Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex

The purinergic receptor P2X(7) is part of a complex signaling mechanism participating in a variety of physiological and pathological processes. Depending on the activation scheme, P2X(7) receptors in vivo are non-selective cation channels or form large pores that can mediate apoptotic cell death. Expression of P2X(7)R in Xenopus oocytes results exclusively in formation of a non-selective cation channel. However, here we show that co-expression of P2X(7)R with pannexin1 in oocytes leads to the complex response seen in many mammalian cells, including cell death with prolonged ATP application. While the cation channel activity is resistant to carbenoxolone treatment, this gap junction and hemichannel blocking drug suppressed the currents induced by ATP in pannexin1/P2X(7)R co-expressing cells. Thus, pannexin1 appears to be the molecular substrate for the permeabilization pore (or death receptor channel) recruited into the P2X(7)R signaling complex.     

Farming for pests? Local and landscape-scale effects of grassland management on rabbit densities

In recent decades in the UK, there has been an increasing trend in numbers of the European wild rabbit, a significant agricultural pest typically associated with grassland habitats. However, the relationship between rabbit abundance and grassland management, in particular grazing, has not been sufficiently explained. We studied rabbit densities in seven pasture-dominated sites in north-east England between autumn and spring in two consecutive years, and used generalised linear mixed models and generalised additive models to explore relationships between habitat and management variables and rabbit abundance at local (field) and landscape scales. At the local scale high rabbit densities were significantly associated with small fields and the very short, homogeneous swards created by intensive sheep grazing during autumn and winter. At the landscape scale, high rabbit numbers were associated with sites with most field margins and a predator removal policy. Our results indicate that landscape management may play a central role in explaining rabbit abundance and distribution in grasslands. We suggest that current pasture management may create favourable conditions for high rabbit densities, and consequently boost numbers of this significant pest species.     

RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells

The ubiquitylation cascade plays an important role in the recruitment of repair factors at DNA double-strand breaks. The involvement of a growing number of ubiquitin E3 ligases adds to the complexity of the DNA damage-induced ubiquitin signaling. Here we use the genetically tractable avian cell line DT40 to investigate the role of HERC2, RNF8 and RNF168 in the DNA damage-induced ubiquitylation pathway. We show that formation of ubiquitin foci as well as cell survival after DNA damage depends on both RNF8 and RNF168. However, we find that RNF8 and RNF168 knockout cell lines respond differently to treatment with camptothecin indicating that they do not function in a strictly linear manner. Surprisingly, we show that HERC2 is required neither for survival nor for ubiquitin foci formation after DNA damage in DT40. Moreover, the E3 ubiquitin ligase activity of HERC2 is not redundant to that of RNF8 or RNF168.     

P2X7 receptor-Pannexin1 complex: pharmacology and signaling

Pannexin 1 (Panx1), an ortholog to invertebrate innexin gap junctions, has recently been proposed to be the pore induced by P2X(7) receptor (P2X(7)R) activation. We explored the pharmacological action of compounds known to block gap junctions on Panx1 channels activated by the P2X(7)R and the mechanisms involved in the interaction between these two proteins. Whole cell recordings revealed distinct P2X(7)R and Panx1 currents in response to agonists. Activation of Panx1 currents following P2X(7)R stimulation or by membrane depolarization was blocked by Panx1 small-interfering RNA (siRNA) and with mefloquine > carbenoxolone > flufenamic acid. Incubation of cells with KN-62, a P2X(7)R antagonist, prevented current activation by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP). Membrane permeabilization to dye induced by BzATP was also prevented by Panx1 siRNA and by carbenoxolone and mefloquine. Membrane permeant (TAT-P2X(7)) peptides, provided evidence that the Src homology 3 death domain of the COOH-terminus of the P2X(7)R is involved in the initial steps of the signal transduction events leading to Panx1 activation and that a Src tyrosine kinase is likely involved in this process. Competition assays indicated that 20 muM TAT-P2X(7) peptide caused 50% reduction in Src binding to the P2X(7)R complex. Src tyrosine phosphorylation following BzATP stimulation was reduced by KN-62, TAT-P2X(7) peptide, and by the Src tyrosine inhibitor PP2 and these compounds prevented both large-conductance Panx1 currents and membrane permeabilization. These results together with the lack Panx1 tyrosine phosphorylation in response to P2X(7)R stimulation indicate the involvement of an additional molecule in the tyrosine kinase signal transduction pathway mediating Panx1 activation through the P2X(7)R.     

Late Campanian to late Maastrichtian bryozoans encrusting on belemnite rostra from the Aktolagay Plateau in western Kazakhstan

Twenty species belonging to fifteen genera of cyclostome and cheilostome bryozoans encrusting belemnite rostra are described from the late Campanian to Maastrichtian of the Aktolagay Plateau, in western Kazakhstan. Due to the moderate to poor preservation of the material, only four cheilostome species are identified down to the species level: Wilbertopora? besoktiensis (Voigt, 1967), ‘Aechmellina’ stenostoma Voigt, 1930, and two new species, ‘Aechmellina’ viskovae and Cheethamia aktolagayensis. All remaining species are left in open nomenclature. Type material of Wilbertopora? besoktiensis from the early Maastrichtian of the Mangyshlak Peninsula in Kazakhstan, has been re-examined. Palaeobiogeographical and implications are discussed. Cheilostomes slightly dominated over cyclostomes in the Aktolagay Plateau fauna encrusting on belemnites in terms of diversity. The dominant colony forms observed were spots and sheets.     

Ancient DNA reveals differences in behaviour and sociality between brown bears and extinct cave bears

Ancient DNA studies have revolutionized the study of extinct species and populations, providing insights on phylogeny, phylogeography, admixture and demographic history. However, inferences on behaviour and sociality have been far less frequent. Here, we investigate the complete mitochondrial genomes of extinct Late Pleistocene cave bears and middle Holocene brown bears that each inhabited multiple geographically proximate caves in northern Spain. In cave bears, we find that, although most caves were occupied simultaneously, each cave almost exclusively contains a unique lineage of closely related haplotypes. This remarkable pattern suggests extreme fidelity to their birth site in cave bears, best described as homing behaviour, and that cave bears formed stable maternal social groups at least for hibernation. In contrast, brown bears do not show any strong association of mitochondrial lineage and cave, suggesting that these two closely related species differed in aspects of their behaviour and sociality. This difference is likely to have contributed to cave bear extinction, which occurred at a time in which competition for caves between bears and humans was likely intense and the ability to rapidly colonize new hibernation sites would have been crucial for the survival of a species so dependent on caves for hibernation as cave bears. Our study demonstrates the potential of ancient DNA to uncover patterns of behaviour and sociality in ancient species and populations, even those that went extinct many tens of thousands of years ago.     

CYP2W1 Is Highly Expressed in Adrenal Glands and Is Positively Associated with the Response to Mitotane in Adrenocortical Carcinoma

Background

Adrenocortical tumors comprise frequent adenomas (ACA) and rare carcinomas (ACC). Human cytochrome P450 2W1 (CYP2W1) is highly expressed in some cancers holding the potential to activate certain drugs into tumor cytotoxins.

Objective

To investigate the CYP2W1 expression in adrenal samples and its relationship with clinical outcome in ACC.

Material and Methods

CYP2W1 expression was investigated by qRT-PCR in 13 normal adrenal glands, 32 ACA, 25 ACC, and 9 different non-adrenal normal tissue samples and by immunohistochemistry in 352 specimens (23 normal adrenal glands, 33 ACA, 239 ACC, 67 non-adrenal normal or neoplastic samples).

Results

CYP2W1 mRNA expression was absent/low in normal non-adrenal tissues, but high in normal and neoplastic adrenal glands (all P<0.01 vs non-adrenal normal tissues). Accordingly, CYP2W1 immunoreactivity was absent/low (H-score 0–1) in 72% of non-adrenal normal tissues, but high (H-score 2–3) in 44% of non-adrenal cancers, in 65% of normal adrenal glands, in 62% of ACAs and in 50% of ACCs (all P<0.001 vs non-adrenal normal tissues), being significantly increased in steroid-secreting compared to non-secreting tumors. In ACC patients treated with mitotane only, high CYP2W1 immunoreactivity adjusted for ENSAT stage was associated with longer overall survival and time to progression (P<0.05 and P<0.01, respectively), and with a better response to therapy both as palliative (response/stable disease in 42% vs 6%, P<0.01) or adjuvant option (absence of disease recurrence in 69% vs 45%, P<0.01).

Conclusion

CYP2W1 is highly expressed in both normal and neoplastic adrenal glands making it a promising tool for targeted therapy in ACC. Furthermore, CYP2W1 may represent a new predictive marker for the response to mitotane treatment.     

Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium

The ability for long-range communication through intercellular calcium waves is inherent to cells of many tissues. A dual propagation mode for these waves includes passage of IP3 through gap junctions as well as an extracellular pathway involving ATP. The wave can be regenerative and include ATP-induced ATP release via an unknown mechanism. Here, we show that pannexin 1 channels can be activated by extracellular ATP acting through purinergic receptors of the P2Y group as well as by cytoplasmic calcium. Based on its properties, including ATP permeability, pannexin 1 may be involved in both initiation and propagation of calcium waves.     

Preferential pathways for light-trapping involving β-ligated chlorophylls

The magnesium atom of chlorophylls (Chls) is always five- or six-coordinated within chlorophyll-protein complexes which are the main light-harvesting systems of plants, algae and most photosynthetic bacteria. Due to the presence of stereocenters and the axial ligation of magnesium the two faces of Chls are diastereotopic. It has been previously recognized that the α-configuration having the magnesium ligand on the opposite face of the 17-propionic acid moiety is more frequently encountered and is more stable than the more seldom β-configuration that has the magnesium ligand on the same face [T.S. Balaban, P. Fromme, A.R. Holzwarth, N. Krauβ, V.I. Prokhorenko, Relevance of the diastereotopic ligation of magnesium atoms in chlorophylls in Photosystem I, Biochim. Biophys. Acta (Bioenergetics), 1556 (2002) 197-207; T. Oba, H. Tamiaki, Which side of the π-macrocycle plane of (bacterio)chlorophylls is favored for binding of the fifth ligand? Photosynth. Res. 74 (2002) 1-10]. In photosystem I only 14 Chls out of a total of 96 are in a β-configuration and these occupy preferential positions around the reaction center. We have now analyzed the α/β dichotomy in the homodimeric photosystem II based on the 2.9 Å resolution crystal structure [A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 Å resolution: role of quinones, lipids, channels and chloride, Nature Struct. Mol. Biol. 16 (2009) 334-342] and find that out of 35 Chls in each monomer only 9 are definitively in the β-configuration, while 4 are uncertain. Ab initio calculations using the approximate coupled-cluster singles-and-doubles model CC2 [O. Christiansen, H. Koch, P. Jørgensen, The second-order approximate coupled cluster singles and doubles model CC2, Chem. Phys. Lett. 243 (1995) 409-418] now correctly predict the absorption spectra of Chls a and b and conclusively show for histidine, which is the most frequent axial ligand of magnesium in chlorophyll-protein complexes, that only slight differences (< 4 nm) are encountered between the α- and β-configurations. Significant red shifts (up to 50 nm) can, however, be encountered in excitonically coupled β-β-Chl dimers. Surprisingly, in both photosystems I and II very similar “special” β-β dimers are encountered at practically the same distances from P700 and P680, respectively. In purple bacteria LH2, the B850 ring is composed exclusively of such tightly coupled β-bacteriochlorophylls a. A statistical analysis of the close contacts with the protein matrix (< 5 Å) shows significant differences between the α- and β-configurations and the subunit providing the axial magnesium ligand. The present study allows us to conclude that the excitation energy transfer in light-harvesting systems, from a peripheral antenna towards the reaction center, may follow preferential pathways due to structural reasons involving β-ligated Chls.