藏东南色季拉山沟壑区土壤氮素空间分布特征

以西藏东南部色季拉山海拔3950—4350 m为研究区,采用30×50 m网格采样法,以地统计学半变异函数为工具,研究了色季拉山森林生态系统沟谷与坡面上土壤氮素空间变异特征及模型。结果表明:土壤全氮、硝态氮和铵态氮含量均表现为0—10 cm10—20 cm,两个层次上空间变异性表现为全氮和铵态氮0—10 cm10—20 cm,而硝态氮表现为10—20 cm0—10cm;不同海拔高度土壤氮含量表现为随着海拔高度的升高而增加,但这种海拔梯度效应并未达显著水平(P0.05);沟谷区土壤氮含量高于坡面,这可能与植被残体在沟谷区的堆积分解促进氮循环有关;土壤全氮、铵态氮和硝态氮均具有中等程度的空间依赖性,其中土壤全氮空间变异符合指数模型,块金值/基台值为50%;土壤铵态氮和硝态氮含量空间变异分布均符合高斯模型,块金值/基台值分别为70.91%和37.45%;该区域土壤全氮、铵态氮和硝态氮含量空间依赖性表现为:硝态氮全氮铵态氮,即土壤硝态氮更易受到空间结构因素的影响,而铵态氮含量空间变化则主要受随机因素的影响。     

3′-Phosphoadenosine-5′-Phosphate Phosphatase Activity Is Required for Superoxide Stress Tolerance in Streptococcus mutans

Aerobic microorganisms have evolved different strategies to withstand environmental oxidative stresses generated by various reactive oxygen species (ROS). For the facultative anaerobic human oral pathogen Streptococcus mutans, the mechanisms used to protect against ROS are not fully understood, since it does not possess catalase, an enzyme that degrades hydrogen peroxide. In order to elucidate the genes that are essential for superoxide stress response, methyl viologen (MV)-sensitive mutants of S. mutans were generated via ISS1 mutagenesis. Screening of approximately 2,500 mutants revealed six MV-sensitive mutants, each containing an insertion in one of five genes, including a highly conserved hypothetical gene, SMU.1297. Sequence analysis suggests that SMU.1297 encodes a hypothetical protein with a high degree of homology to the Bacillus subtilis YtqI protein, which possesses an oligoribonuclease activity that cleaves nano-RNAs and a phosphatase activity that degrades 3′-phosphoadenosine-5′-phosphate (pAp) and 3′-phosphoadenosine-5′-phosphosulfate (pApS) to produce AMP; the latter activity is similar to the activity of the Escherichia coli CysQ protein, which is required for sulfur assimilation. SMU.1297 was deleted using a markerless Cre-loxP-based strategy; the SMU.1297 deletion mutant was just as sensitive to MV as the ISS1 insertion mutant. Complementation of the deletion mutant with wild-type SMU.1297, in trans, restored the parental phenotype. Biochemical analyses with purified SMU.1297 protein demonstrated that it has pAp phosphatase activity similar to that of YtqI but apparently lacks an oligoribonuclease activity. The ability of SMU.1297 to dephosphorylate pApS in vivo was confirmed by complementation of an E. coli cysQ mutant with SMU.1297 in trans. Thus, our results suggest that SMU.1297 is involved in superoxide stress tolerance in S. mutans. Furthermore, the distribution of homologs of SMU.1297 in streptococci indicates that this protein is essential for superoxide stress tolerance in these organisms.Streptococcus mutans, a gram-positive bacterium with a low G+C content, is widely considered the primary etiological agent of dental caries, a common human infectious disease (16, 23). S. mutans is also an important agent of infective endocarditis, as a large number of cases of viridans streptococcus-induced endocarditis are caused by S. mutans (18). During colonization of the oral cavity, S. mutans encounters various environmental stresses, including nutritional limitation, temperature fluctuation, osmotic shock, low pH conditions, radiation, toxins, and variations in oxygen tension (21). Despite these harsh conditions, S. mutans has developed multiple mechanisms for successful survival in the human host by forming diverse and densely populated biofilms on the tooth surface (4). The extraordinary ability of S. mutans to adapt and flourish in the diverse and adverse environment of the oral cavity emphasizes the fundamental importance of the need for detailed analyses of the molecular mechanisms of stress tolerance response in this organism.S. mutans is a facultative anaerobic organism, but it can tolerate aerobic conditions for colonization and survival. Like other streptococci, it does not possess cytochromes and therefore cannot carry out energy-conserving oxidative phosphorylation (2). However, irrespective of the growth conditions, S. mutans derives the energy for growth through fermentation of glucose and other sugars (26). This can lead to unwanted consequences, especially when the organism is exposed to aerobic conditions in the oral cavity. If the molecular oxygen is not fully reduced by the four-electron reduction step to water, it can undergo one- or two-electron reductions to form reactive superoxide radicals, hydroxyl radicals, and hydrogen peroxide, collectively known as reactive oxygen species (ROS) (19). These radicals, when accumulated in large amounts, can trigger oxidation of lipid, protein, and nucleic acid inside the cell, ultimately leading to cellular death (19, 20).Aerobic bacteria have developed multiple strategies to adapt and protect against ROS insults (19). These strategies include (i) enzymes that scavenge ROS, such as superoxide dismutases (SOD), catalases, and peroxidases; (ii) protein repair systems, such as thioredoxin; (iii) DNA damage repair enzymes such as RecA; and (iv) proteins that regulate intracellular iron level to ameliorate the generation of ROS. Although streptococci contain SOD, NADH oxidase, glutathione reductase, and other proteins to counter ROS threats, they do not contain catalase, a key protective enzyme against oxidative radicals. Therefore, the defense strategy against damage by ROS is significantly different in streptococci than in other bacteria. For example, the growth of S. mutans in planktonic or biofilm mode can influence the respiratory rates as well as the activities of the protective enzymes, such as SOD and NADH oxidase (31).Apart from studies related to the physiology of oxidative stress in S. mutans, very little information is available on the oxidative-stress response and its regulation in this organism. Many key regulatory genes, including members of the OxyR and SoxR families, which are involved in sensing and responding to ROS attacks, are not encoded in the genome of S. mutans (2). Instead, S. mutans has a PerR homolog, which has been shown to be involved in hydrogen peroxide stress response in this organism (21). The luxS gene of S. mutans, which encodes an enzyme that synthesizes the intercellular signaling molecule AI-2, is also involved in the oxidative-stress response (52). However, the exact mechanism by which LuxS participates in the oxidative-stress response is currently unknown. Furthermore, a recent investigation suggests that a two-component signal transduction system, ScnRK, is necessary for counteracting ROS in S. mutans (11).The major focus of this study was to identify the genes that are involved in the defense against superoxide stress of S. mutans strain UA159. Toward this end, a library of mutants was generated by insertion mutagenesis, and the mutants were screened for their sensitivity to methyl viologen (MV), a superoxide-generating compound. This study enabled the identification of five loci that are potentially involved in superoxide tolerance. One of the identified loci is SMU.1297, which encodes a protein homologous to YtqI of Bacillus subtilis. The biochemical characterization of SMU.1297 and its role in superoxide stress tolerance response are presented.     

Binding of genistein to human serum albumin demonstrated using tryptophan fluorescence quenching

Genistein is an isoflavone and phytoestrogen that is a potent inhibitor of cell proliferation and angiogenesis. This study was designed to investigate the binding of genistein to human serum albumin (HSA) under physiological conditions with drug concentrations in the range of 6.7 × 10⁻⁶ to 2.0 × 10⁻⁵ mol L⁻¹ and HSA concentration at 1.5 × 10⁻⁶ mol L⁻¹. Fluorescence quenching methods in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to determine the binding mode, the binding constant and the protein structure changes in the presence of genistein in aqueous solution. Changes in the CD spectra and FT-IR spectra were observed upon ligand binding, and the degree of tryptophan fluorescence quenching change did significantly in the complexes. These data have proved the change in protein secondary structure accompanying ligand binding. The change in tryptophan fluorescence intensity was used to determine the binding constants. The thermodynamic parameters, the enthalpy change (ΔH) and the entropy change (ΔS) were calculated to be −22.24 kJ mol⁻¹and 19.60 J mol⁻¹ K⁻¹ according to the van’t Hoff equation, which indicated that hydrophobic and electrostatic interactions play the main role in the binding of genistein to HSA.     

Probing the binding of scutellarin to human serum albumin by circular dichroism, fluorescence spectroscopy, FTIR, and molecular modeling method

The binding of scutellarin with human serum albumin (HSA) was investigated at four temperatures, 296, 303, 310, and 318 K, by fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR), and molecular modeling study at pH 7.40. The binding parameters were determined by Scatchard's procedure, which are approximately consistent with the results of Stern-Volmer equation. The thermodynamic parameters were calculated according to the dependence of enthalpy change on the temperature as follows: DeltaH degrees is a small negative value (-8.55 kJ/mol), whereas DeltaS degrees is a positive value (65.15 J/mol K). Quenching of the fluorescence HSA in the presence of scutellarin was observed. Data obtained by fluorescence spectroscopy and CD experiment, FT-IR experiment, and molecular modeling method suggested that scutellarin can strongly bind to the HSA and the primary binding site of scutellarin is located in site I of HSA. It is considered that scutellarin binds to site I (subdomain II) mainly by a hydrophobic interaction and there are hydrogen bond interactions between the scutellarin and the residues Arg222 and Arg257.     

Interaction of wogonin with bovine serum albumin

The binding of wogonin with bovine serum albumin (BSA) was investigated at different temperatures by fluorescence, circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR) at pH7.40. The association constants K were determined by Stern-Volmer equation based on the quenching of the fluorescence of BSA in the presence of wogonin, which were in agreement with the constants calculated by Scatchard plots. The thermodynamic parameters were calculated according to the Van't Hoff equation and the result indicated that DeltaH(0) and DeltaS(0) had a negative value (-12.02 kJ/mol) and a positive value (58.72 J/mol K), respectively. On the basis of the displacement experimental and the thermodynamic results, it is considered that wogonin binds to site I (subdomain IIA) of BSA mainly by hydrophobic interaction. The studied results by FT-IR and CD experiment indicated that the secondary structures of protein have been perturbed by the interaction of wogonin with BSA.     

Purification of a bioactive recombinant human Reg IV expressed in Escherichia coli

Regenerating gene (Reg) IV is a newly discovered member of the regenerating gene family belonging to the calcium (C-type) dependent lectin superfamily. Reg IV is highly expressed in the gastrointestinal tract and markedly up-regulated in colon adenocarcinoma, pancreatic cancer, gastric adenocarcinoma, and inflammatory bowel disease. However, the physiological and pathological functions of Reg IV are largely unknown, partly due to the limited access of the bioactive protein. We report here the first expression and purification of Reg IV proteins using a prokaryotic system. Human Reg IV was expressed in Escherichia coli as an insoluble protein which was identified in the fraction of inclusion body after ultrasonication of the bacteria. After the protein aggregate was solubilized by guanidine–HCl, it was refolded by sucrose and arginine-assisted procedures and purified using cation-exchange chromatography. The protein identity and purity of the final preparation were confirmed by analysis of the protein mass and immune specificity in SDS–PAGE, Western blotting, and HPLC assay. The biological activity of the protein was determined by the HCT116 and HT29 cell proliferation assays. The highly purified bioactive human Reg IV should aid in further characterization of its physiological and pathological functions.