Biochemical isolation and characterization of the tubulovesicular LC3-positive autophagosomal compartment.

Autophagosomes and their precursors are best defined by electron microscopy but may also be traced in living cells based on the distribution of specific autophagy molecules. LC3, the most commonly examined autophagy marker in mammalian cells, labels structures that are frequently manifested as dots or rings using light microscopy; however, the nature of these structures is not entirely clear. We reported here a novel approach to examine the LC3-positive compartment in cell-free lysates, which revealed that they were actually tubulovesicular structures with considerable heterogeneity. Using affinity purification, we isolated these membranes for electron microscopy, which indicated that they possessed ultrastructural features consistent with autophagosomal membranes at various maturation stages. Further biochemical and proteomics analyses demonstrated the presence of multiple autophagy-related and other functional molecules. The different distribution patterns of Atg5, Atg16, Atg9, and p62/SQSTM1 on the LC3-positive compartment provided new clues on how these molecules might be involved in the dynamics of the autophagosomal membranes. Finally, several morphologically unique groups of LC3-positive membranes were categorized. Their topological configurations suggested that double-membrane vesicles could be derived from single membrane compartments via different means, including tubule-to-vesicle conversion, whose presence was supported by live cell imaging. These findings thus provide new information on the dynamics of the autophagosomal compartment.

Bid-independent mitochondrial activation in tumor necrosis factor alpha-induced apoptosis and liver injury.

The death receptor apoptosis pathway is intimately connected with the mitochondrial apoptosis pathway. Bid is a BH3-only pro-death Bcl-2 family protein and is the major molecule linking the two pathways. Bid-mediated mitochondrial activation occurs early and is responsible for the prompt progress of tumor necrosis factor alpha (TNF-alpha)-induced apoptosis. However, in both cultured cells and animal models of TNF-alpha-induced injury, later-phase Bid-independent mitochondrial activation could be demonstrated. Consequently, bid-deficient mice are still susceptible to endotoxin-induced liver injury and mortality. Notably, embryonic hepatocyte apoptosis and lethality caused by TNF-alpha in the absence of p65relA cannot be rescued by the simultaneous deletion of bid. Further studies indicate that multiple mechanisms including reactive oxygen species, JNK, and permeability transition are critically involved in Bid-independent mitochondrial activation. Inhibition of these events suppresses TNF-alpha-induced mitochondrial activation and apoptosis in bid-deficient cells. These findings thus indicate that there are at least two sets of mechanisms of mitochondrial activation upon TNF-alpha stimulation. While the Bid-mediated mechanism is rapid and potent, the Bid-independent mechanism progresses gradually and involves multiple players. The critical involvement of Bid-independent mitochondrial activation in TNF-alpha-induced apoptosis demands the intervention of TNF-alpha-mediated tissue injury via multiple avenues.

Expression of X-linked inhibitor-of-apoptosis protein in hepatocellular carcinoma promotes metastasis and tumor recurrence.

UNLABELLED: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Despite significantly improved diagnosis and treatment in recent years, the long-term therapeutic effect is compromised by the frequent recurrence and metastasis, of which the molecular mechanisms are not fully understood. Our initial studies in established HCC cell lines with different metastatic capabilities indicated a correlation of metastasis with the resistance to apoptosis and therefore the ability to survive in stressed conditions. Subsequent investigation revealed that increased expression of X-linked inhibitor-of-apoptosis protein (XIAP) was correlated with the resistance to apoptosis and enhanced invasiveness in vitro, which could contribute to increased metastatic foci in vivo. Furthermore, we found that nearly 90% of clinical samples from advanced HCC patients expressed high levels of XIAP. Patients with XIAP-positive tumors had a significantly increased risk of relapse, which resulted from metastasis after total liver resection and orthotopic liver transplantation. Indeed, XIAP expression could be an independent prognostic factor for predicting disease-free survival rate and overall survival rate of these patients. XIAP expression was also highly correlated with advanced cases that exceeded the Milan criteria and could be a prognostic factor for disease-free survival in these patients as well. CONCLUSION: Our studies have shown an important molecule in controlling HCC metastasis, defined a biomarker that can be used to predict HCC recurrence and patient survival after treatment, and suggest that XIAP can be a molecular target subject to intervention to reduce metastasis and recurrence.

Intranasal vaccination with replication-defective adenovirus type 5 encoding influenza virus hemagglutinin elicits protective immunity to homologous challenge and partial protection to heterologous challenge in pigs.

Influenza A virus (IAV) is widely circulating in the swine population and causes significant economic losses. To combat IAV infection, the swine industry utilizes adjuvanted whole inactivated virus (WIV) vaccines, using a prime-boost strategy. These vaccines can provide sterilizing immunity toward homologous virus but often have limited efficacy against a heterologous infection. There is a need for vaccine platforms that induce mucosal and cell-mediated immunity that is cross-reactive to heterologous viruses and can be produced in a short time frame. Nonreplicating adenovirus 5 vector (Ad5) vaccines are one option, as they can be produced rapidly and given intranasally to induce local immunity. Thus, we compared the immunogenicity and efficacy of a single intranasal dose of an Ad5-vectored hemagglutinin (Ad5-HA) vaccine to those of a traditional intramuscular administration of WIV vaccine. Ad5-HA vaccination induced a mucosal IgA response toward homologous IAV and primed an antigen-specific gamma interferon (IFN-γ) response against both challenge viruses. The Ad5-HA vaccine provided protective immunity to homologous challenge and partial protection against heterologous challenge, unlike the WIV vaccine. Nasal shedding was significantly reduced and virus was cleared from the lung by day 5 postinfection following heterologous challenge of Ad5-HA-vaccinated pigs. However, the WIV-vaccinated pigs displayed vaccine-associated enhanced respiratory disease (VAERD) following heterologous challenge, characterized by enhanced macroscopic lung lesions. This study demonstrates that a single intranasal vaccination with an Ad5-HA construct can provide complete protection from homologous challenge and partial protection from heterologous challenge, as opposed to VAERD, which can occur with adjuvanted WIV vaccines.

Role of toll-like receptors in changes in gene expression and NF-kappa B activation in mouse hepatocytes stimulated with lipopolysaccharide.

The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-kappa B activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.