高静水压诱导水稻变异的初步研究

以高静水压处理水稻粤香占、粤丰占、998、999和毕粳38种子后的当代群体(M1),以及从中筛选到的突变株粤变长、巨无霸、粤丰压变1号-5号、毕籼为材料,研究了静水高压对水稻生长发育及其农艺性状的影响,结果发现：(1)与对照相比,粤香占和998处理当代群体的株高和有效穗数明显增加,但株叶形态没有明显变化;粤丰占、999和毕粳38的处理群体没有明显变化;(2)粤变长和巨无霸从第二代(M2)到第四代(M4)性状稳定,未发现明显分离,但在株高、有效穗数、穗粒数、粒长和千粒重等方面,与对照相比明显不同;(3)在M,代,粤丰压变1号一5号中除粤丰压变2号外的株高都发生了分离,而且粤丰压变4号分离出了大粒型和长芒型的植株;(4)毕籼从Mz代到lVt,代,性状稳定,但其株高、穗长、粒长和穗粒数均比对照显著增加,结实率和千粒重则明显降低;(5)经静水高压处理后的粤丰占种子发芽试验中,出现了双苗和3苗现象。这些结果表明静水高压能够影响水稻的生长发育,并能诱导水稻产生变异。     

Studies on Urate Oxidase of Candida utilis

Some physical and chemical properties of urate oxidase (EC 1.7.3.3) isolated from the cells of Candida utilis were investigated. The molecular weight was estimated to be 1.2 × 10⁵ by the equilibrium sedimentation and gel filtration methods. The isoelectric point was determined as 5.4 by the method of density electrofocusing. The enzyme showed a slight absorption at 410 mμ, and the absorbancy at this wave length was only 3% of that at 280 mμ. Contrary to urate oxidase from swine liver, the enzyme from yeast contained a negligible amount of copper, but it contained iron of nearly one atom per mole of the enzyme protein. The yeast urate oxidase was not inactivated by some chelators. However, it was easily inactivated with certain heavy metal ions such as Hg²⁺, and the inactivated enzyme was reactivated by the addition of thiols, indicating that the enzyme is a sulfhydryl enzyme. The inactivation of the enzyme with urea, on the other hand, was greatly accelerated by the addition of thiols, and some discussion was added to the results obtained.     

Different Pressure Resistance of Lactate Dehydrogenases from Hagfish is Dependent on Habitat Depth and Caused by Tetrameric Structure Dissociation

The effects of high hydrostatic pressure on lactate dehydrogenase (LDH) activities from two species of hagfish were examined. LDH from Eptatretus okinoseanus, a deep-sea species, retained 67% of the original activity even at 100 MPa. LDH activity from Eptatretus burgeri, a shallow-sea species, was completely lost at 50 MPa but recovered to the original value at 0.1 MPa. The tetrameric structure of LDH-A₄ from E. okinoseanus did not change at 50 MPa. In contrast, almost all LDH tetramers from E. burgeri dissociated to dimers and monomers at 50 MPa but reverted to tetramers at 0.1 MPa. These results show that the dissociation of tetramers caused the inactivation of E. burgeri LDH. The difference depends on the number 6 and 10 amino acids. The mechanism of the slight, gradual inactivation of E. okinoseanus LDH at high pressure differs and is probably due to the metamorphosis of its inner structures.     

重组尿酸氧化酶发酵工艺优化

目的:优化并获得重组尿酸氧化酶(rUOX)基因工程大肠杆菌BL21(DE3)/pET-32a-uox高密度发酵的工艺参数。方法:在三角摇瓶中进行培养条件的优化实验,分别考察了pH值、接种量、无机盐、碳源、诱导强度等对工程菌生长和重组蛋白表达的影响,得到了优化的发酵条件;在此基础上放大至NBS BIOFLO 110 14 L发酵罐,通过对诱导时机的优化,利用分批补料发酵的方式,使rUOX在高密度培养的条件下得到高表达。结果:在优化的发酵条件下,菌体密度(D600nm)最终达到50以上,相当于20 g/L干重;可溶性rUOX占菌体总蛋白量的45%,其含量达到3.45 g/L。结论:为规模化制备重组黄曲霉尿酸氧化酶奠定了基础。     

Induction of Oxidative Stress by High Hydrostatic Pressure in Escherichia coli

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in Escherichia coli MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (katE, katF, oxyR, sodAB, and soxS) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized E. coli toward t-butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of E. coli MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of E. coli by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.     

Imbibition Influenced by Hydrostatic Pressure

Pea seeds were soaked in distilled water or in saturated D-mannitol solution either under normal or under high external pressures (10-30 atms.) for approximately 24 hours. Imbibition in distilled water was independent of the hydrostatic pressure. However, imbibition in D-mannitol solution was enhanced by the pressure. It was concluded that hydrostatic pressure caused by turgor could enhance imbibition of the protoplast. Under high turgor, the protoplast may swell even at a high osmotic potential. Turgor seems to be of importance for the physiological activity of the plant cell.     

Evaluation of Structural Changes Induced by High Hydrostatic Pressure in Leuconostoc mesenteroides

Scanning electron microcopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC) were used to evaluate structural changes in Leuconostoc mesenteroides cells as a function of high-hydrostatic-pressure treatment. This bacterium usually grows in chains of cells, which were increasingly dechained at elevated pressures. High-pressure treatments at 250 and 500 MPa also caused changes in the external surface and internal structure of cells. Dechaining and blister formation on the surface of cells increased with pressure, as observed in SEM micrographs. TEM studies showed that cytoplasmic components of the cells were affected by high-pressure treatment. DSC studies of whole cells showed increasing denaturation of ribosomes with pressure, in keeping with dense compacted regions in the cytoplasm of pressure-treated cells observed in TEM micrographs. Apparent reduction of intact ribosomes observed in DSC thermograms was related to the reduction in number of viable cells. The results indicate that inactivation of L. mesenteroides cells is mainly due to ribosomal denaturation observed as a reduction of the corresponding peak in DSC thermograms and condensed interior regions of cytoplasm in TEM micrographs.     

Induction of Shiga Toxin-Converting Prophage in Escherichia coli by High Hydrostatic Pressure

Since high hydrostatic pressure is becoming increasingly important in modern food preservation, its potential effects on microorganisms need to be thoroughly investigated. In this context, mild pressures (<200 MPa) have recently been shown to induce an SOS response in Escherichia coli MG1655. Due to this response, we observed a RecA- and LexA-dependent induction of lambda prophage upon treating E. coli lysogens with sublethal pressures. In this report, we extend this observation to lambdoid Shiga toxin (Stx)-converting bacteriophages in MG1655, which constitute an important virulence trait in Stx-producing E. coli strains (STEC). The window of pressures capable of inducing Stx phages correlated well with the window of bacterial survival. When pressure treatments were conducted in whole milk, which is known to promote bacterial survival, Stx phage induction could be observed at up to 250 MPa in E. coli MG1655 and at up to 300 MPa in a pressure-resistant mutant of this strain. In addition, we found that the intrinsic pressure resistance of two types of Stx phages was very different, with one type surviving relatively well treatments of up to 400 MPa for 15 min at 20°C. Interestingly, and in contrast to UV irradiation or mitomycin C treatment, pressure was not able to induce Stx prophage or an SOS response in several natural Stx-producing STEC isolates.     

Response of Pathogenic Vibrio Species to High Hydrostatic Pressure

Vibrio parahaemolyticus ATCC 17802, Vibrio vulnificus ATCC 27562, Vibrio cholerae O:1 ATCC 14035, Vibrio cholerae non-O:1 ATCC 14547, Vibrio hollisae ATCC 33564, and Vibrio mimicus ATCC 33653 were treated with 200 to 300 MPa for 5 to 15 min at 25°C. High hydrostatic pressure inactivated all strains of pathogenic Vibrio without triggering a viable but nonculturable (VBNC) state; however, cells already existing in a VBNC state appeared to possess greater pressure resistance.     

高静水压培养对内皮酯酶表达的影响

探讨高静水压培养对内皮脂酶表达的影响及其机制．分大气压(0 mmHg)和高于1个标准大气压的递增压力(120、150、180 mmHg)培养人脐静脉内皮细胞,用蛋白酶体抑制剂MG132进行干预．RT-PCR检测内皮脂酶 mRNA的表达,间接免疫荧光技术和流式细胞术检测内皮脂酶蛋白的表达．结果显示,高静水压培养明显促进内皮脂酶mRNA和蛋白质的表达,180 mmHg培养24 h内皮脂酶mRNA的表达较对照组上调2.2倍(P < 0.001),内皮脂酶蛋白表达较对照组上调2.54倍(P < 0.001)．蛋白酶体抑制剂MG132干预明显抑制180 mmHg培养诱导的内皮脂酶 mRNA的表达,MG132干预组内皮脂酶mRNA表达约为180 mmHg培养组的50% (P < 0.05)．结果说明,高静水压上调内皮脂酶 mRNA和蛋白质的表达,其机制可能与核因子-κB活化有关．     

New Mathematical Modeling Approach for Predicting Microbial Inactivation by High Hydrostatic Pressure

A new primary model based on a thermodynamically consistent first-order kinetic approach was constructed to describe non-log-linear inactivation kinetics of pressure-treated bacteria. The model assumes a first-order process in which the specific inactivation rate changes inversely with the square root of time. The model gave reasonable fits to experimental data over six to seven orders of magnitude. It was also tested on 138 published data sets and provided good fits in about 70% of cases in which the shape of the curve followed the typical convex upward form. In the remainder of published examples, curves contained additional shoulder regions or extended tail regions. Curves with shoulders could be accommodated by including an additional time delay parameter and curves with tails shoulders could be accommodated by omitting points in the tail beyond the point at which survival levels remained more or less constant. The model parameters varied regularly with pressure, which may reflect a genuine mechanistic basis for the model. This property also allowed the calculation of (a) parameters analogous to the decimal reduction time D and z, the temperature increase needed to change the D value by a factor of 10, in thermal processing, and hence the processing conditions needed to attain a desired level of inactivation; and (b) the apparent thermodynamic volumes of activation associated with the lethal events. The hypothesis that inactivation rates changed as a function of the square root of time would be consistent with a diffusion-limited process.     

高压力对限制性内切酶活性的影响

以质粒ｐＳＰＯＲＴ１为底物研究了高压力对Ⅱ型限制性内切酶ＨｉｎｄⅢ和ＸｂａⅠ反应的影响。高压力未引起两酶底物碱基特异性的改变,但二者反应活力均随压力升高而逐渐下降,ＸｂａⅠ对高压力更敏感。引起ＨｉｎｄⅢ和ＸｂａⅠ限制性内切酶活性完全丧失的压力分别为２００和１８０ＭＰａ,半失活压力分别为１３０和７５ＭＰａ。     

斑马鱼尿酸氧化酶基因的克隆与原核表达

目的探讨斑马鱼尿酸氧化酶基因的克隆与原核表达的方法,研究重组斑马鱼尿酸氧化酶(ZUO)的理化性质与生物学功能。方法用PCR的方法克隆斑马鱼尿酸氧化酶基因,用pET28a质粒进行原核表达。结果成功克隆了斑马鱼尿酸氧化酶基因并完成了测序鉴定,该基因在大肠杆菌表达系统中得到有效表达且具有体外氧化尿酸的生物学功能。目的蛋白为胞内可溶性蛋白,占可溶蛋白总量的50%左右。最佳诱导条件为27℃16 h,粗酶最佳溶解pH为9.16,在此缓冲液溶解条件下,粗酶比活性为1.94 U/mg。获得的重组ZUO产量达64.8 U/g湿菌,高于直接从动物肝脏组织萃取的尿酸氧化酶。结论斑马鱼尿酸酶基因能在大肠杆菌表达系统中表达,其溶解pH比基因工程表达的重组猪尿酸氧化酶略低,对后续开发痛风治疗新药有利。     

Growth of Streptococcus faecalis under High Hydrostatic Pressure and High Partial Pressures of Inert Gases

Growth of Streptococcus faecalis in a complex medium was inhibited by xenon, nitrous oxide, argon, and nitrogen at gas pressures of 41 atm or less. The order of inhibitory potency was: xenon and nitrous oxide > argon > nitrogen. Helium appeared to be impotent. Oxygen also inhibited streptococcal growth and it acted synergistically with narcotic gases. Growth was slowed somewhat by 41 atm hydrostatic pressure in the absence of narcotic gases, but the gas effects were greater than those due to pressure. In relation to the sensitivity of this bacterium to pressure, we found that the volume of cultures increased during growth in a volumeter or dilatometer, and that this dilatation was due mainly to glycolysis. A volume increase of 20.3 ± 3.6 ml/mole of lactic acid produced was measured, and this value was close to one of 24 ml/mole lactic acid given for muscle glycolysis, and interestingly, close to the theoretic volume increase of activation calculated from the depression of growth rate by pressure.     

Effects of High Hydrostatic Pressure on Coastal Bacterial Community Abundance and Diversity

Hydrostatic pressure is an important parameter influencing the distribution of microbial life in the ocean. In this study, the response of marine bacterial populations from surface waters to pressures representative of those under deep-sea conditions was examined. Southern California coastal seawater collected 5 m below the sea surface was incubated in microcosms, using a range of temperatures (16 to 3°C) and hydrostatic pressure conditions (0.1 to 80 MPa). Cell abundance decreased in response to pressure, while diversity increased. The morphology of the community also changed with pressurization to a predominant morphotype of small cocci. The pressure-induced community changes included an increase in the relative abundance of Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, and Flavobacteria largely at the expense of Epsilonproteobacteria. Culturable high-pressure-surviving bacteria were obtained and found to be phylogenetically similar to isolates from cold and/or deep-sea environments. These results provide novel insights into the response of surface water bacteria to changes in hydrostatic pressure.     

超高静压协同中温对凝结芽孢杆菌芽孢灭活动力学规律的研究

研究了超高静压协同中温对凝结芽孢杆菌芽孢在磷酸缓冲液和牛奶(经超高温灭菌)中灭活的动力学规律,并对超高静压的升压过程及相应的灭活效果进行了研究.结果表明,升压过程对凝结芽孢杆菌芽孢灭活的影响不能忽略,且随压力增加这种效果越强,最高使其下降1.77个数量级;凝结芽孢杆菌芽孢在牛奶中比在磷酸缓冲液中有更高的抗性;在3种拟合模型(线性、Weibull和Log-logistic模型)中,线性模型不适合模拟这些存活曲线,而Log-logistic模型能更好地模拟这些存活曲线,其次是Weibull模型.     

Morphological and Physiological Characterization of Listeria monocytogenes Subjected to High Hydrostatic Pressure

High hydrostatic pressure is a new food preservation technology known for its capacity to inactivate spoilage and pathogenic microorganisms. That inactivation is usually assessed by the number of colonies growing on solid media after treatment. Under normal conditions the method does not permit recovery of damaged cells and may underestimate the number of cells that will remain viable and grow after a few days in high-pressure-processed foodstuffs. This study investigated the damage inflicted on Listeria monocytogenes cells treated by high pressure for 10 min at 400 MPa in pH 5.6 citrate buffer. Under these conditions, no cell growth occurred after 48 h on plate count agar. Scanning electron microscopy, light scattering by flow cytometry, and cell volume measurements were compared to evaluate the morphological changes in cells after pressurization. All these methods revealed that cellular morphology was not really affected. Esterase activity, as assessed either by enzymatic activity assays or by carboxy fluorescein diacetate fluorescence monitored by flow cytometry, was dramatically lowered, but not totally obliterated, under the effects of treatment. The measurement of propidium iodide uptake followed by flow cytometry demonstrated that membrane integrity was preserved in a small part of the population, although the membrane potential measured by analytical methods or evaluated by oxonol uptake was reduced from −86 to −5 mV. These results showed that such combined methods as fluorescent dyes monitored by flow cytometry and physiological activity measurements provide valuable indications of cellular viability.     

超高静压协同中温对凝结芽孢杆菌芽孢灭活动力学规律的研究

研究了超高静压协同中温对凝结芽孢杆菌芽孢在磷酸缓冲液和牛奶(经超高温灭菌)中灭活的动力学规律, 并对超高静压的升压过程及相应的灭活效果进行了研究。结果表明, 升压过程对凝结芽孢杆菌芽孢灭活的影响不能忽略, 且随压力增加这种效果越强, 最高使其下降1.77个数量级; 凝结芽孢杆菌芽孢在牛奶中比在磷酸缓冲液中有更高的抗性; 在3种拟合模型(线性、Weibull和Log-logistic模型)中, 线性模型不适合模拟这些存活曲线, 而Log-logistic模型能更好地模拟这些存活曲线, 其次是Weibull模型。