斑蝥素对草地贪夜蛾Sf9细胞膜完整性和膜电位的影响

为明确斑蝥素对昆虫细胞膜的作用及其机理, 本研究利用草地贪夜蛾Spodoptera frugiperda的卵巢细胞系Sf9细胞作为实验材料, 采用透射电子显微技术(transmission electron microscope , TEM)、 激光共聚焦显微镜(laser scanning confocal microscope, LSCM)结合荧光探针FDA/PI及DiBAC4(3)技术研究斑蝥素(cantharidin, CTD)对Sf9细胞膜完整性及膜电位（membrane potential, MP）的影响。结果表明： 32 μmol/L CTD处理6 h和12 h后, 电镜观察均未发现细胞膜结构破损; FDA/PI染色后, 32 μmol/L CTD处理0.5 h后细胞FDA荧光强度比对照显著降低(P<0.05), 碘化丙啶（propidium iodide, PI）染色的细胞比例与对照无显著性差异(P≥0.05)。32 μmol/L CTD处理140 s后即引起MP发生显著性去极化(P<0.05); 64 μmol/L CTD处理瞬时MP发生显著性去极化(P<0.05); 32 μmol/L CTD处理3 h内及64 μmol/L CTD处理2 h 内MP仍保持显著性去极化(P<0.05), 之后去极化程度降低; 32 μmol/L CTD处理6 h及64 μmol/L CTD处理3 h时MP去极化与对照组相比已无显著性差异(P≥0.05)。结果说明, CTD处理短时间内可引起Sf9细胞膜电位去极化并维持一段时间, 同时导致细胞活性发生不可逆下降, 但未对细胞膜结构完整性产生破坏。     

Variations in the content of free and bound amino acids during dormancy and the first cell cycle in Helianthus tuberosus tuber explants

Abstract

Qualitative and quantitative analysis of free and bound amino acids and amides during dormancy and the most important phases of the first cell cycle was carried out in tubers of Helianthus tuberosus.

In the dormant tuber arginine was confirmed to be the most abundant amino acid. A high amount of asparagine was also present; on the contrary glutamine was found in very low concentrations. During the progression of dormancy, all the free amino acids and amides declined while aspartic and glutamic acid increased.

During the G₁ phase of the first cell cycle induced by 2,4-D, all the free amino acids and amides decreased with the exception of glutamic acid.

At 18, 20, 24 h of activation with 2,4-D, corresponding to the S phase and the beginning of mitosis, bound amino acids were also determined. In these phases of the cell cycle they increased reaching a maximum at 20 h; on the other hand the free amino acid and amide content, especially aspartic acid, asparagine and arginine, decreased with the exception of glutamic acid, alanine and phenylalanine.     

The measurement of water relations of plant cell culture. I. Water release in response to centrifuge-induced water potentials

Water loss by cell suspensions during centrifugation is well defined by simple physical principles. The major factors affecting water release during centrifugation are: duration of centrifogation, depth of the cell mass, density of cells, relative centripetal acceleration and centripetal force. Water release during centrifugation was best described by an exponential decay process with a decay constant that increases with acceleration from 0.31 ± 0.01 to 0.66 ± 0.12 min^?1 (mean ± SE) between 4 825 and 19 300 m s^?2, respectively. The cell mass relative water content (RWC) at equilibrium was not a function of rate of water loss and was constant for each acceleration. A centripetal force was generated by the mass of the cells being accelerated away from the axis of rotation. This force generated a pressure that removed some of the cell wall and symplast water, by compression at contact points between the cells and by compression of the cytoplasm. Pressure induced by centripetal forces ranging from ?0.02 to ?0.23 MPa gave a linear relationship (r² > 0.99) between force and RWC. The slope (0.900 MPa) was proportional to the cell wall modulus of elasticity (±). and the intercept was interpreted to give the mass of the cells at full turgor without interstitial water (RWC=1). This interpretation is supported by the findings, of two independent experiments. Centrifuged cells suspended at 100% relative humidity for over 48 h reached the same water content as predicted by the intercept. Interstitial water was labelled with solutions of polyethylene glycol (PEG. M_r 8 000), the diameter of which was too large to enter the pores of plant cell walls. Centripetal accelerations greater than 10 900 m s^?2 removed PEG-labelled water to levels below 0.9% of cell water content. Removal of interstitial water and other loosely bound water provided a convenient method for determination of growth, RWC and ±. The centrifugal methods provide the foundation for new quantitative methods for cell culture water relations analyses.     

Characterization of a membrane bound β-glucosidase responsible for the activation of oat leaf saponins

Oat leaves contain a β-glucosidase (= avenacosidase) specific for the cleavage of the C-26 bound glucose moiety of the oat saponins avenacosides A and B. This transformation activates the fungitoxicities of the avenacosides. Evidence is presented that this enzyme is bound to the tonoplast membrane. The solubilized enzyme showed a pH optimum of 6.0–7.0, a temperature optimum around 40°, a molecular weight of 68 000±3000 and a K_m of 183 (±16) μM. The enzyme is inhibited by Hg²⁺ (10^-2 M) but not by Cu²⁺ (10^-2 M).     

Fate of [carbonyl-14C]methabenzthiazuron in an arid region soil — effect of organic amendment,soil disturbance and fumigation

[Carbonyl-¹⁴C] methabenzthiazuron (MBT) was applied to an arid region soil at a rate of 5mg kg⁻¹ soil to give a¹⁴C content of 2400 KB kg⁻¹ soil. After 15 weeks of incubation at 22°C and 50% of the maximum water holding capacity of the soil, 7.2% of the applied¹⁴C was mineralized to¹⁴CO₂. Where the soil was amended with wheat straw, total mineralization increased to 17.3%. Soil disturbance caused a significant increase while chloroform fumigation caused a significant decrease in the rate of¹⁴CO₂ production, both from amended and unamended soils. These results suggest that MBT is degraded mainly through microbial co-metabolism. Wheat straw amendment resulted in increased transformation of MBT into soil humus. In unamended soil, a major portion of¹⁴C was recovered in fulvic acid and in fractions extracted with organic solvents. Recovery of¹⁴C in non-extractable bound residues (humins) increased as incubation progressed and seemed to be derived from the fulvic acid fraction, which showed a concomitant decrease. More than 99% of the residual¹⁴C in unamended soil consisted of unaltered MBT; the remainder occurred as 1-methyl-1 (benzthiazolyl) urea. In amended soil, a relatively higher percentage of the extractable¹⁴C was found in the metabolite. Small amounts of three unidentified¹⁴C-labelled compounds were also observed. In amended soil, disturbance caused a decrease in extractable-¹⁴C whereas fumigation caused a significant increase, as compared to the untreated control. The effects were more pronounced when the soils were reated at an early stage of incubation. In general, soil disturbance increased the availability of MBT for further transformations while chloroform fumigation decreased the process.     

The isolation and identification of the prosthetic group released from a bound form of abscisic acid

A bound form of abscisic acid, herein called adduct, has been isolated from peas and barley and has been shown to release methyl abscisate upon mild acid hydrolysis. Mass spectrometry of the methyl abscisate released by ²H₂SO₄ in ²H₂O showed that the remainder of adduct was linked as an enolate of the ketone of the moiety that releases methyl abscisate. Some evidence is adduced to suggest that adduct is a metabolic precursor of abscisic acid. The implications of the distribution of adduct between roots and shoots for the plant's response to stress is discussed.