Decreased abundance and diversity of culturable Pseudomonas spp. populations with increasing copper exposure in the sugar beet rhizosphere

Recent studies have indicated that culturable bacteria constitute highly sensitive bioindicators of metal-induced stress in soil. We report the impact of different copper exposure levels characteristic of contaminated agricultural soils on culturable Pseudomonas spp. in the rhizosphere of sugar beet. We observed that the abundance of Pseudomonas spp. was much more severely affected than that of the general population of culturable heterotrophic bacteria by copper. For diversity assessment, Pseudomonas isolates were divided into operational taxonomic units based on amplified ribosomal DNA restriction analysis and genomic PCR fingerprinting by universally primed PCR. Copper significantly decreased the diversity of Pseudomonas spp. in the rhizosphere and significantly increased the frequency of copper-resistant isolates. Concomitant chemical and biological analysis of copper in the rhizosphere and in bulk soil extracts indicated no rhizosphere effect and a relatively low copper bioavailability in the studied soil, suggesting that the observed effects of copper may occur at lower total concentrations in other soils. We conclude that culturable Pseudomonas sensu stricto constitutes a highly sensitive and relevant bioindicator group for the impact of copper in the rhizosphere habitat, and suggest that continued application of copper to agricultural soils poses a significant risk to successful rhizosphere colonization by Pseudomonas spp.     

Redox thermodynamics, acid-base equilibria and salt-induced effects for the cucumber basic protein. General implications for blue-copper proteins

 The reduction potential of the basic blue-copper protein from cucumber peels (CBP) was determined through voltammetric techniques in different conditions of temperature, pH and ionic composition of the medium. The most notable properties of CBP include a positive entropy change upon reduction, a low-pH protonation and detachment of a metal-binding histidine in the reduced protein, and specific binding interactions with a number of anions present in common laboratory buffers, which influence to some extent the redox thermodynamics. The enthalpy and entropy changes accompanying reduction of the Cu(II) center were compared with those for other blue-copper proteins and correlated with spectroscopic data, structural properties and theoretical calculations. This allows some general considerations to be offered regarding the determinants of the reduction potential in this protein class. It emerges, in line with previous studies of the electronic structure of blue-copper sites, that the enthalpic contribution to the reduction potential is mainly modulated by the metal-binding interactions in the trigonal N₂S ligand set, and particularly by the Cu-cysteinate bond, while the entropy term is mainly affected by solvation properties and possibly by the weak axial bond to copper. The role of solvent exposure of the metal site in the active-site protonations in reduced blue-copper proteins is discussed. Finally, it is shown that the Nernst-Debye-Huckel model qualitatively accounts for the ionic strength dependence of the reduction potential. Received: 20 December 1996 / Accepted: 26 March 1997     

Applications of primary human hepatocytes in the evaluation of pharmacokinetic drug-drug interactions: Evaluation of model drugs terfenadine and rifampin

The utility of primary human hepatocytes in the evaluation of drug-drug interactions is being investigated in our laboratories. Our initial approach was to investigate whether drug-drug interactions observed in humans in vivo could be reproduced in vitro using human hepatocytes. Two model drugs were studied: terfenadine and rifampin, representing compounds subjected to drug-drug interactions via inhibitory and induction mechanisms, respectively. Terfenadine was found to be metabolized by human hepatocytes to C-oxidation and N-dealkylation products as observed in humans in vivo. Metabolism by human hepatocytes was found to be inhibited by drugs which are known to be inhibitory in vivo, Ki values for the various inhibitors were derived from the in vitro metabolism data, resulting in the following ranking of inhibitory potency: For the inhibition of C-oxidation, ketoconazole > itraconazole > cyclosporin ~ troleandomycin > erythromycin > naringenin. For the inhibition of N-dealkylation, itraconazole ketoconazole > cyclosporin naringenin erythromycin troleandomycin. Rifampin induction of CYP3A, a known effect of rifampin in vivo, was also reproduced in primary human hepatocytes. Induction of CYP3A4, measured as testosterone 6-hydroxylation, was found to be dose-dependent, treatment duration-dependent, and reversible. The induction effect of rifampin was observed in hepatocytes isolated from all 7 human donors studied, with ages ranging from 1.7 to 78 years. To demonstrate that the rifampin-induction of testosterone 6-hydroxylation could be generalized to other CYP3A4 substrates, we evaluated the metabolism of another known substrate of CYP3A4, lidocaine. Dose-dependent induction of lidocaine metabolism by rifampin is observed. Our results suggest that primary human hepatocytes may be a useful experimental system for preclinical evaluation of drug-drug interaction potential during drug development, and as a tool to evaluate the mechanism of clinically observed drug-drug interactions.     

Characteristics of Arsenic Accumulation by Pteris and non-Pteris Ferns

This research was conducted to understand the mechanisms of arsenic hyperaccumulation in Pteris vittata by comparing the characteristics of arsenic accumulation in Pteris and non-Pteris ferns. Seven Pteris (P.vittata, P. Cretica Rowerii, P. Cretica Parkerii, P. Cretica Albo-lineata, P. Quadriavrita, P. Ensiformis and P. Dentata) and six non-Pteris (Arachnoides simplicor, Didymochlaena truncatula, Dryopteris atrata, Dryopteris erythrosora, Cyrtomium falcatum, and Adiantum hispidulum) ferns were exposed to 0, 1 and 10 mgL⁻¹ arsenic as sodium arsenate for 14-d in hydroponic systems. As a group, the Pteris ferns were more efficient in arsenic accumulation than the non-Pteris ferns, with P. vittata being the most efficient followed by P. cretica. When exposed to 10 mg L⁻¹ As, arsenic concentrations in the fronds and roots of P. vittata were 1748 and 503 mg kg⁻¹. Though not all Pteris ferns were efficient in accumulating arsenic, none of the non-Pteris ferns was an efficient As accumulator (the highest concentration being 452 mg kg⁻¹). The fact that frond arsenic concentrations in the control were highly correlated with those exposed to As (r ² = 0.76–0.87) may suggest that they may be used as a preliminary tool to screen potential arsenic hyperaccumulators. Our research confirms that the ability of P. vittata to translocate arsenic from the roots to the fronds (73–77% As in the fronds), reduce arsenate to arsenite in the fronds (>50% AsIII in the fronds), and maintain high concentrations of phosphate in the roots (48–53% in the roots) all contributed to its arsenic tolerance and hyperaccumulation.     

低温胁迫对辣子草水浸提液化感作用的影响

采用蚕豆根尖细胞微核技术,研究了低温胁迫下辣子草（Gatinsoga parviflora Cav．）水浸提液的化感作用。结果表明：常温条件下,辣子草水浸提液使蚕豆根尖细胞有丝分裂指数下降,微核率和畸变率增大;低温处理后辣子草水浸提液对有丝分裂指数表现为“低浓度促进,高浓度抑制”效应,但显著提高了微核率和畸变率。这说明低温胁迫改变了辣子草的化感作用。     

Cu(II) complexes with square pyramidal (N2S)CuCl2 chromophore: Jahn-Teller distortion and subsequent effect on spectral and structural properties

One monomeric neutral Cu(II) complex [(pmtpm)CuCl₂] (1) is reported by Lindoy and Livingstone [8]. Two new complexes namely, μ-Cl bridged binuclear Cu(II) complex [{(pmtpm)Cu(Cl)}₂ μ-Cl](ClO₄) (2) and a bis μ-Cl bridged binuclear Cu(II) complex [{(pmtpm)Cu}₂(μ-Cl)₂](ClO₄)₂ (3) derived from a tridentate Schiff base ligand, 2-pyridyl-N-(2′-methylthiophenyl)methyleneimine (pmtpm) were synthesized and characterized by various spectroscopic methods and by X-ray crystallography. (N₂S)CuCl₂ chromophore(s) of distorted square pyramidal coordination geometries around Cu(II) ion(s) have been observed for all the complexes 1-3. The equatorial sites of the square plane comprise two N and a thioether S donor atoms of the pmtpm ligand as well as one Cl⁻ ion (terminal in 1 and 2, and bridging in 3) while the remaining axial site is occupied by a terminal Cl⁻ ion (for 1) or a bridging Cl⁻ ion (for 2 and 3). The equatorial Cu-Cl distances are much shorter [1: 2.2511(4) Å, 2: 2.2307(12) Å, 3: 2.2513(12) Å] than the axial Cu-Cl distances [1: 2.4394(4) Å, 2: 2.5597(9) Å, 3: 2.7037(12) Å]. The correlation of an axial Cu-Cl bond elongation with a lower g_|| value in the solid state EPR spectrum and a blue shifted ligand field transition in the solid and solution phase absorption spectrum has been observed.     

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target <Emphasis Type="Italic">lox</Emphasis>-Sites

Cre/lox recombination in vivo has become an important tool to induce chromosomal rearrangements like deletions. Using a combination of Ds transposition and Cre/lox recombination in two independent experiments on chromosomes 6 and 7 of tomato, two sets of somatic deletions up to a size of 200 kb were obtained. The efficiency of somatic deletion decreased with increasing deletion size. The largest germinally transmitted deletion had a size of only 55 kb. The results show that Cre-mediated deletion in somatic cells is less efficient when the lox sites are separated over larger distances. A further drop of the deletion efficiency after germinal transmission of the larger deletions can be explained by the probable loss of genes that are of vital importance to gametophyte function. Plasmid rescue of an 8.4 kb circularised deleted DNA showed that the Cre-mediated deletion takes place in tomato as expected. Since the circular Cre-deleted DNA could only be PCR amplified in plant cells where the deletion was not complete, the double-stranded DNA circle is assumed to be instable.     

Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells

Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death ) and inhibits the integration of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways.