induces distinctive histopathological lesions on the intestinal mucosa, known as the A/E lesions. These lesions are characteristic of enteric pathogens such as enteropathogenic E. coli (EPEC), responsible for severe childhood diarrhea in developing countries (14, 38), enterohemorrhagic E. coli (EHEC), causing hemorrhagic colitis and hemolytic-uremic syndrome, a diarrheagenic E. coli strain of rabbits (RDEC-1), strains of Hafnia alvei isolated from children with diarrhea, and Citrobacter rodentium, causing transmissible colonic hyperplasia in mice (4,16,53). A/E lesions have also been associated with diarrhea in different animal species such as rabbits, calves, dogs, cats, lambs, pigs, and tamarins (8,9,22,32,37,55).A/E lesions result from intimate bacterial adherence to the apical surfaces of enterocytes and activation of several chromosomal gene products that interact with components of the host cell, leading to host cell protein phosphorylation, effacement of target brush borders, and disruption of the underlying actin cytoskeleton (11, 38). The genes are clustered in a chromosomal pathogenicity island called the locus of enterocyte effacement (LEE). Its location and size vary in different strains. In EPEC strain E2348/69 and EHEC O157:H7 strains, the LEE is inserted in the selC locus at about 82 min on the E. coli K-12 chromosome, but its size varies from 35 kb for EPEC to 43 kb for EHEC. In strains of serotype O26:H-, the...

The development of neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington disease is strongly agedependent. Discovering drugs that act on the high rate of aging in older individuals could be a means of combating these diseases. Reduction of the activity of the mitochondrial enzyme CLK-1 (also known as COQ7) slows down aging in Caenorhabditis elegans and in mice. Clioquinol is a metal chelator that has beneficial effects in several cellular and animal models of neurodegenerative diseases as well as on Alzheimer disease patients. Here we show that clioquinol inhibits the activity of mammalian CLK-1 in cultured cells, an inhibition that can be blocked by iron or cobalt cations, suggesting that chelation is involved in the mechanism of action of clioquinol on CLK-1. We also show that treatment of nematodes and mice with clioquinol mimics a variety of phenotypes produced by mutational reduction of CLK-1 activity in these organisms. These results suggest that the surprising action of clioquinol on several age-dependent neurodegenerative diseases with distinct etiologies might result from a slowing down of the aging process through action of the drug on CLK-1. Our findings support the hypothesis that pharmacologically targeting aging-associated proteins could help relieve age-dependent diseases.

Genetic analysis has shown that the slower than normal rhythmic defecation behavior of the clk-1 mutants of Caenorhabditis elegans is the result of altered lipoprotein metabolism. We show here that this phenotype can be suppressed by drugs that affect lipoprotein metabolism, including drugs that affect HMG-CoA reductase activity, reverse cholesterol transport, or HDL levels. These pharmacological effects are highly specific, as these drugs affect defecation only in clk-1 mutants and not in the wild-type and do not affect other behaviors of the mutants. Furthermore, drugs that affect processes not directly related to lipid metabolism show no or minimal activity. Based on these findings, we carried out a compound screen that identified 190 novel molecules that are active on clk-1 mutants, 15 of which also specifically decrease the secretion of apolipoprotein B (apoB) from HepG2 hepatoma cells. The other 175 compounds are potentially active on lipid-related processes that cannot be targeted in cell culture. One compound, CHGN005, was tested and found to be active at reducing apoB secretion in intestinal Caco-2 cells as well as in HepG2 cells. This compound was also tested in a mouse model of dyslipidemia and found to decrease plasma cholesterol and triglyceride levels.Thus, target processes for pharmacological intervention on lipoprotein synthesis, transport, and metabolism are conserved between nematodes and vertebrates, which allows the use of C. elegans for drug discovery.

A correction to this paper has been published and can be accessed via a link at the top of the paper.