匍茎翦股颖对Cu2+、Zn2+、Cd2+与Pb2+ 胁迫的生长响应与阈限浓度

采用砂培法,研究了匍茎翦股颖对Cu2+、Zn2+、Cd2+与Pb2+胁迫的生长响应及阈限浓度,结果表明:种子萌发率随着4种重金属浓度的增加而下降。对株高的影响是当重金属浓度小于100mg/L时会促进株高生长,高于100mg/L则产生抑制作用。Cu2+显著抑制根系生长,并随浓度的增加抑制效应愈加显著;在Cu2+浓度为600mg/L时匍茎翦股颖的根长比对照下降了93.75%。Cu2+、Zn2+、Pb2+浓度小于200mg/L时会促进地上生物量的增加,但高于200mg/L时,地上生物量会随着3种重金属的增加而减少。Cu2+、Zn2+浓度小于100mg/L或Cd2+、Pb2+浓度小于200mg/L会增加叶绿素的含量,高浓度会降低叶绿素的含量;Cd2+在浓度为600mg/L时显著降低叶绿素含量,与对照相比,下降了43.55%。匍茎翦股颖生长的综合效应分析表明,匍茎翦股颖对Cu2+胁迫最敏感,具有较低的阈限浓度,而Zn2+胁迫对匍茎翦股颖的生长影响最小,阈限浓度相对较高。     

Inhibition of Ca2+-dependent K+ channels by lead in one-step inside-out vesicles from human red cell membranes

Pb2+ modified the apparent threshold sensitivity to Ca2+ of individual K+ channels with a biphasic time-course. At first, the sensitivity to Ca2+ was lowered with the result of a decrease of the fraction of activated vesicles at a given Ca2+ concentration. Later, Pb2+ increased the sensitivity to Ca2+ and the fraction of activated vesicles. The increase of Pb2+ concentration increased the extent of the initial inhibition but decreased its duration. The inhibitory effect was not observed when the addition of Ca2+ preceded the addition of Pb2+. The presence of Mg2+ in the incubation medium was also required. In the absence of Mg2+, Pb2+ decreased the rate of uptake of 86Rb, but no decrease in the fraction of activated vesicles could be demonstrated.     

Enhanced and selective adsorption of Pb2+ and Cu2+ by EDTAD-modified biomass of baker's yeast

Enhanced and selective removal of Pb2+ and Cu2+ in the presence of high concentration of K+, Na+, Ca2+ and Mg2+ were achieved by adsorption on biomass of baker's yeast modified with ethylenediaminetetraacetic dianhydride (EDTAD). The modified biomass was found to have high adsorption capacities and fast rates for Pb2+ and Cu2+, and it also displayed consistently high levels of metal uptake over the pH range from 2.7 to 6.0. From Langmuir isotherm, the adsorption capacities for Pb2+ and Cu2+ were found to be 192.3 and 65.0 mg g(-1), respectively, which are about 10 and 14 times higher than that of the unmodified biomass. Competitive biosorption experiments showed that the co-ions of K+, Na+, Ca2+ and Mg2+ had little effects on the uptake of Pb2+ and Cu2+ even at the concentration of 1.0 mol L(-1). The adsorbed Pb2+ and Cu2+ on the modified biomass could be effectively desorbed in an EDTA solution, and the regenerated biomass could be reused repeatedly with little loss of the adsorption capacity.     

Influence of heavy metal ions on the electrophysical properties of Anacystis nidulans and Escherichia coli cells]

The influence of heavy metal ions (Ag+, Cu2+, Cd2+, Pb2+, Mn2+, Zn2+, Gd3+, 1 microM-1 mM) on Anacystis nidulans and Escherichia coli cells has been studied by means of electrophoresis and electro-orientation spectroscopy methods. It has been shown that changes of cell electrophoretic mobility (EM) and low-frequency (20 Hz) electro-orientation effect (EOE) observed with the increase of metal cation concentration characterize the adsorption of these ions on surface layers of cell envelopes. The degree and the character of these changes depend on cation valency and the initial value of cell EM. At the same time different changes of EM and EOE as a result of the multivalent cation adsorption allows to conclude that in that case the anisotropy of the cell surface increases. Cell damages were determined by changes in high-frequency EOE of cells which indicated the disturbance of barrier properties of their cytoplasmic membrane. Toxic effects of Ag+, Cu2+, Cd2+ ions on cells of both species and of Pb2+ on E.coli cells were observed. By toxic effects on the cytoplasmic membrane these ions could be placed in the order: for A.nidulans cells--Ag+ greater than Cu2+ greater than Cd2+; for E.coli cells Ag+ greater than Cu2+ greater than Cd2+ greater than Pb2+. Higher toxicity of heavy metals on E.coli cells seems to be connected with the more negative charge of deep layers of the cell surface.     

Metal ion-dependent hydrolysis of RNA phosphodiester bonds within hairpin loops. A comparative kinetic study on chimeric ribo/2'-O-methylribo oligonucleotides.

Several chimeric ribo/2'- O -methylribo oligonucleotides were synthesized and their hydrolytic cleavage studied in the presence of Mg2+, Zn2+, Pb2+and the 1,4,9-triaza-cyclododecane chelate of Zn2+(Zn2+[12]aneN3) to evaluate the importance of RNA secondary structure as a factor determining the reactivity of phosphodiester bonds. In all the cases studied, a phosphodiester bond within a 4-7 nt loop was hydrolytically more stable than a similar bond within a linear single strand, but markedly less stable than that in a double helix. With Zn2+and Zn2+[12]aneN3, the hydrolytic stability of a phosphodiester bond within a hairpin loop gradually decreased on increasing the distance from the stem. A similar but less systematic trend was observed with Pb2+. Zn2+- and Pb2+-promoted cleavage was observed to be considerably more sensitive to the secondary structure of the chain than that induced by Zn2+[12]aneN3. This difference in behaviour may be attributed to bidentate binding of uncomplexed aquo ions to two different phosphodiester bonds. Mg2+was observed to be catalytically virtually inactive compared with the other cleaving agents studied.     

Structural equilibria in RNA as revealed by 19F NMR

We have incorporated 5-fluorouridine into several sites within a 19-mer RNA modelled on the translational operator of the MS2 bacteriophage. The 19F NMR spectra demonstrate the different chemical shifts of helical and loop fluorouridines of the hairpin secondary structure. Addition of salt gives rise to a species in which the loop fluorouridine gains the chemical shift of its helical counterparts, due to the formation of the alternative bi-molecular duplex form. This is supported by UV thermal melting behaviour which becomes highly dependent on the RNA concentration. Distinct 19F NMR signals for duplex and hairpin forms allow the duplex-hairpin equilibrium constant to be determined under a range of conditions, enabling thermodynamic characterisation and its salt dependence to be determined. Mg2+ also promotes duplex formation, but more strongly than Na+, such that at 25 degrees C, 10 mM MgCl2 has a comparable duplex-promoting effect to 300 mM NaCl. A similar effect is observed with Sr2+, but not Ca2+ or Ba2+. Additional hairpin species are observed in the presence of Na+ as well as Mg2+, Ca2+, Sr2+ and Ba2+ ions. The overall, ensemble average, hairpin conformation is therefore salt-dependent. Electrostatic considerations are thus involved in the balance between different hairpin conformers as well as the duplex-hairpin equilibrium. The data presented here demonstrate that 19F NMR is a powerful tool for the study of conformational heterogeneity in RNA, which is particularly important for probing the effects of metal ions on RNA structure. The thermodynamic characterisation of duplex-hairpin equilibria will also be valuable in the development of theoretical models of nucleic acid structure.     

Physicochemical characterization of the microbial Fe2+ chelator proferrorosamine from Pseudomonas roseus fluorescens.

The microbial chelating compound proferrorosamine A, produced by Pseudomonas roseus fluorescens, formed a complex with Fe2+ of which the apparent stability constant was found to be 10(23). The following order of increasing stability constants of metal complexes with proferrorosamine was established as: Ba2+, Ca2+, Mg2+, Mn2+ less than Hg2+ less than Zn2+ less than Pb2+ less than Co2+ less than Cu2+ congruent to Fe2+ less than Ni2+. Only Ni(2+)-proferrorosamine had a stability constant which was established as: Ba2+, Ca2+, Mg2+, Mn2+ less than Hg2+ less than Zn2+ less than Pb2+ less than Co2+ less than Cu2+ congruent to Fe2+ less than Ni2+. Only Ni(2+)-proferrorosamine had a stability constant which was ca 32 times higher than Fe(2+)-proferrorosamine. Because of the production of proferrorosamine the growth of Ps. roseus fluorescens was not inhibited in iron limiting media by the addition of 0.15 mmol/l of the weaker chemical Fe2+ chelator 2,2'-dipyridyl. This contrasted with the proferrorosamine-negative mutant K2 and Ps. stutzeri, which only produces Fe(3+)-chelating siderophores. Furthermore, it was found that proferrorosamine was able to dissolve Fe2+ from stainless steel. These results show that proferrorosamine is a strong and selective Fe2+ chelator which could be used as an alternative for the toxic 2,2'-dipyridyl to control lactic acid fermentations.     

Influence of alkali metal cations on the transport of calcium by phosphatidate in multi-phase systems

The effects of alkali metal cations on the rates at which Ca2+ and phosphatidic acid were cotransported from aqueous to hydrocarbon medium were examined. The alkali metal cations remained in the aqueous phase yet specifically influenced the transport of Ca2+ into the hydrocarbon solvent. For the physiological cations, Na+ and K+, there were critical concentration ranges in which small changes in concentration effected sharp changes in transport rates. The maximal rate observed with Na+ was an order of magnitude greater than that with K+; however, unlike Na+, K+ promoted low levels of transport below the critical concentration range. Li+ effected only low levels of transport even at high concentrations, whereas Rb+ and Cs+ induced transport at rates proportional to their concentrations. These results are discussed in terms of a classical ionophore model for the complex composed of a neutral phosphatidic acid dimer bridged by Ca2+.     

Characterization of an unusual folding pattern in a catalytically active guanine quadruplex structure

In the presence of certain metal ions, DNA and RNA can form guanine quadruplex structures, which have been proposed to play a functional role in a variety of biological processes. An 18-nucleotide DNA oligomer, PS2.M, d(GTG3TAG3CG3T2G2), was previously reported to bind hemin and the resulting complex exhibited peroxidase activity. It was proposed that PS2.M folds unimolecularly into an antiparallel quadruplex with unusual, single-base loops and terminal guanines positioned in adjacent quartets. Here we describe structural and stability properties of PS2.M alone in different buffers and metal ions, using gel electrophoresis, circular dichroism (CD), ultraviolet (UV)-visible spectroscopies, and one-dimensional 1H nuclear magnetic resonance (NMR). Native gel behavior of PS2.M in the presence of either Na+ or Pb2+ suggests the formation of unimolecular structures but, in the presence of K+, both unimolecular and multistranded structures are observed. In the presence of Pb2+ ions, PS2.M forms a unimolecular quadruplex containing three guanine quartets. CD titrations reveal that binding of Pb2+ ions to PS2.M is stoichiometric, and a single lead cation suffices to fully fold PS2.M. The PS2.M-Na+ system also forms a similar unimolecular quadruplex. In the presence of K+, the PS2.M-K+ system forms mixed species. With increasing time and PS2.M concentration, the contribution of unimolecular species decreases while that of multimolecular species increases, and this behavior is independent of buffer media. These results suggest that the catalytically active form, studied in the presence of K+, may be a parallel, multistranded quadruplex rather than an antiparallel, unimolecular quadruplex.     

RNA-metabolism in Streptomyces hydrogenans. Effect of 20beta-hydroxysteroid-dehydrogenase inducing dienediol on RNA-content and RNA-profile.

For the isolation of RNA and polysomes, logarithmically growing cells were disrupted by grinding with kieselgur. Other methods failed to produce undergraded m-RNA as shown by 10-50% sucrose gradient-centrifugation and electrophoresis in 2% mixed agarose-acrylamide gel columns. RNA was extracted by a modified phenol method or in presence of DEP. After the application of dienediol, the amount of acid precipitable RNA decreased by 20 to 50% in 20 min. Incorportation of precursors into RNA was much slower immediately after induction but increased during the next two hours. Three hours after induction there was no difference in the RNA-content of induced and control cultures. Degradation of stable as well as of unstable RNA was observed. Simultaneous addition of the inducer and rifamycin inhibited the production of 20beta-STDH suggesting the synthesis of special m-RNA. The effects of some other antibiotics have also been studied. The existence of a specific m-RNA in the induced system was established from differences in the 3H/14C quotients of gel fractions in the region between light r-RNA and t-RNA.     

Effects of ribosomal wash factors and spermidine on endogenous and exogenous mRNA stimulated protein synthesis in the wheat germ cell-free system.

Differential effects of Mg2+, spermidine, and reticulocyte ribosomal wash factors on the translation of endogenous, myeloma, and globin mRNA's have been observed in studies with the wheat germ cell-free protein synthesizing system. Spermidine stimulated globin mRNA translation but not the translation of endogenous wheat germ messages, and the polyamine actually inhibited the translation of myeloma mRNA. Ribosomal wash factors, on the other hand, stimulated endogenous and myeloma mRNA dependent protein synthesis in an Mg2+-dependent fashion but inhibited globin mRNA translation. The combination of ribosomal wash factors and spermidine was either stimulatory or inhibitory depending on the Mg2+ concentration and the message. It was further observed that translation of exogenous myeloma mRNA proceeded for only 60 min at 25 degrees C under all conditions tested in this study, while translation of endogenous wheat germ messages continued for longer periods of time. No differential effects of spermidine on the synthesis of high molecular weight myeloma proteins were observed.     

Relationship between intact cell ATP levels and glucocorticoid receptor-binding capacity in the AtT-20 cell

A study was made on the correlation between the degree of membrane fusion and surface tension increase of phosphatidic acid membranes caused by divalent cations. Membrane fusion was followed by the Tb3+/dipicolinic acid assay, monitoring the fluorescent intensity for mixing of the internal aqueous contents of small unilamellar lipid vesicles. The surface tension and surface potential of monolayers made of the same lipids as used in the fusion experiments were measured as a function of divalent cation concentration. It was found that the 'threshold' concentration to induce massive vesicle membrane fusion was the same for Ca2+ and Mg2+, and that the surface tension increase in the monolayer, induced by changing divalent cation concentration from zero to a concentration which corresponds to its threshold value, inducing vesicle membrane fusion, was approximately the same: 6.3 dyn/cm for both Ca2+ and Mg2+. Both the divalent cation's threshold concentrations as well as the surface tension change corresponding to the threshold concentration for the phosphatidic acid membrane were smaller than those for the phosphatidylserine membrane. The different fusion capability of these divalent cations for phosphatidic acid and phosphatidylserine membranes is discussed in terms of the different ion binding capabilities of these ions to the membranes.     

Cytogenetical Toxical Effects of Heavy Metals on Vicia faba and Inquires into the Vicia Micro nucleus

Under the treatment of heavy metal ions Pb2+, Cd2+ and Hg2+, the interval of mitotic stage was shortened, and the time of interphase prolonged so that the cell cycle was prolonged in the root-tip cells of broadbean (Vicia faba L. ). With the increase of concentrations of Pb2+, Cd2+ and Zn2+ below 1.0, 0.01 and 10 ppm respectively, the mitosis index (IM) rose in root-tip cells, but IM decreased when the root-tips were treated with the some heavy metal ions above the above-mentioned respective concentrations. IM was inhibited in Hg2+ of any concentrations. Within the concentration of Pb2+, Cd2+, Hg2+ and Zn2+ below 1.0, 0. 50, 5.0, 100.0 ppm respectively, the frequency of micronucleus (MCNF) rose as the concentrations were increased, and lowered as the respective concentrations exceeded those stated above. Similar changes occurred in the frequency of chromosomal aberrations (CAF) when the concentration of Pb2+, Cd2+, Hg2+, Zn2+ were below or above 5.0, 5.0, 0.50, 100.0 ppm respectively. Mn2+ had no significant effects to them. By data processing with the method of gray system control and computer aided drawing to IM, MCNF and CAF, it was shown that the three parameters varied tremendously in different dose-effect ranges. All of which suggested that in order to obtain a reliable results in the environmental monitoring and hazardous material detection, genetoxicity inspection should be carried out under the optimal condition when (1) the concentration of the heavy metal to be detected does not seriously inhibit mitosis and (2) CAF and MCNF is in positive correlation.     

Effects of Pb2+ on the transient outward potassium current in acutely dissociated rat hippocampal neurons

Modulation of the voltage-dependent transient outward potassium current (IA) by Pb2+ was studied in acutely dissociated rat hippocampal pyramidal cells from the CA1 region at postnatal ages 7-14 days using the conventional whole-cell patch-clamp technique. In the presence of different concentrations of external Pb2+, the initial delay and activation time of IA were concentration-dependently lengthened. In particular, the initial delay was even longer in 1 mM Pb2+, showing no signs of saturation. Pb2+ also slowed the inactivation of IA, for decay time constants in the presence of Pb2+ were increased under the same experimental protocols. The activation curves, which were reasonably fitted by a single Boltzmann function, illustrated that Pb2+ increased the voltage threshold of IA and shifted the normalized activation current-voltage curves to more depolarizing voltage commands. Moreover, Pb2+ significantly affected the steady-state inactivation of IA. The application of Pb 2+ made the curves of the steady-state inactivation of IA shift to more depolarizing voltages with little change in the slopes factors. In brief, the results demonstrated that Pb2+ is a dose- and voltage-dependent, reversible blocker of IA currents of hippocampal CA1 neurons. The observations were fitted by the revised "Kuo and Chen type model", which postulates a Pb2+-selective site near the pore of the IA channel and that modulation of the IA channel by Pb2+ is the result of the competitive influences of Pb2+ on opening and inactivating different pathways.     

Interaction of metal ions with lupin seed conglutin gamma

Various metal ions were capable of aggregating and precipitating conglutin gamma, an oligomeric glycoprotein purified from Lupinus albus seeds, at neutral pH values. The most effective metal ions, at 60-fold molar excess to the protein, were Zn2+, Hg2+ and Cu2+; a lower influence on the physical status of conglutin gamma was observed with Cr3+, Fe3+, Co2+, Ni2+, Cd2+, Sn2+, and Pb2+, while Mg2+, Ca2+ and Mn2+ had no effect at all. The insolubilisation of the protein with Zn2+, which is fully reversible, strictly depended on both metal concentration and pH. with middle points of the sharp transitions at three-fold molar excess and pH 6.5, respectively. Conglutin gamma is also fully retained on a metal affinity chromatography column at which Zn2+ and Ni2+ were complexed. A drop of pH below 6.0 and the use of chelating agents, such as EDTA and imidazole, fully desorbed the protein. A slightly lower binding to immobilised Cu2+ and Co2+ and no binding with Mg2+, Cd2+ and Mn2+ were observed. The role of the numerous histidine residues of conglutin gamma in the binding of Zn2+ is discussed.     

Metal ion requirements for sequence-specific endoribonuclease activity of the Tetrahymena ribozyme

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena thermophila acts as an enzyme, catalyzing sequence-specific cleavage of RNA substrates. We have now examined the metal ion requirements of this reaction. Mg2+ and Mn2+ are the only metal ions that by themselves give RNA enzyme activity. Atomic absorption spectroscopy indicates that Zn, Cu, Co, and Fe are not present in amounts equimolar to the RNA enzyme and when added to reaction mixtures do not facilitate cleavage. Thus, these ions can be eliminated as cofactors for the reaction. While Ca2+ has no activity by itself, it alleviates a portion of the Mg2+ requirement; 1 mM Ca2+ reduces the Mg2+ optimum from 2 to 1 mM. These results, combined with studies of the reactivity of mixtures of metal ions, lead us to postulate that two classes of metal ion binding sites are required for catalysis. Class 1 sites have more activity with Mn2+ than with Mg2+, with the other divalent ions and Na+ and K+ having no activity. It is not known if ions located at class 1 sites have specific structural roles or are directly involved in active-site chemistry. Class 2 sites, which are presumably structural, have an order of preference Mg2+ greater than or equal to Ca2+ greater than Mn2+ and Ca2+ greater than Sr2+ greater than Ba2+, with Zn2+, Cu2+, Co2+, Na+, and K+ giving no detectable activity over the concentration range tested.     

重金属胁迫对真菌生长及发酵液pH的影响

【背景】矿区废渣堆重金属污染严重,废渣堆分布着一些耐重金属的微生物。【目标】探究重金属胁迫对真菌生长及发酵液pH的影响。【方法】从金川矿区废渣堆采集土样,分离培养具有产酸能力的真菌,采用形态学与分子生物学技术鉴定这些菌株,并测定其产酸能力及其对Pb~(2+)、Cd~(2+)和Zn~(2+)的耐受性。【结果】形态学及18S rRNA基因序列分析获得黑曲霉ZJ-I (Aspergillus niger ZJ-I)和产黄青霉ZJ-V (Penicilium chrysogenum ZJ-V)两个产酸菌株。未加重金属培养时,与不接种真菌对照相比,上述2个菌株的发酵液pH分别下降0.58和0.69;添加重金属处理后,随着重金属浓度的增加,pH变化幅度变小,不同浓度Pb~(2+)使A.nigerZJ-I发酵液pH值分别下降0.53、0.39、0.34和0.39,使P. chrysogenum ZJ-V发酵液pH值分别下降0.21、0.23、0.14和0.09;不同浓度Cd~(2+)使A. niger ZJ-I发酵液pH值分别下降0.75、0.43、0.39和0.32,使P. chrysogenum ZJ-V发酵液pH值分别下降0.62、0.46、0.38和0.49;不同浓度Zn~(2+)可使A.nigerZJ-I发酵液pH分别下降0.87、0.61、0.57和0.43,使P. chrysogenum ZJ-V发酵液pH分别下降1.1、0.34、0.44和0.49;低浓度的Zn~(2+)对菌株A.niger ZJ-I和P. chrysogenum ZJ-V产酸都有促进作用,低浓度的Cd~(2+)对A. niger ZJ-I产酸有促进作用。当Cd~(2+)、Zn~(2+)与Pb~(2+)的浓度分别超过200、400、2 000 mg/L时,3种不同浓度的重金属对菌株A. niger ZJ-I的抑制率达到80%以上,抑制效果显著;当Cd~(2+)、Zn~(2+)与Pb~(2+)浓度分别超过200、1 000、2 000 mg/L时,3种不同浓度的重金属对菌株P.chrysogenumZJ-V抑制率达到80%以上,抑制效果显著。【结论】两株真菌均具有产酸能力和一定的重金属耐受性,菌株P. chrysogenum ZJ-V发酵液产酸性能与重金属耐受能力都要优于ZJ-I,菌株ZJ-V具备潜在的淋洗重金属污染土壤的能力。     

Estimation of polyamine binding to macromolecules and ATP in bovine lymphocytes and rat liver.

To estimate the polyamine distribution in bovine lymphocytes and rat liver, the binding constants (K) for DNA, RNA, phospholipid, and ATP were determined under the conditions of 10 mM Tris-HCl, pH 7.5, 2 mM Mg2+, and 150 mM K+. The binding constants of spermine for calf thymus DNA, Escherichia coli 16 S rRNA, phospholipid in rat liver microsomes and ATP were 1.15 x 10(2), 6.69 x 10(2), 2.22 x 10(2), and 5.95 x 10(2) M-1, respectively. From these binding constants and experimentally determined cellular concentrations of macromolecules, ATP, and polyamines, spermine distribution in the cells was estimated. In bovine lymphocytes, the mols of spermine bound to DNA, RNA, phospholipid, and ATP were 0.79, 3.7, 0.23, and 4.3 per 100 mol of phosphate of macromolecules or ATP, respectively. In rat liver, they were 0.19, 1.0, 0.05, and 0.97/100 mol of phosphate of macromolecules or ATP, respectively. The binding constants of spermidine for macromolecules and ATP were smaller than those of spermine, but a similar tendency was observed with spermidine distribution among macromolecules and ATP in the above two cells. The amount of polyamine bound to DNA and phospholipid was significantly lower than that to RNA. When either the Mg2+ or K+ concentration increased, the amount of free spermine and that bound to RNA and ATP increased, but the amount of spermine bound to DNA and phospholipid decreased. The results indicate that most polyamines exist as a polyamine-RNA complex in cells. Under the conditions that globin synthesis is stimulated by spermine in a rabbit reticulocyte cell-free system, the amount of spermine bound to RNA was very close to the value estimated in the cells.