Spontaneous immunity against Bcl-xL in cancer patients

It is well-established that peptide epitopes derived from human tumor-associated Ags can be recognized by CTL in the context of the MHC molecule. However, the vast majority of Ags described are not vital for survival and growth of the tumor cells, and immunoselection of Ag-loss variants during immunotherapy has been demonstrated in several cases. Malfunctions in death pathways observed in human cancers are often due to overexpression of antiapoptotic proteins in the Bcl-2 protein family, i.e., Bcl-2, Mcl-1, and Bcl-xL. These antiapoptotic proteins are implicated in cancer development, tumor progression, and drug resistance. The general overexpression of the antiapoptotic members of the Bcl-2 family in cancer and the fact that down-regulation or loss of expression of these proteins as a means of immune escape would impair sustained tumor growth makes them very attractive targets for anticancer immunotherapy. Recently, we identified spontaneous T cell responses against Bcl-2- and Mcl-1-derived peptides in patients suffering from cancers of different origin. In this study, we demonstrate that Bcl-xL is a target for T cell recognition in cancer patients. Thus, we describe spontaneous HLA-A2-restricted cytotoxic T cell responses against peptide epitopes derived from Bcl-xL by means of ELISPOT and flow cytometry stainings, whereas no responses were detected against any of the Bcl-xL epitopes in any healthy controls. Moreover, Bcl-xL-specific T cells are cytotoxic against HLA-matched cancer cells of different origin. Thus, cellular immune responses against apoptosis inhibitors like the Bcl-2 family proteins appear to represent a general feature in cancer.     

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate–dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21^Cip1 accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.     

Matrix metalloproteinases (MMPs) safeguard osteoblasts from apoptosis during transdifferentiation into osteocytes: MT1-MMP maintains osteocyte viability

Osteoblasts undergo apoptosis or differentiate into either osteocytes or bone-lining cells after termination of bone matrix synthesis. In this study, we investigated the role of matrix metalloproteinases (MMPs) in differentiation of osteoblasts, bone formation, transdifferentiation into osteocytes, and osteocyte apoptosis. This was accomplished by using calvarial sections from the MT1-MMP-deficient mouse and by culture of the mouse osteoblast cell line MC3T3-E1 and primary mouse calvarial osteoblasts. We found that a synthetic matrix metalloprotease inhibitor, GM6001, strongly inhibited bone formation in vitro of both primary osteoblasts and MC3T3 cells by approximately 75%. To further investigate at which level of osteoblast differentiation MMP inhibition was attenuating osteoblast function, we found that neither preosteoblast nor mature osteoblast activity was affected. In contrast, cell survival of osteoblasts forced to transdifferentiate into osteocytes in 3D type I collagen gels were inhibited by more than 50% when exposed to 10 microM GM6001 and to Tissue Inhibitor of Metalloproteinase-2 (TIMP-2), a natural MT1-MMP inhibitor. This shows the importance of MMPs in safeguarding osteoblasts from apoptosis when transdifferentiating into osteocytes. By examination of osteoblasts and osteocytes embedded in calvarial bone in the MT1-MMP deficient mice, we found that MT1-MMP deficient mice had 10-fold higher levels of apoptotic osteocytes than wild-type controls. We have previously shown that MT1-MMP activates latent Transforming Growth Factorbeta (TGF-beta). These findings strongly suggest that MT1-MMP-activated TGF-beta maintains osteoblast survival during transdifferentiation into osteocytes, and maintains mature osteocyte viability. Thus, the interrelationship of MMPs and TGF-beta may play an important role in bone formation and maintenance.     

Importance of the adaptor (membrane fusion) protein hairpin domain for the functionality of multidrug efflux pumps

Drug efflux pumps of Gram-negative bacteria are tripartite export machineries located in the bacterial envelopes contributing to multidrug resistance. Protein structures of all three components have been determined, but the exact interaction sites are still unknown. We could confirm that the hybrid system composed of Pseudomonas aeruginosa channel tunnel OprM and the Escherichia coli inner membrane complex, formed by adaptor protein (membrane fusion protein) AcrA and transporter AcrB of the resistance nodulation cell division (RND) family, is not functional. However, cross-linking experiments show that the hybrid exporter assembles. Exchange of the hairpin domain of AcrA with the corresponding hairpin from adaptor protein MexA of P. aeruginosa restored the functionality. This shows the importance of the MexA hairpin domain for the functional interaction with the OprM channel tunnel. On the basis of these results, we have modeled the interaction of the hairpin domain and the channel tunnel on a molecular level for AcrA and TolC as well as MexA and OprM, respectively. The model of two hairpin docking sites per TolC protomer corresponding with hexameric adaptor proteins was confirmed by disulfide cross-linking experiments. The role of this interaction for functional efflux pumps is discussed.     

The effect of external dummy transmitters on oxygen consumption and performance of swimming Atlantic cod

Decreased critical swimming speed and increased oxygen consumption (     ) was found for externally tagged Atlantic cod Gadus morhua swimming at a high speed of 0·9 body length (total length, L _T) s⁻¹. No difference was found in the standard metabolic rate, indicating that the higher     for tagged cod was due to drag force rather than increased costs to keep buoyancy.     

Allosteric activation of coagulation factor VIIa visualized by hydrogen exchange

Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TF-bound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50A and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases.     

Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB

A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria.     

The 1.4-A crystal structure of the S. pombe Pop2p deadenylase subunit unveils the configuration of an active enzyme

Deadenylation is the first and probably also rate-limiting step of controlled mRNA decay in eukaryotes and therefore central for the overall rate of gene expression. In yeast, the process is maintained by the mega-Dalton Ccr4-Not complex, of which both the Ccr4p and Pop2p subunits are 3′–5′ exonucleases potentially responsible for the deadenylation reaction. Here, we present the crystal structure of the Pop2p subunit from Schizosaccharomyces pombe determined to 1.4Å resolution and show that the enzyme is a competent ribonuclease with a tunable specificity towards poly-A. In contrast to S. cerevisiae Pop2p, the S. pombe enzyme contains a fully conserved DEDDh active site, and the high resolution allows for a detailed analysis of its configuration, including divalent metal ion binding. Functional data further indicates that the identity of the ions in the active site can modulate both activity and specificity of the enzyme, and finally structural superposition of single nucleotides and poly-A oligonucleotides provide insight into the catalytic cycle of the protein.     

Contrasting fire-related resilience of ecologically dominant ants in tropical savannas of northern Australia

This paper examines the role of fire in mediating the relative abundance of two of the world's major ecologically dominant ant genera, Iridomyrmex and Oecophylla, where they coexist across the tropical savanna landscapes of northern Australia. These taxa have contrasting biogeographical histories, which are predicted to lead to contrasting responses to fire. Iridomyrmex is an autochthonous Australian genus that has radiated primarily in the arid zone; as such, its abundance is predicted to be promoted by frequent fire because this maintains an open habitat. In contrast, Oecophylla is a genus of leaf‐nesting ants occurring in the canopies of Old World tropical rainforest, and is a recent arrival to Australia in geological time; the abundance of these ants is predicted to decline under frequent fire. We test these predictions using results from a landscape‐scale fire experiment, where three experimental fire regimes (including no fire) were applied to replicated subcatchments over a 5‐year period. Using sweep nets, ants were sampled in the grass layer (the habitat layer of greatest overlap between Iridomyrmex and Oecophylla) in eucalypt woodland (canopy cover < 30%) and open eucalypt forest (canopy cover about 50%) habitats. A total of 27 species from 11 genera were collected during the study; eight were common enough for statistical analysis, and the abundances of four of these were significantly affected by fire treatment. As predicted, the abundance of Iridomyrmex was promoted by fire, whereas that of Oecophylla declined. These changes occurred only under late‐season (relatively high intensity) fires, and for Oecophylla occurred only in open forest (not woodland) habitat. This fire‐mediated relationship between Iridomyrmex and Oecophylla mirrors the much broader, ecosystem‐wide dynamic between eucalypt‐dominated savanna and rainforest in tropical Australia, with savannas dominated by fire‐resistant sclerophyll elements of Australian origin, and rainforest dominated by fire‐sensitive mesophyll elements of South‐East Asian origin.     

Quantitative determination of Ca2+-dependent Mg2+-ATPase from sarcoplasmic reticulum in muscle biopsies.

The possibility of quantifying the total concentration of Ca2+-dependent Mg2+-ATPase of sarcoplasmic reticulum was investigated by measurement of the Ca2+-dependent steady-state phosphorylation from [gamma-32P]ATP and the Ca2+-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) activity in crude muscle homogenates. The Ca2+-dependent phosphorylation at 0 degree C (mean +/- S.E.) was 40.0 +/- 2.5 (n = 6) and 6.2 +/- 0.7 (n = 4) nmol/g wet wt. in rat extensor digitorum longus (EDL) and soleus muscle, respectively (P less than 0.001). The Ca2+-dependent 3-O-MFPase activity at 37 degrees C was 1424 +/- 238 (n = 6) and 335 +/- 56 (n = 4) nmol/min per g wet wt. in rat EDL and soleus muscle, respectively (P less than 0.01). The molecular activity calculated from these measurements amounted to 35 +/- 5 min-1 (n = 6) and 55 +/- 10 min-1 (n = 4) for EDL and soleus muscle respectively. These values were not different from the molecular activity calculated for purified Ca2+-ATPase (36 min-1). The Ca2+-dependent 32P incorporation in soleus muscle decreased in the order mice greater than rats greater than guinea pigs. In EDL muscles from hypothyroid rats at a 30% reduction of the Ca2+-dependent phosphorylation was observed. The Ca2+-dependent phosphorylation in vastus lateralis muscle from three human subjects amounted to 4.5 +/- 0.8 nmol/g wet wt. It is concluded that measurement of the Ca2+-dependent phosphorylation allows rapid and reproducible quantification of the concentration of Ca2+-dependent Mg2+-ATPase of sarcoplasmic reticulum. Since only 20-60 mg of tissue is required for the measurements, the method can also be used for biopsies obtained in clinical studies.