Polyoxometalate cluster [V12B18O60H6] functionalized with the copper(II) bis-ethylenediamine complex

Three compounds based on the polyoxometalate building block [V₁₂B₁₈O₆₀H₆], (Na)₁₀[(H₂O)V₁₂B₁₈O₆₀H₆]·18H₂O (1), Na₈[Cu(en)₂]₂[V₁₂B₁₈O₆₀H₆](NO₃)₂·14.7H₂O (2), Na₇[Cu(en)₂]₂[V₁₂B₁₈O₆₀H₆](NO₃)·15.5H₂O (3), (en = ethylenediamine), have been hydrothermally synthesized and characterized by single-crystal X-ray diffraction analysis and TGA. Compound 1 consists of polyoxovanadium borate [V₁₂B₁₈O₆₀H₆] clusters which are surrounded by sodium countercations in octahedral sites, stabilized by electrostatic interactions with the oxygen atoms of both vanadium and boron centres. However, compounds 2 and 3 correspond to more complicated structures, constructed from the same polyoxometalate clusters, which are interconnected by [Cu(en)₂]²⁺ moieties via the terminal oxygen atoms of the polyoxoanions, generating one-dimensional structures. The functionalization of this polyoxovanadium borate cluster has been obtained by the use of [Cu(en)₂]²⁺ complex ions, thus demonstrating the capacity of the terminal oxygen atoms of the cluster to bind transition metal centres. The structural stability of the [V₁₂B₁₈O₆₀H₆] cluster permits the formation of functionalized polyoxometalate clusters, generating various crystalline lattices.     

The Arabidopsis Protein CONSERVED ONLY IN THE GREEN LINEAGE160 Promotes the Assembly of the Membranous Part of the Chloroplast ATP Synthase

The chloroplast F₁F_o-ATP synthase/ATPase (cpATPase) couples ATP synthesis to the light-driven electrochemical proton gradient. The cpATPase is a multiprotein complex and consists of a membrane-spanning protein channel (comprising subunit types a, b, b′, and c) and a peripheral domain (subunits α, β, γ, δ, and ε). We report the characterization of the Arabidopsis (Arabidopsis thaliana) CONSERVED ONLY IN THE GREEN LINEAGE160 (AtCGL160) protein (AtCGL160), conserved in green algae and plants. AtCGL160 is an integral thylakoid protein, and its carboxyl-terminal portion is distantly related to prokaryotic ATP SYNTHASE PROTEIN1 (Atp1/UncI) proteins that are thought to function in ATP synthase assembly. Plants without AtCGL160 display an increase in xanthophyll cycle activity and energy-dependent nonphotochemical quenching. These photosynthetic perturbations can be attributed to a severe reduction in cpATPase levels that result in increased acidification of the thylakoid lumen. AtCGL160 is not an integral cpATPase component but is specifically required for the efficient incorporation of the c-subunit into the cpATPase. AtCGL160, as well as a chimeric protein containing the amino-terminal part of AtCGL160 and Synechocystis sp. PCC6803 Atp1, physically interact with the c-subunit. We conclude that AtCGL160 and Atp1 facilitate the assembly of the membranous part of the cpATPase in their hosts, but loss of their functions provokes a unique compensatory response in each organism.The majority of cellular energy is stored in the form of ATP synthesized by the ubiquitous F₁F_o-ATP synthase (F₁ stands for coupling factor 1, F_o for coupling factor o), which is found in the energy-transducing membranes of bacteria, mitochondria, and chloroplasts. The chloroplast F₁F_o-ATP synthase/ATPase (cpATPase) is a rotary motor that is responsible for coupling ATP synthesis (and hydrolysis) to the light-driven electrochemical proton gradient. The cpATPase comprises two physically separable parts, chloroplast coupling factor o (CF_o), which is an integral membrane-spanning proton channel, and chloroplast coupling factor 1 (CF₁), which is located peripheral to the membrane and contains the catalytic site(s) for reversible ATP synthesis (for review, see von Ballmoos et al., 2009). CF_o comprises four different subunit types, designated b (synonymously, I or AtpF), b′ (II or AtpG), c (III or AtpH), and a (IV or AtpI), and contains one each of subunits a, b, and b′ and a ring made up of 14 copies of subunit c. CF₁ comprises five different subunits, α (AtpA), β (AtpB), γ (AtpC), δ (AtpD), and ε (AtpE), and its subunit composition is α₃β₃γδε (for review, see von Ballmoos et al., 2009).The passage of protons through the CF_o motor drives rotation of the ring of c-subunits, which together form a rotor. The c-ring is connected to subunit γ, and rotation of γ causes conformational changes in the catalytic nucleotide-binding sites of the CF₁ motor, resulting in the synthesis and release of ATP (for review, see Okuno et al., 2011). This process is made possible by the fact that CF₁ and CF_o are physically connected by two stalks, a central one containing the ε- and γ-subunits and a peripheral one made up of δ, b, and b′ (for review, see Böttcher and Gräber, 2000; Weber, 2007). There are six nucleotide-binding sites in CF₁, one at each of the αβ-subunit interfaces about halfway along the vertical axis of the hexamer. Three of the sites are located primarily on the β-subunits and are catalytic; the other three are noncatalytic and probably regulatory. While the three-dimensional structure of the α₃β₃ hexamer in chloroplasts has been solved to a resolution of 3.2 Å (Groth and Pohl, 2001), the structure of the entire CF_o has not yet been determined. However, the conformation of the ring-forming part of CF_o from spinach (Spinacia oleracea) chloroplasts has been defined and found to consist of 14 c-units (Vollmar et al., 2009), whereas the c-ring of the ATP synthase from the cyanobacterium Spirulina platensis contains 15 units (Pogoryelov et al., 2009).Similar to other thylakoid multiprotein complexes like PSII and PSI as well as the cytochrome b₆f complex (Cyt b₆f), the assembly of the ATP synthase must be tightly regulated. Moreover, the variable stoichiometry of the constituents of F₁ (three α/β-subunits versus one each of γ, δ, and ε) and F_o (10–15 c-subunits versus one each of a, b, and b′) requires coordination of the expression of the corresponding genes. This is particularly important in eukaryotes, where the genes are located in different compartments, for instance, in the case of the cpATPase, in the plastid (for α, β, ε, a, b, and c) and the nucleus (for b′, γ, and δ).The assembly of ATP synthase has been most extensively studied in Saccharomyces cerevisiae mitochondria, leading to the identification of several factors involved in this process (for review, see Rak et al., 2009). Thus, three proteins in yeast are known to be involved in the assembly of the α₃β₃ hexamer of F₁. Atp11p (Ackerman and Tzagoloff, 1990a; Wang and Ackerman, 1996) and Atp12p (Ackerman and Tzagoloff, 1990a; Wang and Ackerman, 1998) code for mitochondrial proteins that interact with the β- and α-subunits, respectively, to promote their assembly into the oligomeric F₁-ATPase, and the absence of either protein causes the α- and β-subunits to aggregate into insoluble inclusion bodies in the mitochondrial matrix. Lack of the third protein, FORMATION OF MITOCHONDRIAL COMPLEXES1 (Fmc1p), is associated with aggregation of the α- and β-subunits under heat stress, suggesting that Fmc1p is required for correct folding of Atp12p at elevated temperatures (Lefebvre-Legendre et al., 2001). Originally, the c-ring was assumed to form spontaneously (Arechaga et al., 2002), but subsequent studies have indicated that the assembly of this structural component is also a protein-assisted process. Thus, Atp25p is required for both the synthesis of the c-subunit and its oligomerization into a ring structure of the proper size (Zeng et al., 2008). Moreover, Atp10p (Ackerman and Tzagoloff, 1990b), Atp23p (Osman et al., 2007), and OXIDASE ASSEMBLY1 (Oxa1p) (Jia et al., 2007) are involved in F_o assembly in yeast mitochondria.In prokaryotes, two ATP synthase assembly factors have been described in detail. The membrane protein insertase YidC belongs to the Oxa1 family, is required in vitro for the membrane insertion of subunit c, and assists in the formation of the c-ring from monomers (van der Laan et al., 2004; Kol et al., 2008). In bacterial genomes, the atp1/uncI genes typically precede the genes encoding the structural subunits of the F₁F_o-ATP synthase (for review, see Kol et al., 2008). Moreover, in Synechocystis sp. PCC6803, sll1321/atp1 is coordinately expressed with the seven other genes in the ATP synthase operon (Grossman et al., 2010), implying that Sll1321/Atp1 might have a function associated with the ATP synthase. The genes atp1 and uncI code for small proteins; for instance, Synechocystis sp. PCC6803 Sll1321 has 117 amino acids, and Escherichia coli UncI has 130 amino acids. The function of Atp1/UncI has long remained elusive because deletion of uncI in E. coli results merely in a slightly reduced growth yield (Gay, 1984), indicating that the protein is not essential for the formation of the F₁F_o-ATP synthase complex. Similarly, in the alkaliphilic Bacillus pseudofirmus OF4, Atp1/UncI is not absolutely required for ATP synthase function, and a B. pseudofirmus strain deleted for the atp1 gene could still grow nonfermentatively and its purified ATP synthase had a c-ring of normal size (Liu et al., 2013). Recently, a hybrid F₁F_o (F₁ from Bacillus PS3 and F_o from Propionigenium modestum) was expressed in E. coli. In this system, P. modestum Atp1/UncI was found to be indispensable for c-ring formation and coupled ATPase activity (Suzuki et al., 2007). Similarly, functional production of the Na⁺ F₁F_o-ATP synthase from Acetobacterium woodii in E. coli required the A. woodii atp1/uncI gene for proper assembly (Brandt et al., 2013). Moreover, because subunit c monomers, as well as assembled c-rings, can be copurified together with P. modestum UncI/Atp1 (Suzuki et al., 2007) and the oligomerization of P. modestum c-subunits into c₁₁-rings is mediated by Atp1/UncI in vitro (Ozaki et al., 2008), Atp1/UncI seems to play a role in c-ring assembly for some bacterial ATP synthases.In plants and green algae, regulation of the biogenesis of the cpATPase is well understood at the level of translation of CF₁ subunits (Drapier et al., 2007). Thus, synthesis of the nucleus-encoded subunit γ is required for sustained translation of the chloroplast-encoded subunit β, which in turn transactivates the translation of chloroplast-encoded subunit α. Translational down-regulation of subunit β or α, when not assembled, involves the 5′ untranslated regions (UTRs) of their own mRNAs, pointing to control at the level of translation initiation. In addition, a negative feedback exerted by α/β assembly intermediates on the translation of subunit β can be released when subunit γ assembles with α₃β₃ hexamers.Our knowledge of the nature of true assembly factors for the cpATPase is scarce. So far, only the ALBINO3 homolog Alb4 protein, which can functionally substitute for YidC in E. coli, has been shown to play a role in the biogenesis of the cpATPase, possibly by stabilizing or promoting the assembly of CF₁ during its attachment to the CF_o portion (Benz et al., 2009). Thus, Alb4-Oxa1p-YidC represents an ATP synthase assembly factor family that is conserved between prokaryotes, yeast, and plants. For the bacterial Atp1/UncI protein, one homolog exists in yeast, Vma21p, which is an integral membrane protein localized to the endoplasmic reticulum and is required for vacuolar H⁺-ATPase biogenesis (Graham et al., 1998).In this study, we have identified and characterized a knockout mutant for Arabidopsis (Arabidopsis thaliana) CGL160, a protein that displays moderate similarity to prokaryotic Atp1/UncI proteins in its C-terminal domain. AtCGL160 is required for the efficient assembly of the cpATPase, but lack of AtCGL160 in Arabidopsis has more severe effects on cpATPase assembly than those reported in the literature for inactivation of its prokaryotic relatives and can be located to the assembly of c-subunits into the membranous subcomplex. AtCGL160 physically interacts with the c-subunit of CF_o, and, interestingly, Atp1 can replace the C-terminal part of AtCGL160 in such interactions, indicating that the function of Atp1 and CGL160 proteins is conserved.     

Labile carbon retention compensates for CO2 released by priming in forest soils

Increase of belowground C allocation by plants under global warming or elevated CO₂ may promote decomposition of soil organic carbon (SOC) by priming and strongly affects SOC dynamics. The specific effects by priming of SOC depend on the amount and frequency of C inputs. Most previous priming studies have investigated single C additions, but they are not very representative for litterfall and root exudation in many terrestrial ecosystems. We evaluated effects of ¹³C‐labeled glucose added to soil in three temporal patterns: single, repeated, and continuous on dynamics of CO₂ and priming of SOC decomposition over 6 months. Total and ¹³C labeled CO₂ were monitored to analyze priming dynamics and net C balance between SOC loss caused by priming and the retention of added glucose‐C. Cumulative priming ranged from 1.3 to 5.5 mg C g^?1 SOC in the subtropical, and from ?0.6 to 5.5 mg C g^?1 SOC in the tropical soils. Single addition induced more priming than repeated and continuous inputs. Therefore, single additions of high substrate amounts may overestimate priming effects over the short term. The amount of added glucose C remaining in soil after 6 months (subtropical: 8.1–11.2 mg C g^?1 SOC or 41‐56% of added glucose; tropical: 8.7–15.0 mg C g^?1 SOC or 43–75% of glucose) was substantially higher than the net C loss due to SOC decomposition including priming effect. This overcompensation of C losses was highest with continuous inputs and lowest with single inputs. Therefore, raised labile organic C input to soils by higher plant productivity will increase SOC content even though priming accelerates decomposition of native SOC. Consequently, higher continuous input of C belowground by plants under warming or elevated CO₂ can increase C stocks in soil despite accelerated C cycling by priming in soils.     

The Effect of Rhamnolipid Biosurfactant Produced by <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> on Model Bacterial Strains and Isolates from Industrial Wastewater

In this study, the effect of rhamnolipid biosurfactant produced by Pseudomonas fluorescens on bacterial strains, laboratory strains, and isolates from industrial wastewater was investigated. It was shown that biosurfactant, depending on the concentration, has a neutral or detrimental effect on the growth and protein release of model Gram (+) strain Bacillus subtilis 168. The growth and protein release of model Gram (−) strain Pseudomonas aeruginosa 1390 was not influenced by the presence of biosurfactant in the medium. Rhamnolipid biosurfactant at the used concentrations supported the growth of some slow growing on hexadecane bacterial isolates, members of the microbial community. Changes in cell surface hydrophobicity and permeability of some Gram (+) and Gram (−) isolates in the presence of rhamnolipid biosurfactant were followed in experiments in vitro. It was found that bacterial cells treated with biosurfactant became more or less hydrophobic than untreated cells depending on individual characteristics and abilities of the strains. For all treated strains, an increase in the amount of released protein was observed with increasing the amount of biosurfactant, probably due to increased cell permeability as a result of changes in the organization of cell surface structures. The results obtained could contribute to clarify the relationships between members of the microbial community as well as suggest the efficiency of surface properties of rhamnolipid biosurfactant from Pseudomonas fluorescens making it potentially applicable in bioremediation of hydrocarbon-polluted environments.     

Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue

Objectives

To translocation (externalization) of phosphatidylserine lead at least the five negative effects observed during cells cryopreservation: hypoxia, increasing of intracellular Ca²⁺, osmotic disruption of cellular membranes, generation of reactive oxygen species (ROS) and lipid peroxidation. The aim of this study was to test the intensiveness of the phosphatidylserine translocation immediately after thawing and after 45 d xenografting of human ovarian tissue, which was either frozen just after operative removal from patient or cooled before cryopreservation to 5°C for 24 h and then frozen.

Materials and Methods

Ovarian fragments from twelve patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following four groups. Pieces of Group 1 (n=30) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n=30) after operation were cooled to 5°C for 24 h, then frozen after 24 h pre-cooling to 5°C, thawed and just after thawing their quality was analyzed. Group 3 pieces (n=30) were frozen immediately after operation without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was analyzed. Group 4 pieces (n=30) were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was analyzed. The effectiveness of the pre-freezing cooling of tissuewas evaluated by the development of follicles (histology) and by intensiveness of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide).

Results

For groups 1, 2, 3 and 4 the mean densities of follicles per 1 mm³ was 19.0, 20.2, 12.9, and 12.2, respectively (P_{1-2, 3-4} >0.1). For these groups, 99%, 98%, 88% and 90% preantral follicles, respectively were morphologically normal (P_{1-2, 3-4} >0.1). The FACS analysis showed significantly decreased intensiveness of translocation of phosphatidylserine after pre-cooling of frozen tissue (46.3% and 33.6% in Groups 2 and 4, respectively), in contrast with tissue frozen without pre-cooling (77.1% and 60.2 % in Groups 1 and 3, respectively, P_{1, 3-2, 4} <0.05).

Conclusions

Long time (24 h) cooling of ovarian tissue to 5°C before cryopreservation decreased translocation of phosphatidylserine that evidences about increases the viability of the cells in the tissue after thawing.     

Evaluation of the Possible Transmission of BSE and Scrapie to Gilthead Sea Bream (Sparus aurata)

In transmissible spongiform encephalopathies (TSEs), a group of fatal neurodegenerative disorders affecting many species, the key event in disease pathogenesis is the accumulation of an abnormal conformational isoform (PrP^Sc) of the host-encoded cellular prion protein (PrP^C). While the precise mechanism of the PrP^C to PrP^Sc conversion is not understood, it is clear that host PrP^C expression is a prerequisite for effective infectious prion propagation. Although there have been many studies on TSEs in mammalian species, little is known about TSE pathogenesis in fish. Here we show that while gilthead sea bream (Sparus aurata) orally challenged with brain homogenates prepared either from a BSE infected cow or from scrapie infected sheep developed no clinical prion disease, the brains of TSE-fed fish sampled two years after challenge did show signs of neurodegeneration and accumulation of deposits that reacted positively with antibodies raised against sea bream PrP. The control groups, fed with brains from uninfected animals, showed no such signs. Remarkably, the deposits developed much more rapidly and extensively in fish inoculated with BSE-infected material than in the ones challenged with the scrapie-infected brain homogenate, with numerous deposits being proteinase K-resistant. These plaque-like aggregates exhibited congophilia and birefringence in polarized light, consistent with an amyloid-like component. The neurodegeneration and abnormal deposition in the brains of fish challenged with prion, especially BSE, raises concerns about the potential risk to public health. As fish aquaculture is an economically important industry providing high protein nutrition for humans and other mammalian species, the prospect of farmed fish being contaminated with infectious mammalian PrP^Sc, or of a prion disease developing in farmed fish is alarming and requires further evaluation.     

Combined therapy with cyclophosphamide and DNA preparation inhibits the tumor growth in mice

Background

When cyclophosphamide and preparations of fragmented exogenous genomic double stranded DNA were administered in sequence, the regressive effect on the tumor was synergic: this combined treatment had a more pronounced effect than cyclophosphamide alone. Our further studies demonstrated that exogenous DNA stimulated the maturation and specific activities of dendritic cells. This suggests that cyclophosphamide, combined with DNA, leads to an immune response to the tumors that were grafted into the subjects post treatment.

Methods

Three-month old CBA/Lac mice were used in the experiments. The mice were injected with cyclosphamide (200 mkg per 1 kg body weight) and genomic DNA (of human, mouse or salmon sperm origin). The DNA was administered intraperitoneally or subcutaneously. After 23 to 60 days, one million tumor cells were intramuscularly grafted into the mice. In the final experiment, the mice were pre-immunized by subcutaneous injections of 20 million repeatedly thawed and frozen tumor cells. Changes in tumor growth were determined by multiplying the three perpendicular diameters (measured by caliper). Students' t-tests were used to determine the difference between tumor growth and average survival rate between the mouse groups and the controls.

Results

An analysis of varying treatments with cyclophosphamide and exogenous DNA, followed by tumor grafting, provided evidence that this combined treatment had an immunizing effect. This inhibitory effect in mice was analyzed in an experiment with the classical immunization of a tumor homogenate. The strongest inhibitory action on a transplanted graft was created through the following steps: cyclophosphamide at 200 mg/kg of body weight administered as a pretreatment; 6 mg fragmented exogenous DNA administered over the course of 3 days; tumor homogenate grafted 10 days following the final DNA injection.

Conclusion

Fragmented exogenous DNA injected with cyclophosphamide inhibits the growth of tumors that are grafted to mice after this combined treatment.