Two Mechanisms of Cc14-Induced Fatty Liver: Lipid Peroxidation Or Covalent Binding Studied in Cultured Rat Hepatocytes

With cultured hepatocytes it was studied whether CCl₄-induced inhibition of secretion of VLDL and HDL from liver cells is a consequence of covalent binding of CC1₄ metabolites (i.e. CO,; CC1,00) to cell constituents or of membrane damage by lipid peroxidation. Comparing the kinetics of inhibition of lipoprotein secretion with that of CCl₄-bioactivation it was found, that covalent binding of (^HC)-CC1₄ occurred at early time points (5 min) after CC1₄ administration and inhibited the lipoprotein secretion. At 100μM CC1₄ it was depressed by 53% within 60min. Incubations of CC1₄-treated cells with increasing concentrations of vitamin E blocked lipid peroxidation, but lipoprotein secretion was still inhibited. Piperonyl butoxid, a radical scavenger, protected against CCl₄-induced inhibition of lipoprotein section, lipid peroxidation and covalent binding.

These results show that during the early phases of CC1₄ poisoning fat accumulation is the consequence of covalent binding of CC1₄ metabolities to cell structures.     

Effect of Potassium Dihydrogen Phosphate and Bovine Manure Compost on the Degradation of Chlorothalonil in Soil

The effects of potassium dihydrogen phosphate (KH₂PO₄) and bovine manure compost (BMC) on the degradation and metabolism of chlorothalonil were examined in a sandy loam soil under laboratory conditions. In non-sterilized, non-amended soil, chlorothalonil degradation half-life was 8.8 days. However, it was up to 19.0 days in sterilized non-amended soil, suggesting that the degradation rate was about 2-fold slower than that in non-sterilized non-amended soil. Biological mechanisms accounted for 54.7% of chlorothalonil degradation in non-sterilized soil, indicating that the indigenous microorganisms in soil play an important role in the degradation process. In non-sterilized soil, the more acutely toxic metabolite, 4-OH-chlorothalonil (HTI), of chlorothalonil started forming after the second day of incubation. The concentration of HTI reached its maximum level (2.9 μ g g? 1) at 10 days after treatment, and no further degradation of HTI was observed in the following span of 20 days (10–30 days of incubation). However, the degradation rate of chlorothalonil was decreased substantially by the addition of KH₂PO₄ with a half-life of 17.5 days. No formation of HTI was observed before 10 days of incubation and no significant difference of the metabolite concentration at the end of experiment was observed between KH₂PO₄-amended and non-amended treatments, which denoted that the addition of KH₂PO₄ did not reduce the formation of HTI in the longer incubation course. In BMC-amended soil, the degradation rate was about 1.8 times faster than in non-amended soil. At 30 days of incubation, both chlorothalonil and HTI were degraded to a lower level in BMC-amended soil than in non-amended soil. The application of farm litter is a common fertilizing practice in vegetable fields in China, and thus this practice could not only improve soil fertility but also promote the removal of chlorothalonil and its metabolite HTI to further increase safety in crop rotations.     

Compartmentalisation and localisation of the translation initiation factor (eIF) 4F complex in normally growing fibroblasts

Previous observations of association of mRNAs and ribosomes with subcellular structures highlight the importance of localised translation. However, little is known regarding associations between eukaryotic translation initiation factors and cellular structures within the cytoplasm of normally growing cells. We have used detergent-based cellular fractionation coupled with immunofluorescence microscopy to investigate the subcellular localisation in NIH3T3 fibroblasts of the initiation factors involved in recruitment of mRNA for translation, focussing on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). We find that these proteins exist mainly in a soluble cytosolic pool, with only a subfraction tightly associated with cellular structures. However, this "associated" fraction was enriched in active "eIF4F" complexes (eIF4E.eIF4G.eIF4A.PABP). Immunofluorescence analysis reveals both a diffuse and a perinuclear distribution of eIF4G, with the perinuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear stain, partly coincident with vimentin intermediate filaments. All three proteins localise to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion protein vinculin.     

Spectroscopical identification of residues of subunit G of the yeast V-ATPase in its connection with subunit E

A critical point in the V₁ sector and entire V₁V_O complex is the interaction of stalk subunits G (Vma10p) and E (Vma4p). Previous work, using precipitation assays, has shown that both subunits form a complex. In this work, we have analysed the N-terminal segment of subunit G (G_1–59) of the V₁V_O ATPase from Saccharomyces cerevisiae by using nuclear magnetic resonance (NMR) spectroscopy. Analyses of ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra of G_1–59 in the absence and presence of the N-terminal peptides E_1–18 and E_18–38 as well as the produced and purified C-terminal segment (E_39–233) shows specific interactions only with the peptide fragment E_18–38. The binding of this peptide occurs via the residues M₁, V₂, S₃, and K₅ as well for V₂₂, S₂₃, K₂₄, A₂₅ and R₂₆ of G_1–59. The specific E_18–38/G_1–59 binding has been confirmed by fluorescence correlation spectroscopy data. The E_18–38 peptide has been studied by CD spectroscopy and NMR. The 3D structure of this peptide adopts a stable helix-hinge-helix formation in solution. A model structure of the E_18–38/G_1–59 complex reveals the orientation of E_18–38 relative to G_1–59 via salt-bridges of the polar residues and van der Waals forces at the very N-terminus of both segments.     

Linking integrin to IP(3) signaling is important for ovulation in Caenorhabditis elegans

Signals from germ and myoepithelial sheath cells initiate ovulation in Caenorhabditis elegans. The coordinated dilation and contraction of spermatheca lead to subsequent fertilization of oocyte. Either the dominant negative mutant pat-3 beta integrin or disruption of talin expression block ovulation. Talin loss-of-function uncovers roles in cell contractility and migration in C. elegans. Role of beta pat-3 integrins in development and function of Caenorhabditis elegans muscles and gonads, suggesting that the interaction between the cell and the extracellular matrix (ECM) is also important for ovulation. Here, we report that integrin plays an essential role in fertility via IP(3) signaling. Sterility caused by RNAi of pat-3 and ECM molecules was suppressed by increased IP(3) signaling. Our data suggest that the cell-ECM interaction controls ovulation via IP(3) signaling.     

Pex10p functions as an E3 ligase for the Ubc4p-dependent ubiquitination of Pex5p

The Saccharomyces cerevisiae (Sc) PTS1 import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and a Pex4p (Ubc10p)-dependent manner. Both of these modifications require the RING domain-containing protein Pex10p in vivo, but the actual role this protein plays in the ubiquitination of Pex5p has so far, remained enigmatic. Here, we report that the RING domain of Pex10p exhibits E₃ ligase activity in vitro, in combination with the human E₂ enzyme UbcH5a, a homologue of ScUbc4p, but not when ScPex4p was used as an E₂ enzyme in the reaction. We have further characterised Pex10p’s E₃ ligase activity using mutants designed to disturb this activity and show that Pex10p acts as the E₃ ligase for Ubc4p-dependent ubiquitination of Pex5p but not Pex4p-dependent ubiquitination in vivo. These data imply that the two distinct Pex5p modifications require different E₃ ligases, as well as different E₂ enzymes.     

Binding of Drosophila maternal Mamo protein to chromatin and specific DNA sequences

Alterations in chromatin structure dynamically occur during germline development in Drosophila and are essential for the production of functional gametes. We had previously reported that the maternal factor Mamo, which contains both a BTB/POZ domain and C₂H₂ zinc-finger domains and is enriched in primordial germ cells (PGCs), is required for the regulation of meiotic chromatin structure and the production of functional gametes. However, the molecular mechanisms by which Mamo regulates germline development remained unclear. To evaluate the molecular function of Mamo protein, we have investigated the binding of Mamo to chromatin and DNA sequences. Our data show that Mamo binds to chromatin and specific DNA sequences, particularly the polytene chromosomes of salivary gland cells. Overexpression of Mamo affected the organization of polytene chromosomes. Reduction in maternal Mamo levels impaired the formation of germline-specific chromatin structures in PGCs. Furthermore, we found that the zinc-finger domains of Mamo directly bind to specific DNA sequences. Our results suggest that Mamo plays a role in regulating chromatin structure in PGCs.     

Inositolpolyphosphates and their binding proteins —a short review

Since 1983, when it was discovered that inositol 1,4,5-trisphosphate can act as second messenger to release Ca²⁺ from the endoplasmic reticulum, widespread research has focused on the phosphatidylinositol signalling transduction pathway and the host of inositolphosphates formed intracellularly after stimulation thereof. Although the polyphosphates, inositoltetrakisphosphate (InsP₄) and inositolhexakisphosphate (InsP₆), have received their share of attention, a definite physiological role has not been ascribed to them as yet. Different binding proteins for these two polyphosphates have been demonstrated, especially in brain tissue, indicating their possible importance in the cell.InsP₆ is known as one of nature's most powerful antioxidants and has already been demonstrated to possess the abilities to be of use in the industry as well as in the medical profession. As its natural actions are poorly understood and its possible side-effects have not been widely investigated, basic research regarding its cellular and subcellular activities is urgently called for.Recipient of Servier Investigator Award     

Comparison of Site I auxin binding and a 22-kilodalton auxin-binding protein in maize

Several properties of a 43-kilodalton (kDa) auxin-binding protein (ABP) having 22-kDa subunits are shared by a class of auxin binding designated Site I. The spatial distribution of the ABP in the maize (Zea mays L.) mesocotyl corresponds with the distribution of growth induced by naphthalene-1-acetic acid and with the distribution of Site I binding as previously shown by J.D. Walton and P.M. Ray (1981, Plant Physiol. 68, 1334–1338). The greatest abundance of both ABP and Site I activity is at the apical region of the mesocotyl. The ABP and Site I activity co-migrate in isopycnic centrifugation with the endoplasmic-reticulum marker, cytochrome-c reductase. Red light, at low and high fluence, far-red and white light were used to alter the elongation rate of apical 1-cm sections of etiolated maize mesocotyls, the amount of auxin binding, and the abundance of the ABP. Relative changes in auxin binding and the ABP were correlated, but the growth rate was not always correlated with the abundance of the ABP.Abbreviations ABP auxin-binding protein - ER endoplasmic reticulum - FR far-red light - kDa kilodalton - NAA naphthalene-1-acetic acid - PM plasma membrane - R red light - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

The influence of inorganic silicon (Si) on pituitary-thyroid axis

The influence of silicon-treatment on the levels of TSH and thyroid hormones was studied in rats. Concentrations of thyrotropin (TSH), triiodothyronine (T₃), and thyroxine (T₄) were estimated in sera of rats receiving per os a soluble silicon compound—sodium metasilicate nonahydrate (Na₂SiO₃·9H₂O), dissolved in the animals' drinking water. An increase in the TSH level in the tested group was observed, without statistically significant differences in T₃ and T₄ concentrations between the two groups of animals. The results provide evidence for the influence of silicon on the endocrine balance. They could also prove that this chemical element is capable of modifying the rate of some hormones' synthesis.     

Cloning of Dictyostelium eIF6 (p27) and mapping its nucle(ol)ar localization subdomains

Eukaryotic translation initiation factor 6 (eIF6), also termed p27^BBP, is an evolutionary conserved regulator of ribosomal function. The protein is involved in maturation and/or export from the nucleus of the 60S ribosomal subunit. Regulated binding to and release from the 60S subunit also regulates formation of 80S ribosomes, and thus translation. The protein is also found in hemidesmosomes of epithelial cells expressing β4 integrin and is assumed to regulate cross-talk between β4 integrin, intermediate filaments and ribosomes. In the present study we show that the Dictyostelium eIF6 (also called p27^BBP) gene is expressed during growth, down-regulated during the first hours of starvation, and up-regulated again at the end of aggregation. Phagocytosis, and to a lesser extent pinocytic uptake of axenic medium, stimulate gene expression in starving cells. The eIF6 gene is present in single copy and its ablation is lethal. We utilized the green fluorescent protein (GFT) as fusion protein marker to investigate sequences responsible for eIF6 subcellular localization. The protein is found both in cytoplasm and nucleus, and is enriched in nucleoli. Deletion sequence analysis shows that nucle(ol)ar localization sequences are located within the N- and C-terminal subdomains of the protein.     

The Impact of Bioaugmentation on Metal Cyanide Degradation and Soil Bacteria Community Structure

Metal cyanides are significant contaminants of many soils found at the site of former industrial activity. In this study we isolated bacteria capable of degrading ferric ferrocyanide and K₂Ni(CN)₄. One of these bacteria a Rhodococcus spp. was subsequently used to bioaugment a minimal medium broth, spiked with K₂Ni(CN)₄, containing 1 g of either an uncontaminated topsoil or a former coke works site soil. Degradation of the K₂Ni(CN)₄ was observed in both soils, however, bioaugmentation did not significantly impact the rate or degree of K₂Ni(CN)₄ removal. Statistical analysis of denaturing gradient gel electrophoresis profiles showed that the topsoil bacterial community had a higher biodiversity, and its structure was not significantly affected by either K₂Ni(CN)₄ or bioaugmentation. In contrast, profiles from the coke works site indicated significant changes in the bacterial community in response to these additions. Moreover, in both soils although bioaugmentation did not affect rates of biodegradation the Rhodococcus spp. did become established in the communities in broths containing both top and coke works soil. We conclude that bacterial communities from contaminated soils with low biodiversity are much more readily perturbed through interventions such as contamination events or bioaugmentation treatments and discuss the implications of these findings for bioremediation studies.     

Synthesis and characterization of two new triazolate-aliphatic dicarboxylate bridged Zn(II) coordination polymers based on 2D layer motifs with different crystal packing

Two new zinc(II)-triazole-aliphatic dicarboxylate coordination polymers, [Zn(trz)(Hsuc)]_n (1), [Zn₂(trz)₂(tar)]_n (2), have been hydrothermally synthesized by reaction of Zn salt, Htrz with H₂suc and H₂tar, respectively (Htrz = 1,2,4-triazole, H₂suc = succinic acid, H₂tar = tartaric acid).Their structures were determined by single-crystal X-ray diffraction analyses and further characterized by X-ray powder diffraction, elemental analyses, IR spectra and TG analyses. Compound 1 displays a 2D layer structure containing {[Zn₄(trz)₄]⁴⁺}_n layers decorated by the suc ligand. Compound 2 is in a 3D structure formed by the interconnection of 2D {[Zn₄(trz)₄]⁴⁺}_n layers with tar ligand, resulting a 3,4-connected topological network. Due to the different coordination mode and conformation of aliphatic carboxylate ligand, the similar 2D {[Zn₄(trz)₄]⁴⁺}_n layers stack in the -AAA- fashion in 1, while the {[Zn₄(trz)₄]⁴⁺}_n layers hold together in the -ABAB- stacking sequence in 2. Additionally, the two compounds show strong fluorescence in the solid state at room temperature.     

Al4(C5Me4H)4: Structure, reactivity and bonding

The synthesis of Al₄R₄ (R = C₅(CH₃)₄H) (3) and the tetrahedral structure in the solid state are described. These results as well as the ²⁷Al NMR spectra of 3 in solution are in line with the data obtained from DFT calculations. These calculations also support the failed observation of a monomeric AlR species in solution. Monomeric and tetrameric molecules of 3 are discussed with respect to those of (AlCp^∗)₄ (1) and (AlCp)₄ (2). The increasing Al-Al bond strength from 1 to 3 and 2 from X-ray data is also supported by structural and energetic results from DFT calculations.     

Reassimilation of carbon dioxide by Flaveria (Asteraceae) species representing different types of photosynthesis

The capability to reassimilate CO₂ originating from intracellular decarboxylating processes connected with the photorespiratory glycolate pathway and-or decarboxylation of C₄ acids during C₄ photosynthesis has been investigated with four species of the genus Flaveria (Asteraceae). The C₃-C₄ intermediate species F. pubescens and F. anomala reassimilated CO₂ much more efficiently than the C₃ species F. cronquistii and, with respect to this feature, behaved similarly to the C₄ species F. trinervia. Therefore, under atmospheric conditions the intermediate species photorespired with rates only between 10–20% of that measured with F. cronquistii. At low oxygen concentrations (1,5%) the reassimilation potential of F. anomala approached that of F. trinervia and was distinct from that found with F. pubescens. The data are discussed with respect to a possible sequence of events during evolution of C₄ photosynthesis. If compared with related data for C₃-C₄ intermediate species from other genera they support the hypothesis that, during evolution of C₄ photosynthesis, an efficient capacity for CO₂ reassimilation evolved prior to a CO₂-concentrating mechanism.Abbreviations C₃, C₄ assimilated CO₂ initially found in 3-phosphoglycerate (C₃) or malate and aspartate (C₄) - D reassimilation coefficient - R _n , R _t net, total CO₂ evolution as measured with 0.03 and 3% CO₂, respectively - RuBP ribulose-1,5-bisphosphate - TPS true photosynthesis     

Promotion of flowering in apple trees with gibberellin A4 and C-3 epi-gibberellin A4

The proportion of spurs flowering on apple trees (Malus domestica Borkh. cv Golden Delicious) displaying a high degree of alternate-year flowering was increased in the off year by gibberellin A₄ (GA₄) and C-3 epi-GA₄ applied in the previous year. When applied 4.5 weeks after anthesis amounts of GA₄ ranging from 3 to 300 g per spur and 25 or 50 g of C-3 epi-GA₄ per spur were effective. Treatments with GA₄ made seven weeks after anthesis were less effective. A combination of 30 g GA₄ and 30 g zeatin (6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine) promoted flowering at both treatment times, and tended to be more effective than GA₄ alone.Abbreviation GA gibberellin or gibberellin-like substance Contribution No. 618     

Synthesis, crystal structure, magnetic property and oxidative DNA cleavage activity of an octanuclear copper(II) complex showing water-perchlorate helical network

A new octanuclear copper(II) complex has been synthesized and structurally characterized by X-ray crystallography: [Cu(8)(HL)(4)(OH)(4)(H(2)O)(2)(ClO(4))(2)].(ClO(4))(2).2H(2)O (1) (H(3)L=2,6-bis(hydroxyethyliminoethyl)-4-methyl phenol). The complex is formed by the linkage of two terminal bimetallic cationic units and a tetranuclear mu(3)-hydroxo bridged dicubane core by a very short intramolecular hydrogen bond (O-H...O, 1.48(3)A and the angle 175 degrees). The coordination sphere of the terminal copper atoms is square pyramidal, the apical positions being occupied by water and a perchlorate ion. Complex 1 self-assembles to form a new type of water-perchlorate helical network [(H(2)O)(2)(ClO(4))](infinity) involving oxygen atoms of coordinated perchlorate ion and the two lattice water molecules through hydrogen-bonding interaction. The variable temperature-dependent susceptibility measurement (2-300K) of 1 reveals a strong antiferromagnetic coupling, J(1)=-220cm(-1) and J(2)=-98cm(-1) (J(1) and J(2) representing the exchange constant within [Cu(2+)](4) and [Cu(2+)](2) units, respectively). The complex binds to double-stranded supercoiled plasmid DNA giving a K(app) value of 1.2x10(7)M(-1) and displays efficient oxidative cleavage of supercoiled DNA in the presence of H(2)O(2) following a hydroxyl radical pathway.