Die Hieracien der Umgebung von Seckau in Ober-Steiermark

Ohne Zusammenfassung     

Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.     

Investigation of coenzyme Q biosynthesis in human fibroblast and HepG2 cells

Coenzyme Q (CoQ) deficiency occurs in genetic disorders, during aging and various diseases. Diagnosis requires skin fibroblasts in tissue culture. [³H]Mevalonate incorporation was appropriate to measure the rate of CoQ synthesis in fibroblasts and hepatoblastoma cells. [¹⁴C]p-Hydroxybenzoate had limited permeability, but it could be increased with Fugene and cyclodextrin. Inhibition of decaprenyl-4-hydroxybenzoate transferase results in the accumulation of decaprenyl diphosphate, an indicator of enzyme deficiency. Also, analysis of the corresponding mRNAs in this case is useful. In vitro assays to measure trans-prenyltransferase and decaprenyl-4-hydroxybenzoate transferase activities are not available. Neither measurement of methyltransferases is reliable in human cells. In vitro reconstruction of CoQ synthesis, in opposite to cholesterol synthesis, proved to be unsuccessful. Thus, the biochemical characterization of the CoQ biosynthetic system in human cells is restricted to a few reliable analytical procedures.     

Functional HLA-DR T cell epitopes of CEA identified in patients with colorectal carcinoma immunized with the recombinant protein CEA

A baculovirus-produced recombinant CEA (rCEA) protein comprising the extracellular region was used for vaccination of CRC patients with or without GM-CSF as an adjuvant cytokine. Ten patients with a significant proliferative T cell response against rCEA were selected for T cell epitope mapping. Fifteen-aa-long overlapping peptides covering the entire aa sequence of the external domain of CEA were used in a proliferation assay. In six of the patients a repeatable T cell response against at least one peptide was demonstrated. For the first time, nine functional HLA-DR epitopes of CEA were defined. Two of the peptides were recognized by more than one patient, i.e., two and three patients, respectively. Those 15-mer peptides that induced a proliferative T cell response fitted to the actual HLA-DR type (SYFPEITHI). The affinity of the native peptides for the T cell receptor was in the low to intermediate range (scores 6–19). The 15-mer peptides also contained 9-mer peptide sequences that could be predicted to bind to the actual HLA-ABC genotypes (SYFPEITHI/BIMAS). Blocking experiments using monoclonal antibodies indicated that the proliferative T cell response was both MHC class I and II restricted. The defined HLA-DR T cell epitopes were spread over the entire CEA molecule, but a higher frequency was noted towards the C-terminal. Peptides with a dual specificity may form a basis for production of subunit cancer vaccines, but modifications should be done to increase the T cell affinity, thereby optimizing the antitumoral effects of the vaccine.Abbreviations aa amino acid - CRC colorectal carcinoma - GM-CSF granulocyte/monocyte colony stimulating factor - CEA carcino-embryonic antigen - BCP baculovirus control protein - MHC major histocompatibility complex - pp peptide - TAA tumor associated antigen     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

Flow cytometric analysis of mitochondria from CA1 and CA3 regions of rat hippocampus reveals differences in permeability transition pore activation

Mitochondria are important in the pathophysiology of several neurodegenerative diseases, and mitochondrial production of reactive oxygen species (ROS), membrane depolarization, permeability changes and release of apoptogenic proteins are involved in these processes. Following brain insults, cell death often occurs in discrete regions of the brain, such as the subregions of the hippocampus. To analyse mitochondrial structure and function in such subregions, only small amounts of mitochondria are available. We developed a protocol for flow cytometric analysis of very small samples of isolated brain mitochondria, and analysed mitochondrial swelling and formation of ROS in mitochondria from the CA1 and CA3 regions of the hippocampus. Calcium-induced mitochondrial swelling was measured, and fluorescent probes were used to selectively stain mitochondria (nonyl acridine orange), to measure membrane potential (tetramethylrhodamine-methyl-ester, 1,1',3,3,3',3'-hexamethylindodicarbocyanine-iodide) and to measure production of ROS (2',7'-dichlorodihydrofluorescein-diacetate). We found that formation of ROS and mitochondrial permeability transition pore activation were higher in mitochondria from the CA1 than from the CA3 region, and propose that differences in mitochondrial properties partly underlie the selective vulnerability of the CA1 region to brain insults. We also conclude that flow cytometry is a useful tool to analyse the role of mitochondria in cell death processes.     

Diversification in the tropical pacific: comparisons between marine and terrestrial systems and the importance of founder speciation

Patterns of distribution and processes of differentiation haveoften been contrasted between terrestrial and marine biotas.The islands of Oceania offer an excellent setting to explorethis contrast, because the geographic setting for terrestrialand shallow-water, benthic, marine organisms are the same: themyriad islands strewn across the vast Pacific. The size of speciesranges and the geographic distribution of endemism are two biogeographicattributes that are thought to differ markedly between terrestrialand marine biotas in the Pacific. While terrestrial speciesare frequently confined to single islands or archipelagoes throughoutOceania, marine species tend to have wide to very wide distributions,and are rarely restricted to single island groups except forthe most isolated archipelagoes. We explore the conditions underwhich species can reach an island by dispersal and differentiate.Genetic differentiation can occur either through founder speciationor vicariance; these processes are requisite ends of a continuum.We show that founder speciation is most likely when few propagulesenter the dispersal medium and survive well while they travelfar. We argue that conditions favorable to founder speciationare common in marine as well as terrestrial systems, and thatterrestrial-type, archipelagic-level endemism is likely commonin marine taxa. We give examples of marine groups that showarchipelagic level endemism on most Pacific island groups aswell as of terrestrial species that are widespread. Thus boththe patterns and processes of insular diversification are variable,and overlap more between land and sea than previously considered.     

Gates,GAVI, the glorious global funds and more: all you ever wanted to know

Global immunization programmes have achieved some remarkable successes. In 1977, Frank Fenner's Commission declared smallpox to have been eradicated by an 11-year-long intensive campaign. The Expanded Programme on Immunization encompassed six important childhood vaccines and reached over three-quarters of the world's children. Polio eradication has gone remarkably well, with only 10 out of 200 countries reporting residual cases. But amidst all the good news, there is also bad news. Coverage is variable; infrastructure is crumbling; and newer vaccines are not being incorporated in many country programmes. The Bill and Melinda Gates Foundation has introduced a new dynamic here. From their initial gift of $100 million in December 1998, their commitment to date is US$1.5 billion - and rising. At the centre is a Global Children's Vaccine Fund which permitted the launch, in January 2000, of the Global Alliance for Vaccines and Immunization. This is targeted to the 74 poorest countries of the world and is designed to improve vaccination infrastructure, to purchase newer vaccines and to support research and development. Even before we know how successful this programme will be, it has had its imitators. The Global Fund to Fight AIDS, TB and Malaria borrowed many concepts from GAVI. The Global Alliance for Improved Nutrition announced in May 2002 does so as well, and is heavily supported by Gates. Highly effective parasite control programmes antedate all this but will be much strengthened. However, we still face a sizeable budgetary gap both for research and for bringing the best advances to all people who need them.     

Uncoupling protein-2 prevents neuronal death and diminishes brain dysfunction after stroke and brain trauma

Whereas uncoupling protein 1 (UCP-1) is clearly involved in thermogenesis, the role of UCP-2 is less clear. Using hybridization, cloning techniques and cDNA array analysis to identify inducible neuroprotective genes, we found that neuronal survival correlates with increased expression of Ucp2. In mice overexpressing human UCP-2, brain damage was diminished after experimental stroke and traumatic brain injury, and neurological recovery was enhanced. In cultured cortical neurons, UCP-2 reduced cell death and inhibited caspase-3 activation induced by oxygen and glucose deprivation. Mild mitochondrial uncoupling by 2,4-dinitrophenol (DNP) reduced neuronal death, and UCP-2 activity was enhanced by palmitic acid in isolated mitochondria. Also in isolated mitochondria, UCP-2 shifted the release of reactive oxygen species from the mitochondrial matrix to the extramitochondrial space. We propose that UCP-2 is an inducible protein that is neuroprotective by activating cellular redox signaling or by inducing mild mitochondrial uncoupling that prevents the release of apoptogenic proteins.