The type II phosphatidylinositol 4‐kinases (PI4KIIs) produce the lipid phosphatidylinositol 4‐phosphate (PtdIns4P) and participate in a confusing variety of membrane trafficking and signaling roles. ...This review argues that both historical and contemporary evidence supports the function of the PI4KIIs in numerous trafficking pathways, and that the key to understanding the enzymatic regulation is through membrane interaction and the intrinsic membrane environment. By summarizing new research and examining the trafficking roles of the PI4KIIs in the context of recently solved molecular structures, I highlight how mechanisms of PI4KII function and regulation are providing insights into the development of cancer and in neurological disease. I present an integrated view connecting the cell biology, molecular regulation, and roles in whole animal systems of these increasingly important proteins.
The type II phosphatidylinositol 4‐kinases (PI4KIIs) are versatile mediators of membrane trafficking, controlling pathways that impact on disease. Recent advances suggest that the membrane lipid environment regulates enzyme activity and that the complexity of trafficking routes can be explained by the ability of these kinases to promote membrane trafficking in different directions.
Aging is a major risk factor for progression of liver diseases to hepatocellular carcinoma (HCC). Cellular senescence contributes to age-related tissue dysfunction, but the epigenetic basis ...underlying drug-induced senescence remains unclear. macroH2A1, a variant of histone H2A, is a marker of senescence-associated heterochromatic foci that synergizes with DNA methylation to silence tumor-suppressor genes in human fibroblasts. In this study, we investigated the relationship between macroH2A1 splice variants, macroH2A1.1 and macroH2A1.2, and liver carcinogenesis. We found that protein levels of both macroH2A1 isoforms were increased in the livers of very elderly rodents and humans, and were robust immunohistochemical markers of human cirrhosis and HCC. In response to the chemotherapeutic and DNA-demethylating agent 5-aza-deoxycytidine (5-aza-dC), transgenic expression of macroH2A1 isoforms in HCC cell lines prevented the emergence of a senescent-like phenotype and induced synergistic global DNA hypomethylation. Conversely, macroH2A1 depletion amplified the antiproliferative effects of 5-aza-dC in HCC cells, but failed to enhance senescence. Senescence-associated secretory phenotype and whole-transcriptome analyses implicated the p38 MAPK/IL8 pathway in mediating macroH2A1-dependent escape of HCC cells from chemotherapy-induced senescence. Furthermore, chromatin immunoprecipitation sequencing revealed that this hepatic antisenescence state also required active transcription that could not be attributed to genomic occupancy of these histones. Collectively, our findings reveal a new mechanism by which drug-induced senescence is epigenetically regulated by macroH2A1 and DNA methylation and suggest macroH2A1 as a novel biomarker of hepatic senescence that could potentially predict prognosis and disease progression.
•Intravenous contrast (IVC) enhancement is used extensively in routine practice in Irish Radiation Therapy departments.•There are some discrepancies in IVC practice with international ...recommendations.•Training for cannulation and IVC administration procedures should provide RTTs with the skills to implement image enhancement techniques and maximise benefits of IVC where required.
While Computerised Tomography (CT) remains the gold standard in radiation therapy (RT) planning, inferior soft tissue definition remains a challenge. Intravenous contrast (IVC) use during CT planning can enhance soft tissue contrast optimising Target Volume (TV) and Organ at Risk visualisation and delineation. Despite this known benefit, there are no guidelines for when and how to use IVC in RT planning scans in Ireland.
The study aims to examine the patterns of practice in relation to the use of IVC in RT planning scans in Ireland and to determine the level of compliance with international guidelines. Radiation Therapists (RTT) IVC training will also be investigated.
An anonymised online survey was designed based on previously-reported literature. This was distributed to all RT departments in Ireland. The survey contained open, closed and Likert scale questions that investigated IVC practices in each department.
75% (n = 9/12) of Irish departments responded. All responding departments reported using IVC. RTTs cannulated patients in 67% (n = 6/9) of the departments and administration contrast in all departments. Variations from recommended guidelines were found in disease sites where IVC was routinely used and in the assessment of renal functioning prior to contrast administration. IVC training varied in duration and number of supervised procedures required to fulfill competencies.
IVC is used extensively in Irish RT departments. There are variations in IVC practice between departments and with international recommended guidelines.
Pathogenetic mechanisms of amyloid A amyloidosis Simons, J. Paul; Al-Shawib, Raya; Ellmerich, Stephan ...
Proceedings of the National Academy of Sciences - PNAS,
10/2013, Letnik:
110, Številka:
40
Journal Article
Recenzirano
Odprti dostop
Systemic amyloid A (AA) amyloidosis is a serious complication of chronic inflammation. Serum AA protein (SAA), an acute phase plasma protein, is deposited extracellularly as insoluble amyloid fibrils ...that damage tissue structure and function. Clinical AA amyloidosis is typically preceded by many years of active inflammation before presenting, most commonly with renal involvement. Using dose-dependent, doxycycline-inducible transgenic expression of SAA in mice, we show that AA amyloid deposition can occur independently of inflammation and that the time before amyloid deposition is determined by the circulating SAA concentration. High level SAA expression induced amyloidosis in all mice after a short, slightly variable delay. SAA was rapidly incorporated into amyloid, acutely reducing circulating SAA concentrations by up to 90%. Prolonged modest SAA overexpression occasionally produced amyloidosis after long delays and primed most mice for explosive amyloidosis when SAA production subsequently increased. Endogenous priming and bulk amyloid deposition are thus separable events, each sensitive to plasma SAA concentration. Amyloid deposits slowly regressed with restoration of normal SAA production after doxycycline withdrawal. Reinduction of SAA overproduction revealed that, following amyloid regression, all mice were primed, especially for rapid glomerular amyloid deposition leading to renal failure, closely resembling the rapid onset of renal failure in clinical AA amyloidosis following acute exacerbation of inflammation. Clinical AA amyloidosis rarely involves the heart, but amyloidotic SAA transgenic mice consistently had minor cardiac amyloid deposits, enabling us to extend to the heart the demonstrable efficacy of our unique antibody therapy for elimination of visceral amyloid.
Background & Aims
Chronic liver disease is a predisposing factor for development of hepatocellular carcinoma (HCC). Toll‐like receptors play a crucial role in immunity against microbial pathogens and ...recent evidence suggests that they may also be important in pathogenesis of chronic liver disease. The purpose of this study was to determine whether TLR7 and TLR9 are potential targets for prevention and progression of HCC.
Methods
Tissue microarrays containing liver samples from patients with cirrhosis, viral hepatitis and HCC were examined for expression of TLR7 and TLR9 and the data obtained was validated in liver specimens from the hospital archives. Proliferation of human HCC cell lines was studied following stimulation of TLR7 and TLR9 using agonists (imiquimod and CpG‐ODN respectively) and inhibition with a specific antagonist (IRS‐954) or chloroquine. The effect of these interventions was confirmed in a xenograft model and diethylnitrosamine (DEN)/nitrosomorpholine (NMOR)‐induced model of HCC.
Results
TLR7 and TLR9 expression was up‐regulated in human HCC tissue. Proliferation of HuH7 cells in vitro increased significantly in response to stimulation of TLR7. TLR7 and TLR9 inhibition using IRS‐954 or chloroquine significantly reduced HuH7 cell proliferation in vitro and inhibited tumour growth in the mouse xenograft model. HCC development in the DEN/NMOR rat model was also significantly inhibited by chloroquine (P < 0.001).
Conclusion
The data suggest that inhibiting TLR7 and TLR9 with IRS‐954 or chloroquine could potentially be used as a novel therapeutic approach for preventing HCC development and/or progression in susceptible patients.
The type II alpha isoform of phosphatidylinositol 4-kinase has recently been shown to function in the recruitment of adaptor protein-1 complexes to the trans-Golgi network. Here we show that ...phosphatidylinositol 4-kinase IIalpha is also a component of highly dynamic membranes of the endosomal system where it colocalises with protein markers of the late endosome and with endocytosed epidermal growth factor. When phosphatidylinositol 4-kinase IIalpha activity was inhibited in vivo using the monoclonal antibody 4C5G or by depression of endogenous phosphatidylinositol 4-kinase IIalpha protein levels using RNA interference, ligand-bound epidermal growth factor receptor failed to traffic to late endosomes and instead accumulated in vesicles in a sub-plasma membrane compartment. Furthermore, lysosomal degradation of activated epidermal growth factor receptor was dramatically impaired in small inhibitory RNA-treated cells. We demonstrate that phosphatidylinositol 4-kinase IIalpha is necessary for the correct endocytic traffic and downregulation of activated epidermal growth factor receptor.
Phosphoinositide (PI) lipids are intracellular membrane signaling intermediates and effectors produced by localized PI kinase and phosphatase activities. Although many signaling roles of PI kinases ...have been identified in cultured cell lines, transgenic animal studies have produced unexpected insight into the in vivo functions of specific PI 3- and 5-kinases, but no mammalian PI 4-kinase (PI4K) knockout has previously been reported. Prior studies using cultured cells implicated the PI4K2α isozyme in diverse functions, including receptor signaling, ion channel regulation, endosomal trafficking, and regulated secretion. We now show that despite these important functions, mice lacking PI4K2α kinase activity initially appear normal. However, adult Pi4k2aGT/GT animals develop a progressive neurological disease characterized by tremor, limb weakness, urinary incontinence, and premature mortality. Histological analysis of aged Pi4k2aGT/GT animals revealed lipofuscin-like deposition and gliosis in the cerebellum, and loss of Purkinje cells. Peripheral nerves are essentially normal, but massive axonal degeneration was found in the spinal cord in both ascending and descending tracts. These results reveal a previously undescribed role for aberrant PI signaling in neurological disease that resembles autosomal recessive hereditary spastic paraplegia.
Cholesterol is an abundant lipid of the trans-Golgi network (TGN) and of certain endosomal membranes where cholesterol-rich microdomains are important in the organization and compartmentalization of ...vesicular trafficking. Here we describe the development of a rapid method to isolate a cholesterol-rich endomembrane fraction. We show that widely used subcellular fractionation techniques incompletely separate cholesterol-rich membranes, such as the TGN, from organelles, such as late endosomes and lysosomes. To address this issue, we devised a new subcellular fractionation scheme involving two rounds of velocity centrifugation, membrane sonication, and discontinuous sucrose density gradient centrifugation. This strategy resulted in the isolation of a cholesterol and GM1 glycosphingolipid-enriched membrane fraction that was completely cleared of plasma membrane, endoplasmic reticulum, and mitochondria. This buoyant fraction was enriched for the TGN and recycling endosome proteins Rab11 and syntaxin-6, and it was well resolved from cis-Golgi and early and late endosomal membranes. We demonstrate that this technique can give useful insights into the compartmentation of phosphoinositide synthesis, and it facilitates the isolation of cholesterol-rich membranes from a population of TGN-trafficking vesicles.
Phosphatidylinositol (PI) is essential for numerous cell functions and is generated by consecutive reactions catalyzed by CDP-diacylglycerol synthase (CDS) and PI synthase. In this study, we ...investigated the membrane organization of CDP-diacylglycerol synthesis. Separation of mildly disrupted A431 cell membranes on sucrose density gradients revealed cofractionation of CDS and PI synthase activities with cholesterol-poor, endoplasmic reticulum (ER) membranes and partial overlap with plasma membrane caveolae. Cofractionation of CDS activity with caveolae was also observed when low-buoyant density caveolin-enriched membranes were prepared using a carbonate-based method. However, immunoisolation studies determined that CDS activity localized to ER membrane fragments containing calnexin and type III inositol (1,4,5)-trisphosphate receptors but not to caveolae. Membrane fragmentation in neutral pH buffer established that CDP-diacylglycerol and PI syntheses were restricted to a subfraction of the calnexin-positive ER. In contrast to lipid rafts enriched for caveolin, cholesterol, and GM1 glycosphingolipids, the CDS-containing ER membranes were detergent soluble. In cell imaging studies, CDS and calnexin colocalized in microdomain-sized patches of the ER and also unexpectedly at the plasma membrane. These results demonstrate that key components of the PI pathway localize to nonraft, phospholipid-synthesizing microdomains of the ER that are also enriched for calnexin.
Fibrinogen A alpha chain amyloidosis is an autosomal dominant disease associated with mutations in the fibrinogen A alpha chain (
) gene, and it is the most common cause of hereditary renal ...amyloidosis in the UK. Patients typically present with kidney impairment and progress to end-stage renal disease over a median time of 4.6 years.
Six patients presented with proteinuria, hypertension, and/or lower limb edema and underwent detailed clinical and laboratory investigations.
A novel
gene mutation was identified in each case: 2 frameshift mutations F521Sfs*27 and G519Efs*30 and 4 single base substitutions G555F, E526K, E524K, R554H. In 5 subjects, extensive amyloid deposits were found solely within the glomeruli, which stained specifically with antibodies to fibrinogen A alpha chain, and in one of these cases, we found coexistent fibrinogen A alpha chain amyloidosis and anti-glomerular basement membrane antibody disease. One patient was diagnosed with light-chain amyloidosis after a bone marrow examination revealed a small clonal plasma cell population, and laser microdissection of the amyloid deposits followed by liquid chromatography and tandem mass spectrometry identified kappa light chain as the fibril protein.
We report 6 novel mutations in the
gene: 5 were associated with renal fibrinogen A alpha chain amyloidosis and 1 was found to be incidental to light-chain amyloid deposits discovered in a patient with a plasma cell dyscrasia. Clinical awareness and suspicion of hereditary amyloidosis corroborated by genetic analysis and adequate typing using combined immunohistochemistry and laser microdissection and mass spectrometry is valuable to avoid misdiagnosis, especially when a family history of amyloidosis is absent.
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