Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., ...cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca
2+
content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Compared to the emerging embryonic stem cell (ESC) gene network, little is known about the dynamic gene network that directs reprogramming in the early embryo. We hypothesized that Oct4, an ESC ...pluripotency regulator that is also highly expressed at the 1- to 2-cell stages in embryos, may be a critical regulator of the earliest gene network in the embryo.
Using antisense morpholino oligonucleotide (MO)-mediated gene knockdown, we show that Oct4 is required for development prior to the blastocyst stage. Specifically, Oct4 has a novel and critical role in regulating genes that encode transcriptional and post-transcriptional regulators as early as the 2-cell stage. Our data suggest that the key function of Oct4 may be to switch the developmental program from one that is predominantly regulated by post-transcriptional control to one that depends on the transcriptional network. Further, we propose to rank candidate genes quantitatively based on the inter-embryo variation in their differential expression in response to Oct4 knockdown. Of over 30 genes analyzed according to this proposed paradigm, Rest and Mta2, both of which have established pluripotency functions in ESCs, were found to be the most tightly regulated by Oct4 at the 2-cell stage.
We show that the Oct4-regulated gene set at the 1- to 2-cell stages of early embryo development is large and distinct from its established network in ESCs. Further, our experimental approach can be applied to dissect the gene regulatory network of Oct4 and other pluripotency regulators to deconstruct the dynamic developmental program in the early embryo.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background & Aims: Macrophage migration inhibitory factor (MIF) has been shown to play a pivotal role in inflammatory and immune-mediated diseases. This study investigates the role of MIF in gastric ...inflammation. Methods: Expression of MIF was examined in a rat gastric ulcer model induced by acetic acid, and the functional role of MIF in acute gastric ulcer was investigated by administration of a neutralizing anti-MIF antibody. Results: MIF messenger RNA and protein were markedly up-regulated in acute gastric ulcer, which correlated with the accumulation of macrophages (P < 0.001) and neutrophils (P < 0.05) at the site of inflammation. Macrophages, like neutrophils, were the major inflammatory cells infiltrating the ulcer base and they strongly expressed inducible nitric oxide synthase. However, macrophages, not neutrophils, were a rich source of MIF production in acute gastric ulcer. In vivo and in vitro blockade of MIF with the neutralizing anti-MIF antibody significantly inhibited the marked up-regulation of MIF, tumor necrosis factor α, inducible nitric oxide synthase, and intercellular adhesion molecule-1. This was associated with the marked inhibition of macrophage (70% reduced) and neutrophil (60% reduced) accumulation and activation, thus reducing ulcer sizes and attenuating ulceration. Conclusions: This study has shown that MIF was markedly up-regulated during acute gastric ulcer. Inhibition of acute gastric ulcer by blockade of MIF indicates that MIF is a key inflammatory mediator and plays a pathogenic role in gastric inflammation.
GASTROENTEROLOGY 2001;121:619-630
Throughout spermatogenesis, germ cells move progressively from the basal to the adluminal compartment, which is accompanied by continual disassembly and reassembly of intercellular junctions ...suggesting germ cell movement is composed of intermittent phases of junction disassembly and reassembly. A study was performed to correlate the expression of junctional‐complex components (such as zonula occludens‐1 ZO‐1, a tight‐junction component protein) and nonjunctional complex components (such as urokinase‐type plasminogen activator uPA, a serine protease; cathepsin L, a cysteine protease; α2‐macroglobulin, a nonspecific protease inhibitor; and cystatin C, a cysteine protease inhibitor) at the time when inter‐Sertoli tight junctions were established in vitro. This is an attempt to investigate whether the expression of nonjunctional component genes also correlates with the formation of inter‐Sertoli tight junctions in vitro. This is part of an effort to understand the physiologic elements of germ cell movement in the epithelium. Sertoli cells cultured in vitro are known to undergo programmed cell death. To ensure that the changes in target gene expression were not the result of apoptosis, Sertoli cells were cultured in vitro at densities of 0.25, 0.75, and 3 × 106 cells/cm2 for up to 7 days on bicameral culture units coated with Matrigel (Collaborative Research) and were assessed by morphologic analysis and agarose gel electrophoresis. It was noted that many of the Sertoli cells cultured at 3 × 106 cells/cm2 underwent apoptosis by day 7, in contrast to cultures at 0.25 and 0.75 × 106 cells/cm2 illustrating the Sertoli cell number per unit of area may be an important parameter to be considered when studying Sertoli cell function in vitro. Also, it was shown that the expression of ZO‐1 increased significantly between days 2 and 3 prior to the establishment of inter‐Sertoli tight junctions assessed by transepithelial resistance measurement (TER), which illustrates that ZO‐1 can be used as a marker to monitor this cellular event. More interestingly, there was also a transient increase in the expression of uPA and cathepsin L between days 2 and 3 at the time preceding the formation of tight junctions. In Sertoli cells cultured at low density (2 × 104 cells/cm2), when a confluent monolayer of cells could not form, there were no changes in the expression of either ZO‐1, uPA, or cathepsin L throughout the 7‐day culture period. These results show that the establishment of specialized junctions, such as tight junctions between Sertoli cells in vitro, may require the participation of both junctional and nonjunctional complex components.
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BFBNIB, FZAB, GIS, IJS, KILJ, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Mer belongs to the Tyro 3 family of receptor tyrosine kinases (RTKs). Together with Axl and Rse, the three RTKs are believed to play important functional roles in the male gonads because gene ...knockout male mice lacking all of these receptors are infertile. In the present study, postnatal expression of Axl and Rse in mouse testes decreased during maturation while expression of Mer increased age‐dependently during testicular development. To investigate the transcriptional regulation of gene expression in the testis, a ≈ 1.5 kb fragment of the 5′ flanking sequence of Mer was isolated. The sequence lacks a typical TATA or CAAT box. 5′ RACE revealed that the putative major transcriptional start site of Mer is located at +102 bp upstream of the translation initiation site. Using transient transfections of luciferase reporter constructs driven by various lengths of the 5′ flanking sequence, the gene segment −321/+126 showed the highest transcriptional activity in a mouse Sertoli cell line (TM4). DNAase I footprinting experiments revealed four footprints within the region from −321 to −26, including three binding sites for the transcriptional factor Specificity protein 1 (Sp1) and one for an unknown transcriptional factor. Electrophoretic mobility shift assay (EMSA), supershift assay, mutation studies and cotransfection demonstrated that those Sp1 cis‐acting motifs interacted either with Sp1 or Sp1/Sp3, depending on location and the nearby nucleotide sequences. An E2F binding site which down‐regulates Mer transcription, as revealed by EMSA, deletion and mutation studies, was identified downstream in the proximity of the promoter. Taking all of these data together, the study has demonstrated that Sp1, Sp3, E2F and probably another unknown transcriptional factor play a critical role in regulating the proximal promoter activities of Mer.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
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