Stereoselective Interaction Between Tetrahydropalmatine Enantiomers and CYP Enzymes in Human Liver Microsomes

Tetrahydropalmatine (THP), with one chiral center, is an alkaloid that possesses analgesic and many other pharmacological actives. The aim of the present study is to investigate stereoselective metabolism of THP enantiomers in human liver microsomes (HLM) and elucidate which cytochrome P450 (CYP) isoforms contribute to the stereoselective metabolism in HLM. Additionally, the inhibitions of THP enantiomers on activity of CYP enzymes are also investigated. The results demonstrated that (+)‐THP was preferentially metabolized by HLM. Ketoconazole (inhibitor of CYP3A4/5) inhibited metabolism of (?)‐THP or (+)‐THP at same degree, whereas the inhibition of fluvoxamine (inhibitor of CYP1A2) on metabolism of (+)‐THP was greater than that of (?)‐THP; moreover, the metabolic rate of (+)‐THP was 5.3‐fold of (?)‐THP in recombinant human CYP1A2. Meanwhile, THP enantiomers did not show obvious inhibitory effect on the activity of various CYP isoforms (CYP1A2, 2A6, 2C8, 2C9, 2C19, 2E1, and 3A4/5), whereas (?)‐THP, but not (+)‐THP, significantly inhibited the activity of CYP2D6 with the Ki value of 6.42 ± 0.38 μM. The results suggested that THP enantiomers were predominantly metabolized by CYP3A4/5 and CYP1A2 in HLM, and (+)‐THP was preferentially metabolized by CYP1A2, whereas CYP3A4/5 contributed equally to metabolism of (?)‐THP or (+)‐THP. Besides, the inhibition of CYP2D6 by (?)‐THP may cause drug–drug interaction, which should be considered. Chirality 25:43–47, 2013. © 2012 Wiley Periodicals, Inc.     

Structure and function of the CBL–CIPK Ca2+-decoding system in plant calcium signaling

Calcium is a crucial messenger in many growth and developmental processes in plants. The central mechanism governing how plant cells perceive and respond to environmental stimuli is calcium signal transduction, a process through which cellular calcium signals are recognized, decoded, and transmitted to elicit downstream responses. In the initial decoding of calcium signals, Ca²⁺ sensor proteins that bind Ca²⁺ and activate downstream signaling components are implicated, thereby regulating specific physiological and biochemical processes. After calcineurin B-like proteins (CBLs) sense these Ca²⁺ signatures, these proteins interact selectively with CBL-interacting protein kinases (CIPKs), thereby forming CBL/CIPK complexes, which are involved in decoding calcium signals. Therefore, specificity, diversity, and complexity are the main characteristics of the CBL-CIPK signaling system. However, additional CBLs, CIPKs, and CBL/CIPK complexes remain to be identified in plants, and the specific functions of their abiotic and biotic stress signaling will need to be further dissected. Therefore, a much-needed synthesis of recent findings is important to further the study of CBL-CIPK signaling systems. Here, we review the structure of CBLs and CIPKs, discuss the current knowledge of CBL–CIPK pathways that decode calcium signals in Arabidopsis, and link plant responses to a variety of environmental stresses with specific CBL/CIPK complexes. This will provide a foundation for future research on genetically engineered resistant plants with enhanced tolerance to various environmental stresses.     

The Possible Structural Models for Polyglutamine Aggregation: A Molecular Dynamics Simulations Study

Abstract

Huntington's disease is a neurodegenerative disorder caused by a polyglutamine (polyQ) expansion near the N-terminus of huntingtin. Previous studies have suggested that polyQ aggregation occurs only when the number of glutamine (Q) residues is more than 36-40, the disease threshold. However, the structural characteristics of polyQ nucleation in the very early stage of aggregation still remain elusive. In this study, we designed 18 simulation trials to determine the possible structural models for polyQ nucleation and aggregation with various shapes and sizes of initial β-helical structures, such as left-handed circular, right-handed rectangular, and left- and right-handed triangular. Our results show that the stability of these models significantly increases with increasing the number of rungs, while it is rather insensitive to the number of Qs in each rung. In particular, the 3-rung β-helical models are stable when they adopt the left-handed triangular and right-handed rectangular conformations due to the fact that they preserve high β-turn and β-sheet contents, respectively, during the simulation courses. Thus, we suggested that these two stable β-helical structures with at least 3 rungs might serve as the possible nucleation seeds for polyQ depending on how the structural elements of β-turn and β-sheet are sampled and preserved during the very early stage of aggregation.     

Branched Nanowire Based Guanine Rich Oligonucleotides

Abstract

Self-assembly and aggregation of guanine rich sequences can provide useful insights into DNA nanotechnology and telomeric structure and function. In this paper, we designed a guanine rich sequence d(GGCGTTTTGCGG). We found that it can form stable structure in appropriate condition and it exhibits an anomalous CD spectra. This structures can be imaged in ambient environment with a Nanoscope III AFM (Digital Instruments). We found it forms branch structure and long multistrand DNA nanowire after incubation at 37°C for 612 hours in 25 mM TE (pH=8.0) + 5 mM Mg²⁺ + 50 mM K⁺. The ability to self-assemble into branches and long wires not only clearly demonstrate its potential as scaffold structures for nanotechnology, but also give aids to understand telomeric structure further. We have proposed a model to explain how these structures formed.     

Identification of DNA-binding proteins by incorporating evolutionary information into pseudo amino acid composition via the top-n-gram approach

DNA-binding proteins are crucial for various cellular processes and hence have become an important target for both basic research and drug development. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to establish an automated method for rapidly and accurately identifying DNA-binding proteins based on their sequence information alone. Owing to the fact that all biological species have developed beginning from a very limited number of ancestral species, it is important to take into account the evolutionary information in developing such a high-throughput tool. In view of this, a new predictor was proposed by incorporating the evolutionary information into the general form of pseudo amino acid composition via the top-n-gram approach. It was observed by comparing the new predictor with the existing methods via both jackknife test and independent data-set test that the new predictor outperformed its counterparts. It is anticipated that the new predictor may become a useful vehicle for identifying DNA-binding proteins. It has not escaped our notice that the novel approach to extract evolutionary information into the formulation of statistical samples can be used to identify many other protein attributes as well.     

5-Aminolevulinic Acid Ameliorates the Growth, Photosynthetic Gas Exchange Capacity, and Ultrastructural Changes Under Cadmium Stress in Brassica napus L.

Heavy-metal toxicity in soil is one of the major constraints for oilseed rape (Brassica napus L.) production. One of the best ways to overcome this constraint is the use of growth regulators to induce plant tolerance. Response to cadmium (Cd) toxicity in combination with a growth regulator, 5-aminolevulinic acid (ALA), was investigated in oilseed rape grown hydroponically in greenhouse conditions under three levels of Cd (0, 100, and 500 μM) and three levels of foliar application of ALA (0, 12.5, and 25 mg l^?1). Cd decreased plant growth and the chlorophyll concentration in leaves. Foliar application of ALA improved plant growth and increased the chlorophyll concentration in the leaves of Cd-stressed plants. Significant reductions in photosynthetic parameters were observed by the addition of Cd alone. Application of ALA improved the net photosynthetic and gas exchange capacity of plants under Cd stress. ALA also reduced the Cd content in shoots and roots, which was elevated by high concentrations of Cd. The microscopic studies of leaf mesophyll cells under different Cd and ALA concentrations showed that foliar application of ALA significantly ameliorated the Cd effect and improved the structure of leaf mesophyll cells. However, the higher Cd concentration (500 μM) could totally damage leaf structure, and at this level the nucleus and intercellular spaces were not established as well; the cell membrane and cell wall were fused to each other. Chloroplasts were totally damaged and contained starch grains. However, foliar application of ALA improved cell structure under Cd stress and the visible cell structure had a nucleus, cell wall, and cell membrane. These results suggest that under 15-day Cd-induced stress, application of ALA helped improve plant growth, chlorophyll content, photosynthetic gas exchange capacity, and ultrastructural changes in leaf mesophyll cells of the rape plant.     

The critical soil P levels for crop yield, soil fertility and environmental safety in different soil types

Background and aims

Sufficient soil phosphorus (P) is important for achieving optimal crop production, but excessive soil P levels may create a risk of P losses and associated eutrophication of surface waters. The aim of this study was to determine critical soil P levels for achieving optimal crop yields and minimal P losses in common soil types and dominant cropping systems in China.

Methods

Four long-term experiment sites were selected in China. The critical level of soil Olsen-P for crop yield was determined using the linear-plateau model. The relationships between the soil total P, Olsen-P and CaCl₂-P were evaluated using two-segment linear model to determine the soil P fertility rate and leaching change-point.

Results

The critical levels of soil Olsen-P for optimal crop yield ranged from 10.9 mg kg^?1 to 21.4 mg kg^?1, above which crop yield response less to the increasing of soil Olsen-P. The P leaching change-points of Olsen-P ranged from 39.9 mg kg^?1 to 90.2 mg kg^?1, above which soil CaCl₂-P greatly increasing with increasing soil Olsen-P. Similar change-point was found between soil total P and Olsen-P. Overall, the change-point ranged from 4.6 mg kg^?1 to 71.8 mg kg^?1 among all the four sites. These change-points were highly affected by crop specie, soil type, pH and soil organic matter content.

Conclusions

The three response curves could be used to access the soil Olsen-P status for crop yield, soil P fertility rate and soil P leaching risk for a sustainable soil P management in field.     

Effect of aboveground intervention on fine root mass, production, and turnover rate in a Chinese cork oak (Quercus variabilis Blume) forest

Aims

Fine root is an important part of the forest carbon cycle. The growth of fine roots is usually affected by forest intervention. This study aims to investigate the fine root mass, production, and turnover in the disturbed forest.

Methods

The seasonal and vertical distributions of fine root (diameter ≤2 mm) were measured in a Chinese cork oak (Quercus variabilis Blume) forest. The biomass and necromass of roots with diameters ≤1 mm and 1-2 mm in 0-40 cm soil profiles were sampled by using a sequential soil coring method in the stands after clear cutting for 3 years, with the stands of the remaining intact trees as the control.

Results

The fine root biomass (FRB) and fine root necromass (FRN) varied during the growing season and reached their peak in August. Lower FRB and higher FRN were found in the clear cutting stands. The ratio between FRN and FRB increased after forest clear cutting compared with the control and was the highest in June. The root mass with diameter ≤1 mm was affected proportionately more than that of diameter 1-2 mm root. Clear cutting reduced FRB and increased FRN of roots both ≤1 mm and 1-2 mm in diameter along the soil depths. Compared with the control, the annual fine root production and the average turnover rate decreased by 30.7 % and 20.7 %, respectively, after clear cutting for 3 years. The decline of canopy cover contributed to the dramatic fluctuation of soil temperature and moisture from April to October. With redundancy discriminate analysis (RDA) analysis, the first axis was explained by soil temperature (positive) and moisture (negative) in the control stands. Aboveground stand structure, including canopy cover, sprout height, and basal area, influenced FRB and FRN primarily after forest clear cutting.

Conclusions

This study suggested that the reduction of fine root biomass, production, and turnover rate can be attributed to the complex changes that occur after forest intervention, including canopy damage, increased soil temperature, and degressive soil moisture.     

Fusaric acid induction of programmed cell death modulated through nitric oxide signalling in tobacco suspension cells

Fusaric acid (FA) is a nonhost-selective toxin mainly produced by Fusarium oxysporum, the causal agent of plant wilt diseases. We demonstrate that FA can induce programmed cell death (PCD) in tobacco suspension cells and the FA-induced PCD is modulated by nitric oxide (NO) signalling. Cells undergoing cell death induced by FA treatment exhibited typical characteristics of PCD including cytoplasmic shrinkage, chromatin condensation, DNA fragmentation, membrane plasmolysis, and formation of small cytoplasmic vacuoles. In addition, caspase-3-like activity was activated upon the FA treatment. The process of FA-induced PCD was accompanied by a rapid accumulation of NO in a FA dose-dependent manner. Pre-treatment of cells with NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) or NO synthase inhibitor N ^G-monomethyl-arginine monoacetate (L-NMMA) significantly reduced the rate of FA-induced cell death. Furthermore, the caspase-3-like activity and the expression of PAL and Hsr203J genes were alleviated by application of cPTIO or L-NMMA to FA-treated tobacco cells. This indicates that NO is an important factor involved in the FA-induced PCD. Our results also show that pre-treatment of tobacco cells with a caspase-3-specific inhibitor, Ac-DEVD-CHO, can reduce the rate of FA-induced cell death. These results demonstrate that the FA-induced cell death is a PCD and is modulated by NO signalling through caspase-3-like activation.