Oxidized phosphatidylcholine formation and action in oligodendrocytes

Reactive oxygen species play a major role in neurodegeneration. Increasing concentrations of peroxide induce neural cell death through activation of pro-apoptotic pathways. We now report that hydrogen peroxide generated sn-2 oxidized phosphatidylcholine (OxPC) in neonatal rat oligodendrocytes and that synthetic OxPC [1-palmitoyl-2-(5'-oxo)valeryl-sn-glycero-3 phosphorylcholine, POVPC] also induced apoptosis in neonatal rat oligodendrocytes. POVPC activated caspases 3 and 8, and neutral sphingomyelinase (NSMase) but not acid sphingomyelinase. Downstream pro-apoptotic pathways activated by POVPC treatment included the Jun N-terminal kinase proapoptotic cascade and the degradation of phospho-Akt. Activation of NSMase occurred within 1 h, was blocked by inhibitors of caspase 8, increased mainly C18 and C24:1 ceramides, and appeared to be concentrated in detergent-resistant microdomains (Rafts). We concluded that OxPC initially activated NSMase and converted sphingomyelin into ceramide to mediate a series of downstream pro-apoptotic events in oligodendrocytes.     

Apoptosis signal-regulating kinase 1 regulates the expression of caspase-11

Caspase-11 is an inducible caspase involved in the regulation of cell death and inflammation. In the present study, we examined whether apoptosis signal-regulating kinase 1 (Ask1)-mediated signaling pathway is involved in the expression of caspase-11 induced by lipopolysaccharide (LPS). We found that the induction of caspase-11 was suppressed by the inhibitors of NADPH oxidase (Nox) or knockdown of Nox4 that acts downstream of toll-like receptor 4 and generates Ask1-activating reactive oxygen species. Overexpression of dominant negative tumor necrosis factor receptor associate factor 6 also suppressed the induction of caspase-11. Importantly, knockdown or dominant negative form of Ask1 suppressed the induction of caspase-11 following LPS stimulation. Taken together, our results show that Ask1 regulates the expression of caspase-11 following LPS stimulation.     

Calpain-1 Cleaves and Activates Caspase-7

Caspase-7 is an executioner caspase that plays a key role in apoptosis, cancer, and a number of neurodegenerative diseases. The mechanism of caspase-7 activation by granzyme B and caspase-3 has been well characterized. However, whether other proteases such as calpains activate or inactivate caspase-7 is not known. Here, we present that recombinant caspase-7 is directly cleaved by calpain-1 within the large subunit of caspase-7 to produce two novel products, large subunit p18 and p17. This new form of caspase-7 has a 6-fold increase in V_max when compared with the previously characterized p20/p12 form. Zymography revealed that the smaller caspase-7 product (p17) is 18-fold more active than either the caspase-3-cleaved product (p20) or the larger calpain-1 product of caspase-7 (p18). Mass spectrometry and site-directed mutagenesis identified the calpain cleavage sites within the caspase-7 large subunit at amino acid 36 and 45/47. These proteolysis events occur in vivo as indicated by the accumulation of caspase-7 p18 and p17 subunits in cortical neurons undergoing Ca²⁺ dysregulation. Further, cleavage at amino acid 45/47 of caspase-7 by calpain results in a reduction in nuclear localization when compared with the caspase-3 cleavage product of caspase-7 (p20). Our studies suggest the calpain-activated form of caspase-7 has unique enzymatic activity, localization, and binding affinity when compared with the caspase-activated form.Apoptosis is a well-defined cellular destruction pathway that primarily utilizes a family of cysteine proteases, the caspases (1, 2). This cell death program can be initiated by cell death receptor activation (extrinsic pathway) or a variety of drugs or cellular stresses (intrinsic pathway) leading to activation of apical caspase-8, -9, and/or -10 (1, 3, 4). These initiator caspases in turn directly activate the executioner caspases, caspase-3 and -7, which through proteolysis of defined substrates are responsible for the dismantling of the cell and subsequent death (3, 4). Granzyme B, released by cytotoxic T lymphocytes to protect the host from pathogens and tumor cells, can also initiate this apoptotic cascade and therefore is considered an apical caspase mimic (5–7). All caspases, as well as granzyme B, preferentially cleave after aspartic acid residues, with many having well-defined consensus sequences, making substrate cleavage sites easy to predict and establish (3, 4, 7, 8).Caspases exist in a latent form prior to activation. Both the initiator and executioner caspases are synthesized as a single chain protein, which require proteolytic cleavage to become active. Procaspase-7 is expressed as a 303-amino acid residue polypeptide chain. The activation and regulation of executioner caspase-7 by caspases and granzyme B has been extensively studied. Caspase-7 requires cleavage by caspase-3 and caspase-8/-10 or granzyme B, for activation (6, 9). Current evidence suggests that caspase-3 initially cleaves off the first 23 amino acids (propeptide, 2 kDa), followed by caspase-8/-10 or granzyme B cleaving between the large (20 kDa) and small (12 kDa) subunit after amino acid 198 to activate the enzyme. The large subunit containing the catalytic His-237 and Cys-285 (caspase-1 numbering convention), and the small subunit are involved in the formation of the substrate-binding region. In vitro, granzyme B can also activate caspase-7 independently of caspase-3, but this does not appear to occur in vivo (5, 6). Currently, there is no evidence that other classes of proteases play a role in activating or modulating caspase-7 activity.Changes in intracellular Ca²⁺ levels influence apoptosis in a number of cell types (10–13). Because in many of these apoptotic cell models the Ca²⁺-dependent cysteine proteases, calpains, are activated upstream of caspases (14–16), it is possible that calpains may activate and/or modulate caspase activity via direct cleavage. Studies directed at understanding calpains with respect to caspase activation are limited. Calpain-2 was shown to cleave procaspase-9, decreasing its activity (17). In the same study, calpain-2 treatment cleaved procaspase-7 to produce a single, novel fragment, but in this case the effect on enzymatic activity was not investigated (17). To improve our understanding of calpains and the role of calcium in cell death, we carried out studies directed at understanding how calpains activate or modulate caspase activity. We found that calpain treatment produced a large increase in caspase-7 activity. Calpain cleaves procaspase-7 to produce two large subunits of 18.5 and 17.2 kDa, the smaller of which has a robust increase in activity relative to the 20-kDa large subunit produced by caspase-3 cleavage of caspase-7. Both calpain cleavage sites in caspase-7 are identified using mass spectrometry. N-methyl-d-aspartate-induced Ca²⁺-dependent cell death in primary cortical neurons produced calpain-derived caspase-7 cleavage products in vivo. Lastly, the strictly cytosolic localization of the smaller calpain fragment confirms that a previously identified nuclear localization signal (18) is involved in caspase-7 cytosolic/nuclear distribution. Our data suggest that increases in Ca²⁺ leading to activation of calpains may significantly modulate caspase-7 activity and thus, apoptosis.     

Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin‐binding entities

Post‐translational modification with ubiquitin is one of the most important mechanisms in the regulation of protein stability and function. However, the high reversibility of this modification is the main obstacle for the isolation and characterization of ubiquitylated proteins. To overcome this problem, we have developed tandem‐repeated ubiquitin‐binding entities (TUBEs) based on ubiquitin‐associated (UBA) domains. TUBEs recognize tetra‐ubiquitin with a markedly higher affinity than single UBA domains, allowing poly‐ubiquitylated proteins to be efficiently purified from cell extracts in native conditions. More significant is the fact that TUBEs protect poly‐ubiquitin‐conjugated proteins, such as p53 and IκBα, both from proteasomal degradation and de‐ubiquitylating activity present in cell extracts, as well as from existing proteasome and cysteine protease inhibitors. Therefore, these new ‘molecular traps’ should become valuable tools for purifying endogenous poly‐ubiquitylated proteins, thus contributing to a better characterization of many essential functions regulated by these post‐translational modifications.     

Purification of homogeneous forms of fungal peroxygenase

Extracellular peroxygenase from the agaric fungus Agrocybe aegerita is a versatile biocatalyst that oxygenates various substrates by means of hydrogen peroxide. The enzyme is routinely produced in suspensions of soybean meal and has until now been purified by several steps of fast protein liquid chromatography (FPLC) using different ion exchangers. The final protein fraction had a molecular mass of 46 kDa but still consisted of several incompletely separated proteins with slightly differing isoelectric points (pI 5.2, 5.6, 6.1), probably representing differently glycosylated isoforms. This made it difficult to further purify the individual protein forms. Since homogeneous protein fractions are a pre-requisite for X-ray crystallography and specific structure-function studies, an appropriate FPLC procedure was developed starting with pre-purification of crude peroxygenase on SP Sepharose followed by chromatofocusing on a Mono P column and elution with a pH gradient. Three sufficiently separated main protein peaks were eluted from the Mono P column and confirmed to be distinct forms of aromatic peroxygenase with different pIs. All A. aegerita peroxygenase forms oxygenated toluene and naphthalene and no catalytic differences were observed between them. We tested also two devices for preparative isoelectric focusing (Rotofor, IsoPrime systems) for peroxygenase separation but resolution and protein recovery were not sufficient.     

Cost-effectiveness of postural exercise therapy versus physiotherapy in computer screen-workers with early non-specific work-related upper limb disorders (WRULD); a randomized controlled trial

Background

Exercise therapies generate substantial costs in computer workers with non-specific work-related upper limb disorders (WRULD).

Aims

To study if postural exercise therapy is cost-effective compared to regular physiotherapy in screen-workers with early complaints, both from health care and societal perspective.

Methods

Prospective randomized trial including cost-effectiveness analysis; one year follow-up. Participants: Eighty-eight screen-workers with early non-specific WRULD; six drop-outs. Interventions: A ten week postural exercise program versus regular physiotherapy. Outcome measures: Effectiveness measures: Pain: visual analogous scale (VAS), self-perceived WRULD (yes/no). Functional outcome: Disabilities of Arm, Shoulder and Hand- Dutch Language Version (DASH-DLV). Quality of life outcome: EQ-5D. Economic measures: health care costs including patient and family costs and productivity costs resulting in societal costs. Cost-effectiveness measures: health care costs and societal costs related to the effectiveness measures. Outcome measures were assessed at baseline; three, six and twelve months after baseline.

Results

At baseline both groups were comparable for baseline characteristics except scores on the Pain Catastrophizing Scale and comparable for costs. No significant differences between the groups concerning effectiveness at one year follow-up were found. Effectiveness scores slightly improved over time. After one year 55% of participants were free of complaints. After one year the postural exercise group had higher mean total health care costs, but lower productivity costs compared to the physiotherapy group. Mean societal costs after one year (therefore) were in favor of postural exercise therapy [- €622; 95% CI -2087; +590)]. After one year, only self- perceived WRULD seemed to result in acceptable cost-effectiveness of the postural exercise strategy over physiotherapy; however the probability of acceptable cost-effectiveness did not exceed 60%. Considering societal costs related to QALYs, postural exercise therapy had a probability of over 80% to be cost-effective over a wide range of cost-effectiveness ceiling ratios; however based on a marginal QALY-difference of 0.1 over a 12 month time frame.

Conclusion

Although our trial failed to find significant differences in VAS, QALYs and ICERs based on VAS and QALYs at one-year follow-up, CEACs suggest that postural exercise therapy according to Mensendieck/Cesar has a higher probability of being cost-effective compared to regular physiotherapy; however further research is required.

Trial registration

ISRCTN 15872455     

Isolation and characterization of polymorphic microsatellite loci for the northern flicker (Colaptes auratus; Aves)

To investigate the mating system of northern flickers (Colaptes auratus), we developed primers for 14 microsatellite loci and screened them in 68 unrelated adults and their offspring. All markers were highly polymorphic with 9 to +36 alleles per locus. One marker was Z‐chromosome linked; one marker exceeded the size standard range and could not be analysed further. We checked the other 12 markers for Mendelian inheritance in 36 broods for which the social parents were known. Seven markers showed evidence for the presence of null alleles, and three of those showed significant deviation from Hardy–Weinberg equilibrium. Markers were generally unlinked.     

A Distinct Macrophage Population Mediates Metastatic Breast Cancer Cell Extravasation,Establishment and Growth

Background

The stromal microenvironment and particularly the macrophage component of primary tumors influence their malignant potential. However, at the metastatic site the role of these cells and their mechanism of actions for establishment and growth of metastases remain largely unknown.

Methodology/Principal Findings

Using animal models of breast cancer metastasis, we show that a population of host macrophages displaying a distinct phenotype is recruited to extravasating pulmonary metastatic cells regardless of species of origin. Ablation of this macrophage population through three independent means (genetic and chemical) showed that these macrophages are required for efficient metastatic seeding and growth. Importantly, even after metastatic growth is established, ablation of this macrophage population inhibited subsequent growth. Furthermore, imaging of intact lungs revealed that macrophages are required for efficient tumor cell extravasation.

Conclusion/Significance

These data indicate a direct enhancement of metastatic growth by macrophages through their effects on tumor cell extravasation, survival and subsequent growth and identifies these cells as a new therapeutic target for treatment of metastatic disease.