Abstract. Invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi occurs by an actinindependent mechanism distinct from phagocytosis. Clusters of host lysosomes are observed at the site of parasite attachment, and lysosomal markers are detected in the vacuolar membrane at early stages of the entry process. These observations led to the hypothesis that the trypanosomes recruit host lysosomes to their attachment site, and that lysosomal fusion serves as a source of membrane to form the parasitophorous vacuole.Here we directly demonstrate directional migration of lysosomes to the parasite entry site, using time-lapse video-enhanced microscopy of L6E 9 myoblasts exposed to T. cruzi trypomastigotes. BSA-gold-loaded lysosomes moved towards the cell periphery, in the direction of the parasite attachment site, but only when their original position was less than 11-12 Ixm from the invasion site. Lysosomes more distant from the invasion area exhibited only the short multi-directional saltatory movements previously described for lysosomes, regardless of their proximity to the cell margins.Specific depletion of peripheral lysosomes was obtained by microinjection of NRK cells with antibodies against the cytoplasmic domain of lgp 120, a treatment that aggregated lysosomes in the perinuclear area and inhibited T. cruzi entry. The microtubule-binding drugs nocodazole, colchicine, vinblastine, and taxol also inhibited invasion, in both NRK and LrE 9 cells. Furthermore, microinjection of antibodies to the heavy chain of kinesin blocked the acidification-induced, microtubule-dependent redistribution of lysosomes to the host cell periphery, and reduced trypomastigote entry.Our results therefore demonstrate that during T. cruzi invasion of host cells lysosomes are mobilized from the immediately surrounding area, and that availability of lysosomes at the cell periphery and microtubule/kinesin-mediated transport are requirements for parasite entry.

Medical message boards are online resources where users with a particular condition exchange information, some of which they might not otherwise share with medical providers. Many of these boards contain a large number of posts and contain patient opinions and experiences that would be potentially useful to clinicians and researchers. We present an approach that is able to collect a corpus of medical message board posts, de-identify the corpus, and extract information on potential adverse drug effects discussed by users. Using a corpus of posts to breast cancer message boards, we identified drug event pairs using co-occurrence statistics. We then compared the identified drug event pairs with adverse effects listed on the package labels of tamoxifen, anastrozole, exemestane, and letrozole. Of the pairs identified by our system, 75–80% were documented on the drug labels. Some of the undocumented pairs may represent previously unidentified adverse drug effects.

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Ca2؉ -regulated exocytosis, previously believed to be restricted to specialized cells, was recently recognized as a ubiquitous process. In mammalian fibroblasts and epithelial cells, exocytic vesicles mobilized by Ca 2؉ were identified as lysosomes. Here we show that elevation in intracellular cAMP potentiates Ca 2؉ -dependent exocytosis of lysosomes in normal rat kidney fibroblasts. The process can be modulated by the heterotrimeric G proteins G s and G i , consistent with activation or inhibition of adenylyl cyclase. Normal rat kidney cell stimulation with isoproterenol, a ␤-adrenergic agonist that activates adenylyl cyclase, enhances Ca 2؉ -dependent lysosome exocytosis and cell invasion by Trypanosoma cruzi, a process that involves parasite-induced [Ca 2؉ ] i transients and fusion of host cell lysosomes with the plasma membrane. Similarly to what is observed for T. cruzi invasion, the actin cytoskeleton acts as a barrier for Ca 2؉ -induced lysosomal exocytosis. In addition, infective stages of T. cruzi trigger elevation in host cell cAMP levels, whereas no effect is observed with noninfective forms of the parasite. These findings demonstrate that cAMP regulates lysosomal exocytosis triggered by Ca 2؉ and a parasite/host cell interaction known to involve Ca 2؉ -dependent lysosomal fusion.Ca 2ϩ -regulated exocytosis has been traditionally regarded as a specialized process present in only a few cell types. Recently, however, evidence has been accumulating indicating that it is a ubiquitous process observed in a variety of animal cells (1-5). A previous study from our laboratory showed that between 10 and 20% of conventional lysosomes in fibroblasts and epithelial cells fuse with the plasma membrane and release their contents upon elevation of the intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) (6). Lysosome exocytosis is similar to other well characterized forms of Ca 2ϩ -regulated exocytosis in specialized cells, in the sense that it is ATP and temperature-dependent and requires micromolar levels of [Ca 2ϩ ] i (6). These findings are consistent with recent capacitance measurements in Chinese hamster ovary cells, which showed that vesicles in the size range of lysosomes (0.4 -1.5 M) fuse with the plasma membrane in response to 6 -20 M [Ca 2ϩ

SUMMARY:Entactin-1 (nidogen-1) is an ubiquitous component of basement membranes. From in vitro experiments, entactin-1 was assigned a role in maintaining the structural integrity of the basement membrane because of its binding affinity to other components, such as type IV collagen and laminin. Entactin-1 also interacts with integrin receptors on the cell surface to mediate cell adhesion, spreading, and motility. Targeted disruption of the entactin-1 gene in the mouse presented in this study revealed a duplication of the entacin-1 locus. Homozygous mutants for the functional locus lacked entactin-1 mRNA and protein and often displayed seizure-like symptoms and loss of muscle control in the hind legs. The behavior patterns suggested the presence of neurologic deficits in the central nervous system, thus providing genetic evidence linking entactin-1 to proper functions of the neuromuscular system. In homozygous mutants, structural alterations in the basement membranes were found only in selected locations including brain capillaries and the lens capsule. The morphology of the basement membranes in other tissues examined superficially appeared to be normal. These observations suggest that the lost functions of entactin-1 result in pathologic changes that are highly tissue specific. (Lab Invest 2002, 82:1617-1630.