岩溶区不同植被下土壤水溶解无机碳含量及其稳定碳同位素组成特征

自2010年7月至2011年7月对重庆青木关岩溶区典型植被下的土壤水进行了月动态取样,分析了土壤水溶解无机碳含量(DIC浓度)及其稳定碳同位素组成(δ13CDIC值)的时空变化特征,以揭示岩溶土壤系统碳酸盐岩溶蚀作用及其碳汇效应。研究结果表明:草地和针叶林地土壤水的DIC浓度和δ13CDIC值相对较低,分别为59.12 mg/L和-17.22‰,31.47 mg/L和-16.37‰;而旱地、灌丛地、退耕还林地土壤水具有较高的DIC浓度和δ13CDIC值,分别达153.88 mg/L和-12.2‰,221.82 mg/L和-11.9‰,97.30 mg/L和-11.23‰,其中灌丛和退耕还林地的δ13CDIC值与DIC浓度呈正比,且雨季较旱季偏高约4‰—5‰。根据δ13CDIC值,结合各植被类型下土壤水DIC浓度与其相应的土壤碳酸盐含量呈正相关,判断旱地、灌丛地、退耕还林地等岩溶土壤水中的DIC主要来自土壤中碳酸盐岩矿物的碳酸溶蚀,即岩溶土壤中存在着碳酸盐岩碳酸溶蚀作用,从而在一定程度上减少了土壤系统向大气排放的CO2量。     

Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc¹³ localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G₁ arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc¹³ localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal.     

In Vivo Substrate Diversity and Preference of Small Heat Shock Protein IbpB as Revealed by Using a Genetically Incorporated Photo-cross-linker

Small heat shock proteins (sHSPs), as ubiquitous molecular chaperones found in all forms of life, are known to be able to protect cells against stresses and suppress the aggregation of a variety of model substrate proteins under in vitro conditions. Nevertheless, it is poorly understood what natural substrate proteins are protected by sHSPs in living cells. Here, by using a genetically incorporated photo-cross-linker (p-benzoyl-l-phenylalanine), we identified a total of 95 and 54 natural substrate proteins of IbpB (an sHSP from Escherichia coli) in living cells with and without heat shock, respectively. Functional profiling of these proteins (110 in total) suggests that IbpB, although binding to a wide range of cellular proteins, has a remarkable substrate preference for translation-related proteins (e.g. ribosomal proteins and amino-acyl tRNA synthetases) and moderate preference for metabolic enzymes. Furthermore, these two classes of proteins were found to be more prone to aggregation and/or inactivation in cells lacking IbpB under stress conditions (e.g. heat shock). Together, our in vivo data offer novel insights into the chaperone function of IbpB, or sHSPs in general, and suggest that the preferential protection on the protein synthesis machine and metabolic enzymes may dominantly contribute to the well known protective effect of sHSPs on cell survival against stresses.     

Regulation of Phospholipase D Activity and Phosphatidic Acid Production after Purinergic (P2Y6) Receptor Stimulation

Phosphatidic acid (PA) is a lipid second messenger located at the intersection of several lipid metabolism and cell signaling events including membrane trafficking, survival, and proliferation. Generation of signaling PA has long been primarily attributed to the activation of phospholipase D (PLD). PLD catalyzes the hydrolysis of phosphatidylcholine into PA. A variety of both receptor-tyrosine kinase and G-protein-coupled receptor stimulations have been shown to lead to PLD activation and PA generation. This study focuses on profiling the PA pool upon P2Y₆ receptor signaling manipulation to determine the major PA producing enzymes. Here we show that PLD, although highly active, is not responsible for the majority of stable PA being produced upon UDP stimulation of the P2Y₆ receptor and that PA levels are tightly regulated. By following PA flux in the cell we show that PLD is involved in an initial increase in PA upon receptor stimulation; however, when PLD is blocked, the cell compensates by increasing PA production from other sources. We further delineate the P2Y₆ signaling pathway showing that phospholipase Cβ3 (PLCβ3), PLCδ1, DGKζ and PLD are all downstream of receptor activation. We also show that DGKζ is a novel negative regulator of PLD activity in this system that occurs through an inhibitory mechanism with PKCα. These results further define the downstream events resulting in PA production in the P2Y₆ receptor signaling pathway.     

Inhibition of the First Step in Synthesis of the Mycobacterial Cell Wall Core,Catalyzed by the GlcNAc-1-phosphate Transferase WecA,by the Novel Caprazamycin Derivative CPZEN-45

Because tuberculosis is one of the most prevalent and serious infections, countermeasures against it are urgently required. We isolated the antitubercular agents caprazamycins from the culture of an actinomycete strain and created CPZEN-45 as the most promising derivative of the caprazamycins. Herein, we describe the mode of action of CPZEN-45 first against Bacillus subtilis. Unlike the caprazamycins, CPZEN-45 strongly inhibited incorporation of radiolabeled glycerol into growing cultures and showed antibacterial activity against caprazamycin-resistant strains, including a strain overexpressing translocase-I (MraY, involved in the biosynthesis of peptidoglycan), the target of the caprazamycins. By contrast, CPZEN-45 was not effective against a strain overexpressing undecaprenyl-phosphate–GlcNAc-1-phosphate transferase (TagO, involved in the biosynthesis of teichoic acid), and a mutation was found in the tagO gene of the spontaneous CPZEN-45-resistant strain. This suggested that the primary target of CPZEN-45 in B. subtilis is TagO, which is a different target from that of the parent caprazamycins. This suggestion was confirmed by evaluation of the activities of these enzymes. Finally, we showed that CPZEN-45 was effective against WecA (Rv1302, also called Rfe) of Mycobacterium tuberculosis, the ortholog of TagO and involved in the biosynthesis of the mycolylarabinogalactan of the cell wall of M. tuberculosis. The outlook for WecA as a promising target for the development of antituberculous drugs as a countermeasure of drug resistant tuberculosis is discussed.     

The effect of different strengths of extremely low-frequency electric fields on antioxidant status,lipid peroxidation,and visual evoked potentials

The aim of the study was to investigate the effects of extremely low-frequency electric field (ELF EF) on visual evoked potential (VEP), thiobarbituric acid reactive substances (TBARS), total antioxidant status (TAS), total oxidant status (TOS), and oxidant stress index (OSI). Thirty female Wistar rats, aged 3 months, were divided into three equal groups: Control (C), the group exposed to EF at 12 kV/m strength (E12), and the group exposed to EF at 18 kV/m strength (E18). Electric field was applied to the E12 and E18 groups for 14 days (1 h/day). Brain and retina TBARS, TOS, and OSI were significantly increased in the E12 and E18 groups with respect to the control group. Also, TBARS levels were significantly increased in the E18 group compared with the E12 group. Electric fields significantly decreased TAS levels in both brain and retina in E12 and E18 groups with respect to the control group. All VEP components were significantly prolonged in rats exposed to electric fields compared to control group. In addition, all latencies of VEP components were increased in the E18 group with respect to the E12 group. It is conceivable to suggest that EF-induced lipid peroxidation may play an important role in changes of VEP parameters.     

Study on the Interaction Mechanism of 2‐Aminoanthraquinone with Calf Thymus DNA

By utilizing multispectrosopic techniques, the toxic interaction of 2‐aminoanthraquinone (2‐AAQ) with calf thymus deoxyribonucleic acid (ctDNA) was investigated in vitro under simulated physiological conditions. The experimental results proved that 2‐AAQ has a toxic interaction with ctDNA. The binding capacity of DNA with 2‐AAQ is diminishing as the pH value of system increasing in the optimization of experimental condition. Moreover we selected pH 7.4, which is nearly physiological condition to enhance the practical significance. According to the Stern–Volmer equation, the quenching was the static quenching process. And the quenching constant can be derived from the fluorescence quenching spectrogram. Ultraviolet absorption spectra and the change in the fluorescence intensity at different ionic strengths further indicated that there was electrostatic binding between 2‐AAQ and ctDNA. The circular dichroism experiment showed that the DNA conformation varied from B to A conformation. The basic group enhanced after 2‐AAQ embedding. The double helix is more compact, and the DNA conformation changes. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:272‐278, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21487     

Improving the acidic stability of a β-mannanase from Bacillus subtilis by site-directed mutagenesis

β-Mannanase can randomly hydrolyze the (1→4)-β-d-mannosidic linkages in mannans, galactomannans and glucomannans, yielding manno-oligosaccharides. In this study, the β-mannanase (MAN) from Bacillus subtilis B10-02 was overexpressed successfully in B. subtilis 168 as a hexa-histidine tagged, secreted protein. The recombinant enzyme BsMAN6H was not stable under acidic conditions, which restricts its use in food and feed industry. We aimed to improve the acid stability of BsMAN6H by changing several surface-exposed amino acid residues to acidic or neutral ones. Among the mutations, the His54Asp resulted in a shift in the optimal pH from 6.5 to 5.5. Accordingly, the acid stability was improved by a factor of a negative potential on the structure surface around the mutated site. Furthermore, the H54D variant showed the enzyme activity up to 3207.82 U/mL in bioreactors using the cheap Kojac powder as substrate. As a result, a bacterial β-mannanase was produced efficiently with increased acid stability, improving its applicability in the animal feed industry.     

Inhibition of Acid Phosphatase Isoforms Purified from Mature Soybean (Glyczne MAX) Seeds

The four soybean seed acid phosphatase isoforms AP1, AP2, AP3A and AP3B were competitively inhibited by phosphate, vanadate, fluoride and molybdate, using p-nitrophenylphos-phate as substrate. The four isoforms were not significantly affected by compounds that can interact with SH residues or by pyridoxal phosphate. These results indicated that cysteine and lysine residues are not present in the active site of the four soybean seed acid phosphatase isoforms. The inhibition constant values for phosphate, vanadate, fluoride and molybdate at pH 5.0 were respectively: API (250, 12.8, 1.7, 0.05 μM), AP2 (800,10, 500, 0.025 μM), AP3A (250, 24.2,250, 0.032 μM), AP3B (2400, 36.9,750, 0.05 μM).     

A new technique for studying the stylet tracks of homopteran insects in hand-sectioned plant tissue using light or epifluorescence microscopy

Homopteran insects, such as aphids, psyllids and scales, inject a proteinaceous salivary sheath into their host plant tissue during feeding. This sheath, also referred to as a stylet track, remains in the tissue after the stylets are withdrawn, and is useful for studying plant resistance to insects and plant virus transmission. We describe a new method for studying stylet tracks. Hand microtome sectioned plant material was fixed and cleared in ethanol. The stylet tracks were stained with acid fuchsin and counterstained with aniline blue or fast green. The acid fuchsin stained stylet tracks were pink to red under light microscopy, and orange under TRITC epifluorescence. Stylet tracks in unstained sections autofluoresced under DAPI epifluorescence. This new technique is significantly faster and less complex than previous techniques, and permitted visualization of stylet tracks with light or epifluorescence microscopy within 1 hr of collecting fresh plant material. The technique was also applicable to a broad range of homopterans and plant taxa and provided excellent photomicrographs.