盐分胁迫下两个苋菜品种对镉及主要渗透调节物质累积的差异

采用重金属Cd平均含量为2.28 mg·kg-1的污灌菜园土,在外源Na Cl处理下进行盆栽试验,研究花红圆叶苋菜(BRLA)和红柳叶苋菜(ARW)对Cd及主要渗透调节物质吸收和积累的品种间差异。结果显示:随盐胁迫增强,两品种根、叶Cd和Na+含量较对照显著增加(P0.05),且品种ARW的Cd累积量高于品种BRLA。与ARW相比,BRLA根可贮存更多的Na+以减少向叶转运,在0.4%盐处理时BRLA叶K+/Na+比值降幅为39.2%,而ARW达56.9%。盐分促进BRLA根、叶Mg2+含量增加,而ARW根Mg2+则低于对照。盐胁迫后,两品种根、叶脯氨酸含量显著高于对照(P0.05),且BRLA表现较强的脯氨酸合成能力。两品种根、叶总游离氨基酸与可溶性蛋白含量随盐胁迫强度增大呈相反的变化。盐分促进了两品种根部可溶性糖积累。综上,BRLA比ARW具有较低Cd累积性,表现较好的耐盐性,具备较强的渗透响应能力,可为筛选和培育既耐盐又低累积Cd的农作物提供科学参考。     

Thermodynamic and kinetic basis for the relaxed DNA sequence specificity of "promiscuous" mutant EcoRI endonucleases

Promiscuous mutant EcoRI endonucleases produce lethal to sublethal effects because they cleave Escherichia coli DNA despite the presence of the EcoRI methylase. Three promiscuous mutant forms, Ala138Thr, Glu192Lys and His114Tyr, have been characterized with respect to their binding affinities and first-order cleavage rate constants towards the three classes of DNA sites: specific, miscognate (EcoRI*) and non-specific. We have made the unanticipated and counterintuitive observations that the mutant restriction endonucleases that exhibit relaxed specificity in vivo nevertheless bind more tightly than the wild-type enzyme to the specific recognition sequence in vitro, and show even greater preference for binding to the cognate GAATTC site over miscognate sites. Binding preference for EcoRI* over non-specific DNA is also improved. The first-order cleavage rate constants of the mutant enzymes are normal for the cognate site GAATTC, but are greater than those of the wild-type enzyme at EcoRI* sites. Thus, the mutant enzymes use two mechanisms to partially bypass the multiple fail-safe mechanisms that protect against cleavage of genomic DNA in cells carrying the wild-type EcoRI restriction-modification system: (a) binding to EcoRI* sites is more probable than for wild-type enzyme because non-specific DNA is less effective as a competitive inhibitor; (b) the combination of increased affinity and elevated cleavage rate constants at EcoRI* sites makes double-strand cleavage of these sites a more probable outcome than it is for the wild-type enzyme. Semi-quantitative estimates of rates of EcoRI* site cleavage in vivo, predicted using the binding and cleavage constants measured in vitro, are in accord with the observed lethal phenotypes associated with the three mutations.     

Differences in strategies to combat osmotic stress in Burkholderia cenocepacia elucidated by NMR-based metabolic profiling

Aims: To investigate mechanisms of osmotic tolerance in Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc) of closely related strains, which is of clinical as well as environmental importance. Methods and Results: We employed NMR‐based metabolic profiling (metabolomics) to elucidate the metabolic consequences of high osmotic stress for five isolates of B. cenocepacia. The strains differed significantly in their levels of osmotic stress tolerance, and we identified three different sets of metabolic responses with the strains least impacted by osmotic stress exhibiting higher levels of the osmo‐protective metabolites glycine‐betaine and/or trehalose. Strains either increased concentrations or had constitutively high levels of these metabolites. Conclusions: Even within the small set of B. cenocepacia isolates, there was a surprising degree of variability in the metabolic responses to osmotic stress. Significance and impact of the study: The metabolic responses, and hence osmotic stress tolerance, vary between different B. cenocepacia isolates. This study provides a first look into the potentially highly diverse physiology of closely related strains of one species of the Bcc and illustrates that physiological or clinically relevant phenotypes are unlikely to be inferable from genetic relatedness within this species group.     

Quantitative relationships between osmotic potential amd epicotyl growth in Vigna radiata as affected by osmotic stress and cotyledon excision

Ethylene biosynthesis in leaf discs of tobacco ( Nicotiana tabacum L. cv. Xanthi), as measured by the conversion of L-[3,4-¹⁴C]-methionine to ¹⁴C₂H₄, was markedly inhibited by exogenous ethylene. This inhibition was accompanied by a decrease in total (free + conjugated) content of 1-aminocyclopropane-1-carboxylic acid (ACC), most of which appeared in its conjugated inactive form. The autoinhibitory effect of ethylene was reversible and could be relieved by Ag⁺. The Ag⁺-treated leaf discs, with or without ethylene, contained only free ACC at an increased level. The results suggest that in tobacco leaves, the autoinhibition of ethylene production resulted from reduction in the availability of free ACC, through both suppression of ACC formation and increased ACC conjugation.     

Importance of phosphate contacts for sequence recognition by EcoRI restriction enzyme

We have studied the importance of charge and hydrogen-bonding potential of the phosphodiester backbone for binding and cleavage by EcoRI restriction endonuclease. We used 12-mer oligodeoxynucleotide substrates with single substitutions of phosphates by chiral methylphosphonates at each position of the recognition sequence -pGpApApTpTpCp-. Binding was moderately reduced between 4- and 400-fold more or less equally for the R(P) and S(P)-analogues mainly caused by missing charge interaction. The range of cleavage effects was much wider. Four substrates were not cleaved at all. At both flanking positions and in the purine half of the sequence up to the central position, cleavage was more impaired than binding and differences between R(P) and S(P) diastereomeres were more pronounced. These effects are easily interpreted by direct phosphate contacts seen in the crystal structure. For the effects of substitutions in the pyrimidine half of the recognition sequence, more indirect effects have to be discussed.     

Thermodynamic parameters governing interaction of EcoRI endonuclease with specific and nonspecific DNA sequences

Equilibrium binding of EcoRI endonuclease to DNA has been analyzed by nitrocellulose filter and preferential DNA cleavage methods. Association constants for pBR322 and a 34-base pair molecule containing the EcoRI site of this plasmid in a central position were determined to be 1.9 X 10(11) M-1 and 1.0 X 10(11) M-1 at 37 degrees C, respectively, with the stoichiometry of binding being 0.8 +/- 0.1 mol of endonuclease dimer per mol of DNA. In contrast, the affinity of the enzyme for a pBR322 derivative from which the EcoRI site has been deleted is 3.2 X 10(9) M-1 as judged by competitive binding experiments. If it is assumed that each base pair can define the beginning of a nonspecific binding site, this value corresponds to an affinity for nonspecific sites of 7.4 X 10(5) M-1. Furthermore, the affinity of the endonuclease for the EcoRI-methylated sequence is at least three orders of magnitude less than that for the unmodified recognition site. The dependence on temperature and ionic strength of the equilibrium constant governing specific interactions has also been examined. The temperature dependence of the reaction indicates that entropy increase accounts for 70% of the free energy of specific binding at 37 degrees C. Affinity of the endonuclease for the EcoRI site is highly dependent on NaCl concentration. Analysis of this dependence according to the theory of Record and colleagues (Record, T. M., Jr., Lohman, T. M., and deHaseth, P. (1976) J. Mol. Biol. 107, 145-158) has implicated 8 ion pairs in the stability of specific complexes, a value identical with the number of phosphate contacts determined by ethylation interference analysis (Lu, A. L., Jack, W. E., and Modrich, P. (1981) J. Biol. Chem. 256, 13200-13206). Extrapolation to 1 M NaCl suggests that nonelectrostatic interactions account for 40% of the free energy change associated with specific complex formation.     

Regulation of PIKfyve phosphorylation by insulin and osmotic stress

PIKfyve is a protein and lipid kinase that plays an important role in membrane trafficking, including TGN to endosome retrograde sorting and in insulin-stimulated translocation of the GLUT4 glucose transporter from intracellular storage vesicles to the plasma membrane. We have previously demonstrated that PIKfyve is phosphorylated in response to insulin in a PI3-kinase and protein kinase B (PKB)-dependent manner. However, it has been implied that this was not due to direct phosphorylation of PIKfyve by PKB, but as a result of an insulin-induced PIKfyve autophosphorylation event. Here we demonstrate that purified PIKfyve is phosphorylated in vitro by a recombinant active PKB on two separate serine residues, S318 and S105, which flank the N-terminal FYVE domain of the protein. Only S318, however, becomes phosphorylated in intact cells stimulated with insulin. We further demonstrate that S318 is phosphorylated in response to hyperosmotic stress in a PI3-kinase- and PKB-independent manner. Importantly, the effects of insulin and sorbitol were not prevented by the presence of an ATP-competitive PIKfyve inhibitor (YM20163) or in a mutant PIKfyve lacking both lipid and protein kinase activity. Our results confirm, therefore, that PIKfyve is directly phosphorylated by PKB on a single serine residue in response to insulin and are not due to autophosphorylation of the enzyme. We further reveal that two stimuli known to promote glucose uptake in cells, both stimulate phosphorylation of PIKfyve on S318 but via distinct signal transduction pathways.     

Physiological and regulatory convergence between osmotic and nutrient stress responses in microbes

Bacterial cells are regularly confronted with simultaneous changes in environmental nutrient supply and osmolarity. Despite the importance of osmolarity and osmoregulation in bacterial physiology, the relationship between the cellular response to osmotic perturbations and other stresses has remained largely unexplored. Bacteria cultured in hyperosmotic conditions and bacteria experiencing nutrient stress exhibit similar physiological changes, including metabolic shutdown, increased protein instability, dehydration, and condensation of chromosomal DNA. In this review, we highlight overlapping molecular players between osmotic and nutrient stresses. These connections between two seemingly disparate stress response pathways reinforce the importance of central carbon metabolism as a control point for diverse aspects of homeostatic regulation. We identify important open questions for future research, emphasizing the pressing need to develop and exploit new methods for probing how osmolarity affects phylogenetically diverse species.     

A mechanistic link between oxidative stress and membrane mediated amyloidogenesis revealed by infrared spectroscopy

The fully developed lesion of Alzheimer's disease is a dense plaque composed of fibrillar amyloid β-proteins (Aβ) with a characteristic and well-ordered β-sheet secondary structure. Because the incipient lesion most likely develops when these proteins are first induced to form β-sheet structure, it is important to understand factors that induced Aβ to adopt this conformation. In this review, we describe the application of polarized attenuated total internal reflection infrared FT-IR spectroscopy for characterizing the conformation, orientation, and rate of accumulation of Aβ on lipid membranes. We also describe the application and yield of linked analysis, whereby multiple spectra are fit simultaneously with component bands that are constrained to share common fitting parameters. Results have shown that membranes promote β-sheet formation under a variety of circumstances that may be significant to the pathogenesis of Alzheimer's disease.     

Cereal prolamin evolution and homology revealed by sequence analysis

Prolamin mixtures were isolated from oats, rice, normal and high-lysine sorghum, two varieties of pearl millet, two strains of teosinte, and gamma grass and subjected to NH₂-terminal amino acid sequence determinations. In each case (except for rice, whose prolamins apparently have blocked or unavailable NH₂-terminal residues), primarily a single sequence was observed despite significant heterogeneity, suggesting that prolamin homology in each cereal arose through duplication and mutation of a single ancestral gene. Comparisons were then made to prolamin sequences previously determined for wheat, corn, barley, and rye. Within genera, different varieties or subspecies exhibited few differences, but more distantly related genera, subtribes, and tribes showed increasingly large differences. Within the subfamily Festucoideae, no homology was apparent between prolamins of oats and those of the subtribe Triticinae (including wheat, rye, and barley, for which prolamin homology was previously demonstrated). Within the subfamily Panicoideae, corn was shown to be closely related to teosinte but more distantly to Tripsacum. Sorghum was shown to have diverged less from corn than had millet. These comparisons demonstrate that prolamin sequence analyses can successfully predict and clarify evolutionary relationships of cereals.     

A mechanistic link between oxidative stress and membrane mediated amyloidogenesis revealed by infrared spectroscopy

The fully developed lesion of Alzheimer's disease is a dense plaque composed of fibrillar amyloid beta-proteins (Abeta) with a characteristic and well-ordered beta-sheet secondary structure. Because the incipient lesion most likely develops when these proteins are first induced to form beta-sheet structure, it is important to understand factors that induced Abeta to adopt this conformation. In this review, we describe the application of polarized attenuated total internal reflection infrared FT-IR spectroscopy for characterizing the conformation, orientation, and rate of accumulation of Abeta on lipid membranes. We also describe the application and yield of linked analysis, whereby multiple spectra are fit simultaneously with component bands that are constrained to share common fitting parameters. Results have shown that membranes promote beta-sheet formation under a variety of circumstances that may be significant to the pathogenesis of Alzheimer's disease.     

Protein structure and hydration probed by SANS and osmotic stress

Interactions governing protein folding, stability, recognition, and activity are mediated by hydration. Here, we use small-angle neutron scattering coupled with osmotic stress to investigate the hydration of two proteins, lysozyme and guanylate kinase (GK), in the presence of solutes. By taking advantage of the neutron contrast variation that occurs upon addition of these solutes, the number of protein-associated (solute-excluded) water molecules can be estimated from changes in both the zero-angle scattering intensity and the radius of gyration. Poly(ethylene glycol) exclusion varies with molecular weight. This sensitivity can be exploited to probe structural features such as the large internal GK cavity. For GK, small-angle neutron scattering is complemented by isothermal titration calorimetry with osmotic stress to also measure hydration changes accompanying ligand binding. These results provide a framework for studying other biomolecular systems and assemblies using neutron scattering together with osmotic stress.     

Differences between the complexes formed by monomeric fibrin with fragment D and dimer D

The paper is concerned with studies in formation of monomeric fibrin (fm) complexes with fragment D (D) of fibrinogen and dimer D (DD) of stabilized fibrin. The complexes are shown to be essentially different. The fm-D complexes are unstable, their composition is a function of D concentration in the mixture, the ultimate molar D/fm ratio is equal to 3. The fm-DD complexes are quite stable, their composition is constant: the molar DD/fm ratio is equal to 1. In mixtures containing fm, DD and different amounts of D complexes of different composition are formed but the total number of D-units in them approaches 3. A model is suggested showing interaction of fm molecules in protofibril formation with allowance for the retention of binding centres which provide the lateral link between protofibrils.     

Pathogenetic interplay between osmotic and oxidative stress: the hepatic encephalopathy paradigm

Hepatic encephalopathy (HE) defines a primary gliopathy associated with acute and chronic liver disease. Astrocyte swelling triggered by ammonia in synergism with different precipitating factors, including hyponatremia, tumor necrosis factor (TNF)-alpha, glutamate and ligands of the peripheral benzodiazepine receptor (PBR), is an early pathogenetic event in HE. On the other hand, reactive nitrogen and oxygen species (RNOS) including nitric oxide are considered to play a major role in HE. There is growing evidence that osmotic and oxidative stresses are closely interrelated. Astrocyte swelling produces RNOS and vice versa. Based on recent investigations, this review proposes a working model that integrates the pathogenetic action of osmotic and oxidative stresses in HE. Under participation of the N-methyl-D-aspartate (NMDA) receptor, Ca(2+), the PBR and organic osmolyte depletion, astrocyte swelling and RNOS production may constitute an autoamplificatory signaling loop that integrates at least some of the signals released by HE-precipitating factors.     

Glycerol production by yeasts under osmotic and sulfite stress.

The yeasts Saccharomyces cerevisiae, Candida boidinii, Pichia augusta, and Pichia anomala were tested for glycerol production both under osmotic stress and by addition of a sulfite-steering agent. The osmotic pressure was increased by employing glucose concentrations from 50 to 200 g/L and by supplementing with NaCl (40 g/L). Of all the yeasts, S. cerevisiae exhibited the highest level of osmotolerance. The increased osmotic pressure affected glycerol formation the most in C. boidinii. In both Pichia species, glycerol formation was not sufficiently induced when exposed to sugar and salt stress. The addition of 40 g/L Na2SO3 to the medium containing 100 g/L glucose shifted the metabolism of all yeasts towards glycerol formation. Saccharomyces cerevisiae achieved 68.6%, while C. boidinii reached 25.5% of the theoretical glycerol yield, respectively. The highest glycerol yield, 82.3% of the theoretical, was produced by S. cerevisiae under microaerophilic conditions.     

The "instantaneous" deformation of cartilage: effects of collagen fiber orientation and osmotic stress

The present study was undertaken with two objectives in view. The first was to distinguish between the "instantaneous" deformation and creep of articular cartilage when subjected to a step loading in unconfined compression. This was done by observing changes in the specimen's diameter rather than its thickness. The second objective was to investigate experimentally the anisotropic behaviour of cartilage in a compressive loading mode, corresponding to the physiological situation. An apparatus was thus developed and constructed which enabled us to follow the "instantaneous" changes of the surface area of the sample as the latter was being loaded in unconfined compression. Specimens of human articular cartilage from normal femoral heads and condyles were tested. Full thickness specimens were tested with and without the underlying bone, as well as partial thickness specimens, characterizing the different zones of cartilage. Solutions of different ionic strength were used to vary the osmotic stress and specimens covering a considerable range of proteoglycan concentrations were selected. The effects of hydration and proteoglycan removal on the "instantaneous" deformation were also studied. The "instantaneous" deformation was found to be of a strongly anisotropic nature in all zones. The deformation was always smaller along the Indian-ink prick pattern than at 90 degrees to it, and this effect was most pronounced in the superficial zone of cartilage. The results reveal an analogy with the tensile properties of cartilage and indicate that the collagen network is mainly responsible for controlling the "instantaneous" deformation. The proteoglycans play an indirect role by modulating the stiffness of the collagen network through their osmotic pressure.     

Role of DNA conformation & energetic insights in Msx-1-DNA recognition as revealed by molecular dynamics studies on specific and nonspecific complexes

In most of homeodomain–DNA complexes, glutamine or lysine is present at 50th position and interacts with 5th and 6th nucleotide of core recognition region. Molecular dynamics simulations of Msx-1–DNA complex (Q50-TG) and its variant complexes, that is specific (Q50K-CC), nonspecific (Q50-CC) having mutation in DNA and (Q50K-TG) in protein, have been carried out. Analysis of protein–DNA interactions and structure of DNA in specific and nonspecific complexes show that amino acid residues use sequence-dependent shape of DNA to interact. The binding free energies of all four complexes were analysed to define role of amino acid residue at 50th position in terms of binding strength considering the variation in DNA on stability of protein–DNA complexes. The order of stability of protein–DNA complexes shows that specific complexes are more stable than nonspecific ones. Decomposition analysis shows that N-terminal amino acid residues have been found to contribute maximally in binding free energy of protein–DNA complexes. Among specific protein–DNA complexes, K50 contributes more as compared to Q50 towards binding free energy in respective complexes. The sequence dependence of local conformation of DNA enables Q50/Q50K to make hydrogen bond with nucleotide(s) of DNA. The changes in amino acid sequence of protein are accommodated and stabilized around TAAT core region of DNA having variation in nucleotides.