Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels

Cyclophilin D was recently shown to bind to and decrease the activity of F(0)F(1)-ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al. (2009) J Biol Chem, 284, 33982-33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria, an increase in F(0)F(1)-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F(0)F(1)-ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of an effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ～ 2.2-fold lower flux control coefficient of the F(0)F(1)-ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ～ 30% change in F(0)F(1)-ATP synthase activity in fully energized or fully de-energized mitochondria affects the ADP-ATP exchange rate mediated by the ANT in the range 1.38-1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F(0)F(1)-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels.     

Radioprotective role of cyanobacterial phycobilisomes

Cyanobacteria are thought to be responsible for pioneering dioxygen production and the so-called “Great Oxygenation Event” that determined the formation of the ozone layer and the ionosphere restricting ionizing radiation levels reaching our planet, which increased biological diversity but also abolished the necessity of radioprotection. We speculated that ancient protection mechanisms could still be present in cyanobacteria and studied the effect of ionizing radiation and space flight during the Foton-M4 mission on Synechocystis sp. PCC6803. Spectral and functional characteristics of photosynthetic membranes revealed numerous similarities of the effects of α-particles and space flight, which both interrupted excitation energy transfer from phycobilisomes to the photosystems and significantly reduced the concentration of phycobiliproteins. Although photosynthetic activity was severely suppressed, the effect was reversible, and the cells could rapidly recover from the stress. We suggest that the actual existence and the uncoupling of phycobilisomes may play a specific role not only in photo-, but also in radioprotection, which could be crucial for the early evolution of Life on Earth.     

Cysteine racemization during the Fmoc solid phase peptide synthesis of the Nav1.7‐selective peptide – protoxin II

Protoxin II is biologically active peptide containing the inhibitory cystine knot motif. A synthetic version of the toxin was generated with standard Fmoc solid phase peptide synthesis. If N‐methylmorpholine was used as a base during synthesis of the linear protoxin II, it was found that a significant amount of racemization (approximately 50%) was observed during the process of cysteine residue coupling. This racemization could be suppressed by substituting N‐methylmorpholine with 2,4,6‐collidine. The crude linear toxin was then air oxidized and purified. Electrophysiological assessment of the synthesized protoxin II confirmed its previously described interactions with voltage‐gated sodium channels. Eight other naturally occurring inhibitory knot peptides were also synthesized using this same methodology. The inhibitory potencies of these synthesized toxins on Nav1.7 and Nav1.2 channels are summarized. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Pattern of cytokine expression by rat liver epithelial cells supporting long-term culture of human CD34(+)umbilical cord blood cells

Fetal liver is the main site of haematopoiesis during mid-gestation. The adult liver still provides a favourable environment for extramedullary haematopoiesis. Nevertheless, regulation of liver haematopoiesis by cell-cell contacts or by cytokines remains poorly understood. Recently, we have shown that rat liver epithelial cells (RLECs) support long-term survival and multilineage differentiation of adult human CD34(+)and CD34(+)/CD38(-)haematopoietic cells obtained from granulocyte-colony stimulating factor mobilized peripheral blood and from bone marrow respectively. In addition, the importance of physical proximity between haematopoietic cells and RLECs was clearly demonstrated. Here, our findings give evidence that RLECs belonging to the epithelial but non-parenchymal liver compartment also sustain the long-term production of progenitors from human CD34(+)umbilical cord blood cells. Moreover, to better analyse the regulation of haematopoiesis in this RLEC coculture model, we have investigated the cytokine expression by RLECs alone and by RLECs coming from coculture with CD34(+)cells from umbilical cord blood. We demonstrated that a broad spectrum of cytokines acting at different stages of haematopoiesis is produced by RLECs. Interestingly, an upregulation of leukemia inhibitory factor expression by RLECs in presence of CD34(+)haematopoietic cells was observed. These data suggest an important role of cell-cell interactions in the regulation of haematopoiesis.     

Germline polymorphisms of RET and GFRA1 genes in patients with medullary thyroid carcinoma

The association of RET and GFRA1 polymorphisms with a predisposition to sporadic medullary thyroid cancer (MTC) and their effects on the clinical features of hereditary and sporadic MTC were studied in 67 MTC patients (22 hereditary and 45 sporadic), 3 asymptomatic carriers of mutant RET, and 178 healthy control residents of Russia. RET exons 8, 10, 11, 13, 14, 15, and 16 and intron 1 along with the GFRA1 5′-UTR were screened by PCR and subsequent direct sequencing or RFLP analysis. Eight polymorphic variants of RET (exons 11, 13, 14, and 15 and introns 1, 8, 13, and 14) and four GFRA1 polymorphisms were detected. Linkage disequilibrium was found between RET variants G691S and S904S, L769L and IVS8, and S836S and IVS13. In sporadic MTC the allele frequency of only one polymorphic RET variant, L769L, was significantly lower than in the control group. In hereditary MTC a significant overrepresentation of the S836S and underrepresentation of the S904S polymorphic variants were observed as compared to groups with sporadic MTC and the controls. Cosegregation was not found between individual polymorphisms and the phenotype of sporadic MTC. In patients with hereditary MTC whose genotype had the polymorphic L769L and the wild-type S836S variants, the disease manifested 20 years later, on average, than in individuals with polymorphic L769L and S836S or with wild-type L769L (P = 0.01). The results suggest a protective role of the L769L polymorphism in sporadic MTC and a modulating effect of the combination polymorphic L769L with wild-type S836S on the clinical outcome of hereditary MTC.     

Patches of barnacles and ascidians in soft bottoms: Associated motile fauna in relation to the surrounding assemblage

Epibenthic patches dominated by barnacles Balanus crenatus Bruguiere and solitary ascidians Styela spp., Bolthenia echinata (L.) and Molgula spp. in the White Sea shallow subtidal develop on bivalve shells and small stones surrounded with muddy sand. The space between barnacles and ascidians is filled with muddy sediment inhabited by motile taxa. We hypothesized that (i) epibenthic patches and unstructured sediment would attract different motile fauna and (ii) motile fauna of the patches would be affected by local abundances of epibenthic foundation species. Most dominant motile species demonstrated a significant difference in abundance between the two microhabitats. In contrast to the fauna of the sediment, species composition observed in aggregations of barnacles and ascidians was stable across different locations. In the field experiment initially clear bivalve shells after 5 years of exposure developed barnacle clusters with motile fauna similar to that observed in natural aggregations. Amphipods, isopods and bivalves, capitellid polychaets, Cirratulus cirratus (Müller) and Pholoe minuta Fabricius (Polychaeta) dominated in the sediment inside epibenthic patches. The proportion of capitellids, known to be sensitive to organic enrichment, was much higher within the patches than outside. The abundances of motile taxa found in aggregations were mostly determined by the number of barnacles of different size and of their empty shells, the biomass of ascidians, and the effect of location. Different dominant species demonstrated sensitivity to different parameters.Physical structure of the habitat, provided by barnacles and ascidians, as well as their biodeposition activity are regarded as the main factors structuring the motile fauna in the community studied. The spatial pattern observed seems to imply a range of pattern-generating biogenic processes, similar to those previously revealed in patches of filter-feeding bivalves, tube-building worms and seagrass.     

Multiple foundation species shape benthic habitat islands

Pattern generation by foundation species (FS) is a primary structuring agent in marine and terrestrial communities. Prior research, focused on single-species or guild-dominated habitats, stressed the role of facilitation in maintaining community structure. However, many habitats are developed by multiple FS from different guilds. Competition between these FS may provide an additional agent potentially responsible for spatial and temporal patterns. In the White Sea, epibenthic patches formed by barnacles (Balanus crenatus) and solitary ascidians (mainly Styela spp. and Molgula spp.) on small stones and empty bivalve shells (mainly Serripes groenlandicus) produce microhabitats for different sessile taxa. We hypothesized that: (1) several FS would provide habitats for most of other species in the community; (2) different FS promote different assemblages of sessile organisms; (3) the interplay of facilitation and competition best explains observed patterns of abundance and demography in FS; and (4) these interactions shape the whole community, increasing the diversity compared to less heterogeneous patches constituted by single FS. We examined 459 patches and the results generally supported this hypothesis. The number of FS in a patch positively affected species diversity. Most sessile species (72% of individuals) resided on barnacles, ascidians and red algae, except barnacles that dominated the primary substrate. The size structure of barnacles (live individuals and empty shells) and ascidians were interrelated, suggesting long-term patch dynamics whereby ascidians regularly replace barnacles. Following this replacement, we expect consequent changes to the entire dependent assemblage. Evidence for these changes exists in the spatial pattern: most sessile and motile taxa demonstrated significant associations with either FS. Our results indicate that the small-scale patterns observed in patches formed by multiple FS are primarily generated by facilitation of dependent taxa by FS, and facilitation and competition between different FS. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Dedicated to E. A. Ninbourg, our late tutor.