Granzyme B-induced and Caspase 3-dependent Cleavage of Gelsolin by Mouse Cytotoxic T Cells Modifies Cytoskeleton Dynamics

Granule-associated perforin and granzymes (gzms) are key effector molecules of cytotoxic T lymphocytes (Tc cells) and natural killer cells and play a critical role in the control of intracellular pathogens and cancer. Based on the notion that many gzms, including A, B, C, K, H, and M exhibit cytotoxic activity in vitro, all gzms are believed to serve a similar function in vivo. However, more recent evidence supports the concept that gzms are not unidimensional but, rather, possess non-cytotoxic potential, including stimulation of pro-inflammatory cytokines and anti-viral activities. The present study shows that isolated mouse gzmB cleaves the actin-severing mouse protein, cytoplasmic gelsolin (c-gelsolin) in vitro. However, when delivered to intact target cells by ex vivo immune Tc cells, gzmB mediates c-gelsolin proteolysis via activation of caspases 3/7. The NH₂-terminal c-gelsolin fragment generated by either gzmB or caspase 3 in vitro constitutively severs actin filaments without destroying the target cells. The observation that gzmB secreted by Tc cells initiates a caspase cascade that disintegrates the actin cytoskeleton in target cells suggests that this intracellular process may contribute to anti-viral host defense.     

Inefficient clearance of dying cells in patients with SLE: anti-dsDNA autoantibodies,MFG-E8, HMGB-1 and other players

Systemic lupus erythematosus (SLE) is a complex disease resulting from inflammatory responses of the immune system against several autoantigens. Inflammation is conditioned by the continuous presence of autoantibodies and leaked autoantigens, e.g. from not properly cleared dying and dead cells. Various soluble molecules and biophysical properties of the surface of apoptotic cells play significant roles in the appropriate recognition and further processing of dying and dead cells. We exemplarily discuss how Milk fat globule epidermal growth factor 8 (MFG-E8), biophysical membrane alterations, High mobility group box 1 (HMGB1), C-reactive protein (CRP), and anti-nuclear autoantibodies may contribute to the etiopathogenesis of the disease. Up to date knowledge about these key elements may provide new insights that lead to the development of new treatment strategies of the disease.     

DNA ligase 4 stabilizes the ribosomal DNA array upon fork collapse at the replication fork barrier

DNA double-strand breaks (DSB) were shown to occur at the replication fork barrier in the ribosomal DNA of Saccharomyces cerevisiae using 2D-gel electrophoresis. Their origin, nature and magnitude, however, have remained elusive. We quantified these DSBs and show that a surprising 14% of replicating ribosomal DNA molecules are broken at the replication fork barrier in replicating wild-type cells. This translates into an estimated steady-state level of 7–10 DSBs per cell during S-phase. Importantly, breaks detectable in wild-type and sgs1 mutant cells differ from each other in terms of origin and repair. Breaks in wild-type, which were previously reported as DSBs, are likely an artefactual consequence of nicks nearby the rRFB. Sgs1 deficient cells, in which replication fork stability is compromised, reveal a class of DSBs that are detectable only in the presence of functional Dnl4. Under these conditions, Dnl4 also limits the formation of extrachromosomal ribosomal DNA circles. Consistently, dnl4 cells displayed altered fork structures at the replication fork barrier, leading us to propose an as yet unrecognized role for Dnl4 in the maintenance of ribosomal DNA stability.     

Purification of cadmium-binding proteins from related species of terrestrial helicidae (gastropoda,mollusca): A comparative study

Summary Three species of terrestrial Helicidae (Helix pomatia, Cepaea hortensis andArianta arbustorum) were fed cadmium-rich diet in the laboratory. The snails accumulated high amounts of the metal in their hepatopancreas. Most cadmium and some zinc were found, after centrifugation, in the soluble fractions from which a cadmium-binding protein was isolated for each species by ion exchange and gel chromatography. The proteins contained different amounts of cadmium, but little or no zinc, and showed high absorption at 254 nm indicating the presence of cadmium-mercaptide bonds. After gel filtration, a molecular weight of 12000 was found for cadmium-binding proteins fromHelix pomatia andArianta arbustorum, whereas a molecular weight of 10 000 was found for a cadmium-binding protein fromCepaea hortensis. SDS-polyacrylamide gel electrophoresis showed one single band for each protein fromHelix pomatia andArianta arbustorum and suggested a molecular weight of 11000 for both species. Amino acid analysis revealed, for each protein, high amounts of cysteine (12–20%), glycine (15–19%), and serine (12–14%), and moderately elevated contents of lysine (9–13%) and alanine (4–8%), but no methionine and only traces, if any, of aromatic amino acids. The ratios of cadmium to cysteine were 1:5, 1:10 and 1:3 in the proteins fromHelix pomatia,Cepaea hortensis andArianta arbustorum, respectively. Some features of the isolated proteins resembled mammalian metallothioneins. Most characteristics, however, differed from true metallothioneins and were similar to cadmium-binding proteins found in some marine molluscs.     

Developmental profiles of gangliosides in mouse and rat cerebral cortex

Summary Developmental profiles of 11 gangliosides, concentration of lipid- and glycoprotein-bound sialic acid, and activity of AChE of the rat and mouse cerebral cortex were followed from the 7^th day of gestation to the 21^st postnatal day.There are three main changes in ganglioside concentration, which are similar in both species. The first occurs from gestation day 10 until birth: parallel to decreased proliferation, cell migration, and neuroblast differentiation, G_M3 and G_D3 in mouse cortex and G_D3 in the rat's decreases in favor of G_Q1b, G_T1b, and G_D1a.The second occurs from birth until the first postnatal week: Parallel to increased growth and arborization of dendrites and axons as well as synaptogenesis in rats and mice, there is a two-fold rise of G_D1a, whereas G_Q1b and G_T1b remain on a nearly constant level. Concomitantly, G_M3 and G_D3 decreases. The third period of ganglioside changes starts in the second postnatal week, parallel to onset of myelination, and is characterized by an increase of G_M1 in parallel with a decrease of the polysialogangliosides G_T1b and G_Q1b.     

Latent Membrane Protein LMP2A Impairs Recognition of EBV-Infected Cells by CD8+ T Cells

The common pathogen Epstein-Barr virus (EBV) transforms normal human B cells and can cause cancer. Latent membrane protein 2A (LMP2A) of EBV supports activation and proliferation of infected B cells and is expressed in many types of EBV-associated cancer. It is not clear how latent EBV infection and cancer escape elimination by host immunity, and it is unknown whether LMP2A can influence the interaction of EBV-infected cells with the immune system. We infected primary B cells with EBV deleted for LMP2A, and established lymphoblastoid cell lines (LCLs). We found that CD8+ T cell clones showed higher reactivity against LMP2A-deficient LCLs compared to LCLs infected with complete EBV. We identified several potential mediators of this immunomodulatory effect. In the absence of LMP2A, expression of some EBV latent antigens was elevated, and cell surface expression of MHC class I was marginally increased. LMP2A-deficient LCLs produced lower amounts of IL-10, although this did not directly affect CD8+ T cell recognition. Deletion of LMP2A led to several changes in the cell surface immunophenotype of LCLs. Specifically, the agonistic NKG2D ligands MICA and ULBP4 were increased. Blocking experiments showed that NKG2D activation contributed to LCL recognition by CD8+ T cell clones. Our results demonstrate that LMP2A reduces the reactivity of CD8+ T cells against EBV-infected cells, and we identify several relevant mechanisms.     

Nonlinearities make a difference: comparison of two common Hill-type models with real muscle

Compared to complex structural Huxley-type models, Hill-type models phenomenologically describe muscle contraction using only few state variables. The Hill-type models dominate in the ever expanding field of musculoskeletal simulations for simplicity and low computational cost. Reasonable parameters are required to gain insight into mechanics of movement. The two most common Hill-type muscle models used contain three components. The series elastic component is connected in series to the contractile component. A parallel elastic component is either connected in parallel to both the contractile and the series elastic component (model [CC+SEC]), or is connected in parallel only with the contractile component (model [CC]). As soon as at least one of the components exhibits substantial nonlinearities, as, e.g., the contractile component by the ability to turn on and off, the two models are mechanically different. We tested which model ([CC+SEC] or [CC]) represents the cat soleus better. Ramp experiments consisting of an isometric and an isokinetic part were performed with an in situ cat soleus preparation using supramaximal nerve stimulation. Hill-type models containing force–length and force–velocity relationship, excitation–contraction coupling and series and parallel elastic force–elongation relations were fitted to the data. To test which model might represent the muscle better, the obtained parameters were compared with experimentally determined parameters. Determined in situations with negligible passive force, the force–velocity relation and the series elastic component relation are independent of the chosen model. In contrast to model [CC+SEC], these relations predicted by model [CC] were in accordance with experimental relations. In conclusion model [CC] seemed to better represent the cat soleus contraction dynamics and should be preferred in the nonlinear regression of muscle parameters and in musculoskeletal modeling.     

The effect of negative feedback loops on the dynamics of boolean networks

Feedback loops play an important role in determining the dynamics of biological networks. To study the role of negative feedback loops, this article introduces the notion of distance-to-positive-feedback which, in essence, captures the number of independent negative feedback loops in the network, a property inherent in the network topology. Through a computational study using Boolean networks, it is shown that distance-to-positive-feedback has a strong influence on network dynamics and correlates very well with the number and length of limit cycles in the phase space of the network. To be precise, it is shown that, as the number of independent negative feedback loops increases, the number (length) of limit cycles tends to decrease (increase). These conclusions are consistent with the fact that certain natural biological networks exhibit generally regular behavior and have fewer negative feedback loops than randomized networks with the same number of nodes and same connectivity.     

Association of common polymorphisms in GLUT9 gene with gout but not with coronary artery disease in a large case-control study

Background

Serum uric acid (UA) levels have recently been shown to be genetically influenced by common polymorphisms in the GLUT9 gene in two genome-wide association analyses of Italian and British populations. Elevated serum UA levels are often found in conjunction with the metabolic syndrome. Hyperuricemia is the major risk factor for gout and has been associated with increased cardiovascular morbidity and mortality. The aim of the present study was to further elucidate the association of polymorphisms in GLUT9 with gout and coronary artery disease (CAD) or myocardial infarction (MI). To test our hypotheses, we performed two large case-control association analyses of individuals from the German MI Family Study.

Methods and Findings

First, 665 patients with gout and 665 healthy controls, which were carefully matched for age and gender, were genotyped for four single nucleotide polymorphisms (SNPs) within or near the GLUT9 gene. All four SNPs demonstrated highly significant association with gout. SNP rs6855911, located within intron 7 of GLUT9, showed the strongest signal with a protective effect of the minor allele with an allelic odds ratio of 0.62 (95% confidence interval 0.52–0.75; p  =  3.2*10⁻⁷). Importantly, this finding was not influenced by adjustment for components of the metabolic syndrome or intake of diuretics. Secondly, 1,473 cases with severe CAD or MI and 1,241 healthy controls were tested for the same four GLUT9 SNPs. The analyses revealed, however, no significant association with CAD or with MI. Additional screening of genome-wide association data sets showed no signal for CAD or MI within the GLUT9 gene region.

Conclusion

Thus, our results provide compelling evidence that common genetic variations within the GLUT9 gene strongly influence the risk for gout but are unlikely to have a major effect on CAD or MI in a German population.