Ultrastructural and Immunocytochemical Characterization of Autophagic Vacuoles in Isolated Hepatocytes: Effects of Vinblastine and Asparagine on Vacuole Distributions

The interactions between the autophagic and the endocytic degradation pathways were investigated by means of immunogold labeling of autophagic vacuoles (AVs) in ultrathin frozen sections from isolated rat hepatocytes. AVs were identified by their autophagocytosed contents of the degradation-resistant cytosolic enzyme CuZn-superoxide dismutase (SOD). Another cytosolic enzyme, carbonic anhydrase (CAIII), was rapidly degraded in the lysosomes, making the vacuolar CAIII/SOD ratio useful as a rough indicator of the progress of autophagic-lysosomal degradation. Lysosomes could be recognized by the presence of the lysosomal membrane glycoprotein lgp120, which was absent from hepatocytic endosomes. Endocytic inputs into the AVs were detected by the presence of gold-conjugated bovine serum albumin (BSA-gold), taken up by fluid-phase endocytosis. All vacuoles recognized morphologically as AVs were SOD-positive, as were essentially all of the lysosomes (96%). The majority (72%) of the lysosomes also labeled positively for BSA within 2 h of endocytosis. The data are thus compatible with the notion that all lysosomes can engage in both autophagic and endocytic degradation. Lgp120 appeared to distinguish well between lysosomes and nonlysosomal AVs: the lgp120-negative AVs (nonlysosomes) had a CAIII/SOD ratio identical to that of the cytosol, indicating that no degradation had occurred, In the lgp120-positive AVs (lysosomes), the ratio was only 43% of the cytosolic value, consistent with substantial CAIII degradation. Among the nonlysosomal AVs (about one-third of all AVs), one-half were BSA-positive, suggesting that early AVs (autophagasomes) and intermediary AVs (amphisomes) that had fused with endosomes were equally abundant. These morphological data thus support previous biochemical evidence for a prelysosomal meeting of the autophagic and endocytic pathways. The microtubule inhibitor vinblastine inhibited the autophagic influx to the lysosomes, causing an accumulation of autophagosomes and a reduction in average lysosomal size. Vinblastine also inhibited the endocytic flux, thereby precluding the formation of amphisomes and of BSA-positive lysosomes. High concentrations (20 mM) of asparagine induced swelling of amphisomes and of BSA-positive lysosomes, probably reflecting an acidotropic effect of ammonia generated by asparagine deamination. Asparagine also caused an accumulation of autophagosomes, amphisomes, and BSA-negative lysosomes, presumably as a result of impaired fusion with the swollen BSA-positive lysosomes. The two agents thus appear to perturb the autophagic-endocytic-lysosomal vacuole dynamics by different mechanisms, making them useful in the further study of these complex organelle interactions.     

SGT2 and MDY2 interact with molecular chaperone YDJ1 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Sgt2 was thought to be the homologue of vertebrate SGT (small glutamine tetratricopeptide repeat-containing protein). SGT has been known to interact with both Hsp70 and Hsp90. However, it was not clear whether Sgt2 might have a similar capacity. Here, we showed that Ssa1/Ssa2 (yeast heat shock cognate [Hsc]70), Hsc82 (yeast Hsp90), and Hsp104 coprecipitated with Sgt2 from yeast lysates. Another molecular chaperone, Ydj1, known to interact with Ssal and Hsc82, also coprecipitated with Sgt2. Synthetic lethality between SGT2 and YDJ1 was observed after the cells were under stress, although Sgt2 might not interact physically with Ydj1. We also found that Mdy2 interacted with the N-terminal region of Sgt2 and that Mdy2 appeared to interact physically with Ydj1. Mdy2 therefore may mediate the association of Ydj1 and Sgt2. In addition, the mating efficiency of mdy2delta, sgt2delta, and mdy2deltasgt2delta strains was reduced to a similar extent. Compared with mdy2delta and ydj1delta cells, ydj1deltamdy2delta cells, however, showed a further suppression in mating efficiency. Moreover, MDY2 interacted genetically with YDJ1. These results suggest that protein complexes containing Sgt2 and Mdy2 bring molecular chaperones together to carry out certain chaperoning functions.     

Chaperone-like protein p32 regulates ULK1 stability and autophagy

Mitophagy mediates clearance of dysfunctional mitochondria, and represents one type of mitochondrial quality control, which is essential for optimal mitochondrial bioenergetics. p32, a chaperone-like protein, is crucial for maintaining mitochondrial membrane potential and oxidative phosphorylation. However, the relationship between p32 and mitochondrial homeostasis has not been addressed. Here, we identified p32 as a key regulator of ULK1 stability by forming complex with ULK1. p32 depletion potentiated K48-linked but impaired K63-linked polyubiquitination of ULK1, leading to proteasome-mediated degradation of ULK1. As a result, silencing p32 profoundly impaired starvation-induced autophagic flux and the clearance of damaged mitochondria caused by mitochondrial uncoupler. Importantly, restoring ULK1 expression in p32-depleted cells rescued autophagy and mitophagy defects. Our findings highlight a cytoprotective role of p32 under starvation conditions by regulating ULK1 stability, and uncover a crucial role of the p32–ULK1-autophagy axis in coordinating stress response, cell survival and mitochondrial homeostasis.Mitophagy is a selective form of autophagy by which mitochondria are degraded in autolysosomes. p32 is a critical regulator of mitochondrial bioenergetics.¹ It primarily localizes to the mitochondrial matrix, but has also been reported to be present in other subcellular locations.^{2, 3, 4, 5} Many human tumors exhibit higher p32 expression levels than their nonmalignant counterpart tissues.^{6, 7, 8, 9} Depleting p32 in human cancer cells strongly shifts their metabolism from oxidative phosphorylation to glycolysis.¹ Consistently, p32 knockout causes mid-gestation lethality of knockout embryos and defects in oxidative phosphorylation. Mouse embryonic fibroblasts (MEFs) generated from p32 knockout embryos exhibited impaired ATP production and reduced mitochondrial membrane potential, which is in agreement with the observation that p32 silencing leads to increased mitochondrial fragmentation.^{10, 11} Notably, p32 was found to form protein complex with a variety of molecules^{7, 12, 13} and has been suggested that it may act as a multifunctional chaperone protein.^{12, 13, 14}ULK1 has a crucial role in mitophagy induction.¹⁵ Despite the pivotal role of ULK1 in mitochondrial clearance, little is known as how ULK1 itself is regulated. ULK1 is a relatively stable protein and is subject to proteasome-mediated degradation. Post-translational modifications including K63-linked ubiquitylation^{16, 17} and phosphorylation^{18, 19, 20} have been reported to modulate the rates of ULK1 turnover and kinase activity in different cellular contexts. Hsp90 and Cdc37 have been shown to regulate ULK1 stability and activity by forming complex with ULK1, which subsequently influences Atg13-mediated mitophagy.²¹ Here, we found p32 regulates ULK1 stability by forming protein complex with ULK1. The interaction between ULK1 and p32 is crucial for maintaining the steady-state levels and activity of ULK1. We further show that p32 ablation results in a defect in autophagy in EBSS-starved cells, and impairs clearance of dysfunctional mitochondria in cells exposed to mitochondrial uncoupler. Importantly, these autophagy and mitophagy defects can be restored by re-introducing ULK1 into p32-deficient cells, demonstrating ULK1 functions as a crucial downstream effector of p32.     

A forward loop between glioma and microglia: glioma-derived extracellular matrix-activated microglia secrete IL-18 to enhance the migration of glioma cells

The mediators and cellular effectors of inflammation are important constituents of the local environment of tumors. In some occasions, oncogenic changes induce an inflammatory microenvironment that promotes the progression of tumors. In gliomas, the presence of microglia may represent tumor-related inflammation and microglia activation, and subsequent inflammatory responses may influence tumor growth and metastasis. Here, we found that C6 glioma--but not primary astrocyte-derived extracellular matrix (ECM) could activate microglia, including primary microglia and BV-2 cell line, and activated microglia-secreted interleukin (IL)-18, a potent inflammatory cytokine of the IL-1 family, to promote C6 migration. In addition, by coating purified ECM components, it was found that secretion of IL-18 by activated microglia was enhanced when microglia encountered with fibronectin and vitronectin. Furthermore, IL-18-induced C6 migration and microfilament disassembly were antagonized by iNOS inhibitor, guanylate cyclase inhibitor, and protein kinase G inhibitor. Taken together, these results indicate that IL-18 secreted by microglia, which was activated by C6 glioma-derived ECM, enhanced migration of C6 glioma through NO/cGMP pathway.     

Genome Sequence of Acinetobacter baumannii TYTH-1

Acinetobacter baumannii has emerged recently as a major cause of health care-associated infections due to the extent of its antimicrobial resistance and its propensity to cause large nosocomial outbreaks. Here we report the genome sequence of Acinetobacter baumannii TYTH-1 isolated in Taiwan during 2008.     

Discovery of potent antimicrobial peptide analogs of Ixosin-B

Antimicrobial peptides (AMPs) represent the first defense line against infection when organisms are infected by pathogens. These peptides are generally good targets for the development of antimicrobial agents. Peptide amide analogs of Ixosin-B, an antimicrobial peptide with amino acid sequence of QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY, were designed, synthesized and examined for antimicrobial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Within the peptides synthesized, we discovered an 11-mer peptide, KRLRRVWRRWR-amide, which exhibited potent antimicrobial activity while very little hemolytic activity in human erythrocytes was observed even at high dose level (100 μM). With further modifications, this peptide could be developed into a potent antimicrobial agent in the future.     

Loss of thioredoxin function in retinas of mice overexpressing amyloid β

Amyloid β peptides (Aβ) have been implicated in the pathogenesis of age-related macular degeneration (ARMD) and glaucoma. In this study, retinas of mice overexpressing Aβ (Tg) were compared to those of wild-type mice (Wt) and analyzed for oxidative stress parameters. We observed a progressive decrease in all retinal cell layers, which was significantly greater in Tg mice at 14 months and culminated in loss of the outer retina at 18 months of age. We also observed higher levels of reactive oxygen species, glial fibrillary acidic protein, and hydroperoxide in Tg versus Wt mice (14 months). These effects were associated with phosphorylation/activation of the apoptosis signal kinase 1 and the p38 mitogen-activated kinase. Western blotting analysis revealed progressive increases in the levels of thioredoxin 1 and thioredoxin inhibitory protein in Tg compared to Wt mice. No changes were observed in the levels of thioredoxin reductase 1 (TrxR1); however, measurements of TrxR1 activity showed a 42.7±8% reduction in Tg mice versus Wt at 14 months of age. Our data suggest that Aβ-mediated retinal neurotoxicity involves impairment of the thioredoxin system and enhanced oxidative stress, potentially implicating this mechanism in the pathogenesis of ARMD and glaucoma.     

Effects of rosiglitazone on global ischemia-induced hippocampal injury and expression of mitochondrial uncoupling protein 2

We investigate the effect of rosiglitazone, a ligand for peroxisome proliferator-activated receptor-gamma (PPARgamma) with anti-inflammatory and anti-oxidative actions, on hippocampal injury and its roles in mitochondrial uncoupling protein 2 (UCP2) expression caused by transient global ischemia (TGI) in rats. Increased UCP2 expression was observed in mitochondria of hippocampal CA1 2-24h after TGI/reperfusion, with maximal expression levels at 6-18h. Administration of rosiglitazone to hippocampus 30min prior to the onset of TGI further enhanced mitochondrial UCP2 expression 2-6h following TGI/reperfusion. Rats subjected to TGI/reperfusion displayed a significant increase in lipid peroxidation, based on increased malondialdehyde (MDA) levels, in hippocampal CA1 mitochondria 2-6 h after reperfusion. Rosiglitazone significantly attenuated TGI/reperfusion-induced lipid peroxidation and suppressed hippocampal CA1 neuronal death based on the surviving neuronal counts. In conclusion, our results provide correlative evidence for the "PPARgamma-->UCP2-->neuroprotection" cascade in ischemic brain injury.     

In vitro culture period but not the passage number influences the capacity of chimera production of inner cell mass and its deriving cells from porcine embryos

Mammalian embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass (ICM) of the blastocyst. These cells are able to proliferate continuously without differentiation in vitro under suitable conditions. Their capacity of pluripotency in differentiation will be resumed when they are reintroduced into host embryos, when they will contribute to the embryonic development to form chimeric individuals. Manipulation of ES cells has been mainly established from studies in the mouse, and is powerful in the production of transgenic animals. Porcine ICM-derived cell lines possess the same cellular morphology and in vitro behavior as those of murine ES cells, but have lower efficiency in chimera formation when reintroduced into host embryos. This study was to determine the influences of passage number and the duration of in vitro culture on the capacity of porcine ICM-derived cells in the generation of chimeric embryos. The results showed that when passage number of porcine ICM-derived cells was less than 15, there were no detrimental effects on its integration ability. Extending the culture time up to 6 days in each passage of porcine ICM-derived cells impaired its integration capacity into the host blastocyst. Porcine ICM-derived cells cultured for more than 4 days in each passage should not be used for blastocyst injection if high efficiency of chimera production is to be achieved.     

Characterization of CalS9 in the biosynthesis of UDP-xylose and the production of xylosyl-attached hybrid compound

The gene cluster of calicheamicin contains calS9, which encodes UDP-GlcA decarboxylase that converts UDP-GlcA to UDP-xylose. calS9 was cloned in pET32a(+) and expressed in Escherichia coli BL21 (DE3) to characterize its putative function. The reaction product was analyzed by high-performance liquid chromatography (HPLC) and electrospray ionization-mass spectrometry. The deoxysugar biosynthesis of Streptomyces sp. KCTC 0041BP was inactivated by gene replacement to generate Streptomyces sp. GerSM2 mutant, which was unable to produce dihydrochalcomycin. calS7, calS8, and calS9 UDP-xylose biosynthetic genes were cloned in an integrative plasmid pSET152 to generate pBPDS, which was heterologously expressed in Streptomyces sp. GerSM2. Finally, novel glycosylated product, 5-O-xylosyl-chalconolide derivative, in the conjugal transformants was isolated and analyzed by HPLC and liquid chromatography–mass spectrometry.