Cold resistance in the lesser mealworm Alphitobius diaperinus (Panzer) (Coleoptera: Tenebrionidae)

We have investigated cold resistance, measured by the supercooling point (SCP) temperature, in life stages of the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae), collected in Brittany poultry houses. Mean SCP values drastically increased during the insect ontogeny: egg (-26.1 C), first instar larvae (-21.6 C), last instar larvae (-15.5 C), pupae (-11.6 C), teneral adults (-12.0 C) and mature adults (-13.1 C). Nymphal metamorphosis and adult maturation did not promote substantial decrease of freezing resistance. The SCP values reflect the physiological states of the developmental stages especially the absence of ice nucleating agents: (i) lower SCP values in egg and unfed newly-emerged larvae I (i.e. -25.1 C), (ii) higher SCP values in fed larvae (i.e. -14.7 C), pupae and adults most likely due to the presence of ice nucleation sites in the gut. A tropical species, A. diaperinus, seems not to use its potential cold hardiness even in winter to remain in this warm habitat in temperate regions.     

CO2激光预处理对UV-B辐射引起的小麦幼苗脂质过氧化伤害的防护作用

用20 mW·mm－2CO2激光对小麦萌发的种子分别照射1 min、3 min、5 min、7 min,待其长至幼苗期,在光背景（PAR）90 μmol·m－2·s－1条件下,用3.10 kJ·m－2 UV-B照射7 h·d－1,然后对其丙二醛（MDA）、还原型谷胱甘肽（GSH）、超氧化物岐化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）以及抗坏血酸（AsA）等含量进行测定.结果发现,CO2激光预处理可提高小麦GSH和AsA含量以及SOD、CAT、POD酶活性,降低MDA含量,从而抑制了由UV-B辐射引起小麦的脂质过氧化作用,以处理5 min为最适时间.     

抗氧化剂抗脂质过氧化机制的ESR研究

以NADH诱导的心肌线粒体损伤体系为模型,在体外用ESR研究了谷胱甘肽(GSH)、超氧化物歧化酶(SOD)及辣根过氧化物酶(HRP)的抗氧化机制.结果表明,低浓度的GSH可部分抑制体系中自由基的生成;SOD与HRP以适当比例共同作用,可完全抑制体系中自由基的生成,较好地防止脂质过氧化的发生.     

一些含-CH2CH2N(CH3或C2H5)2基团的药物对辐照红细胞膜中硫代巴比妥酸反应物及超弱化学发光强度的影响

用硫代巴比妥酸反应的荧光分析法和超微弱发光法检测了十种含—CH_2CH_2N(CH_3或C_2H_5).基团的临床应用药物对辐照膜脂过氧化的影响,结果表明绝大多数被试药物有较强的抗膜脂过氧化作用.此外,黄嘌吟-黄嘌吟氧化酶系统的实验表明部分被试药物具有较强的减少O_2的能力.对药物结构与其抗膜脂过氧化效率之间的关系进行了初步探讨,结果提示:具有多元环及一正电中心的—CH_2CH_2N(CH_3或C_2H_5)_2化合物可能多为膜脂过氧化的抑制物.     

基于紫外及荧光光谱方法的中药抗氧化应激作用研究

糖尿病引起患者全身多部位发生慢性并发症,严重危害患者的生命安全,成为其致死致残的主要原因。糖尿病患者体内的高血糖环境引发的组织内氧化应激反应增加是糖尿病并发症的重要发病机制之一,而蛋白非酶糖基化反应和脂质过氧化反应过程与氧化应激密切相关。从糖尿病并发症的发病机理出发,以氧化应激为基点,运用光谱法对17种中药提取物抗氧化应激反应的活性进行综合考察。利用大鼠肝脏体外脂质自氧化反应结合紫外光谱法对中药提取物抑制脂质过氧化反应的能力进行评价,中药黄芩和槐米具有较强的抑制活性,富含黄酮类、生物碱类及木脂素类提取物表现出较好的脂质过氧化反应抑制作用,而皂苷类成分的活性相对较弱。以牛血清白蛋白/葡萄糖(果糖)体系为糖基化筛选模型,利用荧光光谱法对体外避光孵育反应体系进行研究,中药槐米、黄芩和知母表现出了较强的非酶糖基化反应抑制能力。由17种中药提取物抗氧化应激反应的结果分析,富含黄酮类成分中药的抗氧化应激作用强于生物碱类、木脂素类和萜类的,皂苷类中药的活性相对较弱;而体外综合活性较好的中药槐米和黄芩可作为下一步研究的主要对象。本研究为进一步的药理活性研究提供了一定的理论指导,为开发防治糖尿病并发症的中药新药奠定基础。     

Solute accumulation in heat seedlings during cold acclimation: contribution to increased freezing tolerance

Accumulation of sugars, amino acids and glycinebetaine in leaf tissues during cold acclimation was simultaneously monitored and compared in three wheat cultivars that have different freezing tolerance. Freezing tolerance was the decreasing order of cv. Norstar (NO) > cv. Chihokukomugi (CH) > or = cv. Haruyutaka (HA). During cold acclimation, there was a significant increase in osmotic concentration in the three cultivars. The increase was largely due to the increase in soluble sugars and the extent of the increase was the greatest in NO and the least in HA. While there was a considerable increase in glucose, fructose and sucrose during the first week of cold acclimation, the increase in raffinose occurred only after the second week. The total sugar content was the order of NO > CH > HA after 4 weeks of cold acclimation. Proline increased in all cultivars after 1 week of cold acclimation but a prolonged cold acclimation resulted in different profiles: no further increase occurred in HA while an additional increase occurred in other two cultivars. In all three cultivars, a noticeable increase of glycinebetaine occurred only after the second week of cold acclimation with the amount being the order of NO > CH > HA. It is concluded that a substantial part of the increase in osmotic concentration during cold acclimation was due to the increase in sugars, but the extent of the contribution of each compatible solute is cultivar-specific and can be associated with the degree of the maximum freezing tolerance attainable.     

MR spectroscopic imaging of glutathione in the white and gray matter at 7 T with an application to multiple sclerosis

Detection of glutathione (GSH) is technically challenging at clinical field strengths of 1.5 or 3 T due to its low concentration in the human brain coupled with the fact that conventional single-echo acquisitions, typically used for magnetic resonance (MR) spectroscopy acquisitions, cannot be used to resolve GSH given its overlap with other resonances. In this study, an MR spectral editing scheme was used to generate an unobstructed detection of GSH at 7 T. This technique was used to obtain normative white (WM) and gray matter (GM) GSH concentrations over a two-dimensional region. Results indicated that GSH was significantly higher (P<.001) in GM relative to WM in normal subjects. This finding is consistent with previous radionuclide experiments and histochemical staining and validates this 7 T MR spectroscopy technique. To our knowledge, this is the first study to report normative differences in WM and GM glutathione concentrations in the human brain. Glutathione is a biomarker for oxidative status and this non-invasive in vivo measurement of GSH was used to explore its sensitivity to oxidative state in multiple sclerosis (MS) patients. There was a significant reduction (P<.001) of GSH between the GM in MS patients and normal controls. No statistically significant GSH differences were found between the WM in controls and MS patients. Reduced GSH was also observed in a MS WM lesion. This preliminary investigation demonstrates the potential of this marker to probe oxidative state in MS.     

Cryopreservation of Arabidopsis thaliana shoot tips

Development of a successful shoot tip cryopreservation method for Arabidopsis thaliana L. will enable researchers to use molecular tools to study processes important for successful cryopreservation in this model organism. We demonstrate that Arabidopsis can be successfully cryopreserved using either plant vitrification solution 2 (PVS2) or plant vitrification solution 3 (PVS3) as cryoprotectants prior to rapidly cooling shoot tips in liquid nitrogen (LN). Shoot tip regrowth after PVS2 cryoprotectant treatment was improved after cold acclimation treatments of 8 or 18 days. All of the shoots tips regrew after LN exposure when cryoprotected with PVS3 for 60 min at 22 degree C. In addition, shoot tips could be cryopreserved using a two-step cooling procedure with PGD (polyethylene glycol-glucose-dimethyl sulfoxide) as a cryoprotectant. The high levels of shoot formation after LN exposure of Arabidopsis shoot tips makes this a desirable system in which molecular tools can be used to examine how alterations in biochemical, metabolic and developmental processes affect regrowth after cryoprotective treatments.     

低能N+ 离子预处理对水稻UV-B辐射的当代损伤效应的影响

采用3种不同注量的N离子对供试材料进行了离子注入预处理, 再用10.08 kJ/(m2d1)增强UV-B辐射对其幼苗进行辐照试验, 测定了过氧化物酶（POD）活性、 过氧化物歧化酶（SOD）活性、 过氧化氢酶（CAT）活性、 丙二醛（MDA）含量、 还原型谷胱甘肽（GSH）含量和可溶性糖含量的变化。 研究结果表明: 经N离子注入预处理的水稻材料受到UV-B辐射时其POD和SOD活性明显升高, 并在注量为2.0×1017 ions/cm2时活性达到最强; CAT活性和MDA含量明显降低, GSH含量显著增加, 而可溶性糖的含量则变化程度很小。 从这些研究结果可以看出, 经过一定注量离子注入预处理的水稻材料当受到UV B辐射时能产生较强的抗氧化能力和防御能力,而且抗氧化系统优于碳水化合物系统而先于表达。 由此可见, 离子注入预处理对水稻UV-B辐射的生物学效应的影响相当明显。 The seedlings of rice (xindao18) were exposed to UV B (10.08 kJ/(m2d1 )) irradiation follo wing the pretreatment with three different implantation dosages of low energy N+ ions. Changes in the levels of the superoxide (POD),superoxide dismutase (SOD),catalase (CAT), malondialdehyde (MDA),glutathione (GSH) and soluble sugar were measured. The result showed that the UV-B irradiation on the seedlings of rice pretreated with low energy ions implantation could lead to increase activities in POD and SOD,and the maximum appeared on the dose of 2.0×1017 ions/cm2. Meanwhile,it made the content of GSH increased, and caused the activity of CAT and the content of MDA to be decreased. But there was no obvious change in soluble sugar. It was suggested that the rice pretreated by low energy ion implantation could enhance the antioxidation capacity and defensive ability when irradiated by UV-B, and the antioxidation system could be induced earlier than carbohydrate system. Therefore, the biological effects of UV-B irradiation on rice pretreated by low energy ion implantation were quite obvious.     

Antioxidant systems in supporting environmental and programmed adaptations to low temperatures

Hetero and endothermic adaptive responses arising as a result of natural responses to environmental cues include antioxidant systems that support adaptations to environmental low temperatures in the broadest sense. These temperatures induce phase changes in energy production and consequently changes in the concentration of reactive oxygen species (ROS). The latter may lead to oxidative stress and the impairment of cellular homeostasis and antioxidant defence systems (ADS) scavenge the ROS so generated. In endotherms the ADS responds to oxidative pressure during acute cold stress conditions, this response is tissue specific and does not extend to prevent other oxidative damage. The early acute phase of cold exposure is accompanied by a significant depletion in redox equivalents. Under such conditions it is questionable if ADS has the capacity to neutralize elevated levels of ROS since there is also an increased energy demand and enhanced ATP consumption. Prolonged exposure to cold leads to ADS adaptation. Hibernators and freeze-tolerant species elevate their ADS before hibernation or freezing in order to prepare for and cope with re-awakening. The involvement of ROS and the role of the ADS in organisms subjected to low temperatures are features intercalated into physiological mechanisms of homestasis. The exact mechanisms for ADS regulation have not been fully defined and are the subject of many ongoing intriguing scientific investigations.     

Recovery of embryos of Zizania palustris following exposure to liquid nitrogen

Factors affecting survival of embryos of Zizania palustris after exposure to liquid nitrogen were investigated. Moisture content was the most critical factor. Embryos desiccated to a moisture content between 0.36 and 0.56 g water per g dry weight had highest survival after liquid nitrogen exposure (56 +/- 4% and 52 +/- 9% respectively). The recovery of embryos in the light following liquid nitrogen exposure also influenced survival. Survival increased from 35 +/- 4% to 56 +/- 4% when embryos containing 0.36 g water per g dry weight were recovered in the dark compared to light. Embryos exposed to desiccation and freezing stresses were subject to increased levels of lipid peroxidation. Light exacerbated lipid peroxidation in recovering embryos. Furthermore, catalase and peroxidase, enzymes involved in reducing reactive oxygen species, increased in activity in response to stress, and were further up-regulated in tissues recovered in the light. This study suggests that there are a number of factors influencing the survival of tissues exposed to liquid nitrogen and recovery procedures that reduce oxidative stress should be employed.     

Persistence of pierisin-1 activities in the adult cabbage white butterfly,Pieris rapae,during storage after killing

Crude extracts from larvae, pupae and adults of cabbage white butterflies, Pieris rapae and Pieris brassicae, and green-veined butterfly, Pieris napi, have an ability to induce apoptosis in the human cancer cell lines. As apoptosis inducing protein, pierisin-1 and -2 have been isolated from pupae of P. rapae and P. brassicae, respectively, and shown to exhibit DNA ADP-ribosylating activity. Although the highest activity was detected in the late phase of larvae and early phase of pupae, certain activity was found in adult butterflies. In order to investigate distribution of substances having pierisin-like activities in butterflies, many species need to be analyzed. However, fresh samples of larvae and pupae are hard to obtain, especially if samples are of scarce species or from overseas. The usage of adult butterflies is practical to examine the distribution of pierisin-like activity in many species. In this study, we examined the cytotoxicity of crude extracts from adults of P. rapae against HeLa cells and DNA ADP-ribosylation ability during storage for 1, 2 and 8 weeks at room temperature after killing adult butterflies after eclosion. Body weights decreased to 18% for 8 weeks through dehydration. Cytotoxicity of samples from butterfly kept for 1, 2 and 8 weeks decreased to 47, 39 and 22%, respectively, of the control value. DNA ADP-ribosylating activity of the samples also decreased to 30, 27 and 23%. Similar reduction was observed on western blot analysis with anti-pierisin-1 antibody. Fortunately, these results suggest that cytotoxic and DNA ADP-ribosylating activity persists to some extent in the body after killing, at least for 8 weeks. Thus, butterfly adult samples kept for two months at room temperature can still be useful for examination of the presence of substance having pierisin-like activity.     

Toxicity of nano- and micro-sized ZnO particles in human lung epithelial cells

This is the first comprehensive study to evaluate the cytotoxicity, biochemical mechanisms of toxicity, and oxidative DNA damage caused by exposing human bronchoalveolar carcinoma-derived cells (A549) to 70 and 420 nm ZnO particles. Particles of either size significantly reduced cell viability in a dose- and time-dependent manner within a rather narrow dosage range. Particle mass-based dosimetry and particle-specific surface area-based dosimetry yielded two distinct patterns of cytotoxicity in both 70 and 420 nm ZnO particles. Elevated levels of reactive oxygen species (ROS) resulted in intracellular oxidative stress, lipid peroxidation, cell membrane leakage, and oxidative DNA damage. The protective effect of N-acetylcysteine on ZnO-induced cytotoxicity further implicated oxidative stress in the cytotoxicity. Free Zn²⁺ and metal impurities were not major contributors of ROS induction as indicated by limited free Zn²⁺ cytotoxicity, extent of Zn²⁺ dissociation in the cell culture medium, and inductively-coupled plasma-mass spectrometry metal analysis. We conclude that (1) exposure to both sizes of ZnO particles leads to dose- and time-dependent cytotoxicity reflected in oxidative stress, lipid peroxidation, cell membrane damage, and oxidative DNA damage, (2) ZnO particles exhibit a much steeper dose–response pattern unseen in other metal oxides, and (3) neither free Zn²⁺ nor metal impurity in the ZnO particle samples is the cause of cytotoxicity.     

Purification and characterization of uridine phosphorylase from the ice-nucleating bacterium, Pantoea agglomerans NBRC12686

The ice-nucleating bacterium, Pantoea agglomerans NBRC12686 responds to a decrease in temperature with the induction of proteins, which are classified as cold-induced proteins. When the temperature of the strain NBRC12686 culture was lowered from 30 degree C to 12 degree C, the viability after freezing treatment significantly improved. By the use of SDS-polyacrylamide gel electrophoresis and high-performance liquid chromatography (HPLC), we analyzed the cold acclimation response in strain NBRC12686. After a shift from 30 degree C to 12 degree C, several proteins and saccharides were synthesized. After 48 h of cold acclimation, the induction level of proteins increased. In addition, ribose-1-phosphate was fractionated by HPLC using a TSK gel Sugar AXG column. Cell-free extracts were prepared from a cold acclimation culture (30 degree C to 12 degree C) and a non-cold acclimation culture (30 degree C), and then subjected to SDS-PAGE. A protein of approximately 29.7-kDa was present in the cold acclimation culture but was not present in the non-cold acclimation culture. The 29.7-kDa protein was purified by various chromatographies. We found that apparent molecular mass of the protein was approximately 119-kD constructed of 4 subunits of 29.7-kDa each. Based on the analysis of the N-terminal amino acid sequences of proteins, the 29.7-kDa protein had 83 percent identity with that of uridine phosphorylase (UPase) obtained from Escherichia coli K-12. We confirmed that the 29.7-kDa protein was novel, judged by molecular mass different from the already-known UPase or cryoprotectants. The cryoprotective activity of UPase of 29.7-kDa protein for LDH was approximately 30 percent at 5.0 microgram per ml of the protein. Furthermore, UPase had a high level of cryoprotective activity even after treating at 70 degree C for 30 min, but had no activity after treating at 100 degree C. We could elucidate that UPase from strain NBRC12686 had a cryoprotective activity as well as an enzyme activity, and it seems that UPase works in two different mechanisms for freezing tolerance.     

Biochemistry and physiology of overwintering in the mature larva of the pine needle gall midge,Thecodiplosis japonensis (Diptera: cecidomyiidae) in Korea

The pine needle gall midge, Thecodiplosis japonensis, overwinters in the soil as a third instar mature larva. The metabolic and physiological compensations and adjustments during its overwintering and acclimation were studied. Field-sampled larvae in 1997/98 winter showed a significant increase in whole-body trehalose by January (5.71 +/- 0.09 vs. 9.41 +/- 0.42 mg/g wet weight) along with a more significant decrease in whole-body glycogen (16.25 +/- 0.18 vs. 5.65 +/- 0.45 mg/g wet weight). Afterwards, there was a partial reconversion of trehalose to glycogen. Moreover, trace amounts of glycerol and steady content of glucose as potential cryoprotectants were found during the overwintering period. Temperature acclimation of field-sampled larvae affects interconversion between trehalose and glycogen. Trehalose accumulation does not affect the larval supercooling capacity. The mean supercooling point of the larvae remained nearly constant at about -20 degree he winter and was unchanged after temperature acclimation. Low temperature survival experiment suggested that the larvae adopt a freeze-avoiding strategy for overwintering.     

Some attributes of cold hardiness of the gregarious ectoparasitoid Colpoclypeus florus (Hymenoptera: Eulophidae)

Cold hardiness of diapause and non-diapause larvae of the parasitoid Colpoclypeus florus Walker (Hymenoptera: Eulophidae) was examined in the laboratory. Mean supercooling point (SCP) for diapausing larvae was -26.7 degree C and for non-diapausing larvae immediately after their larval development, was -16.2 degree C. Mean SCP for non-diapause larvae at the prepupa stage was -19.1 degree C. A short period of acclimation (1 week at 5 degree C) had no influence on the mean SCPs of both diapause and non-diapause larvae. Pre-freeze mortality for diapause and non-diapause larvae was also studied. A constant exposure of diapause larvae to -6 degree C resulted in high mortality (70.7 percent) after a period of 40 days. In contrast, 6 days at -6 degree C were sufficient to cause the same level of mortality in non-diapause larvae. After exposure of 15 days at -9 degree C, mortality for non-diapause larvae was 70 percent, whereas after 20 days at the same temperature mortality of diapause larvae was 25 percent. The importance of these findings for the cryobiology of C. florus is discussed.     

Combined effects of ultrasound and antioxidants on the quality maintenance of bay scallop (Argopecten irradians) adductor muscles during cold storage

The combined effects of ultrasound and the antioxidants of bamboo leaves (AOB) on the quality maintenance of the adductor muscle of scallops (AMSs) during cold storage was investigated. Ultrasound power at 350 W coupled with AOB solution (2% w/v) (UAOB-350) was applied to treat the AMSs according to Taylor diagram analysis. The microstructure, oxidative changes (lipid and protein oxidation), total numbers of colonies, total volatile basic nitrogen, and texture of the AMSs during 6 days of cold storage were analysed. The results indicated that UAOB-350 treatment could effectively retard protein and lipid oxidation and bacterial growth and maintain better microstructure and texture characteristics than AOB solution treatment alone, prolonging the shelf life of the AMSs by 2 days during storage at 4 °C. These results indicate that the UAOB-350 combination method has promising potential to maintain the quality and extend the shelf life of AMSs during cold storage.     

辐照诱变对小麦生理学效应的影响

以小麦品种小偃81为材料, 利用低能N+离子, 60Co-γ射线以及紫外线UV-C等为诱变源, 对种子胚部进行辐照处理。 研究其对小麦发芽势、 发芽率、 根长及苗高等生理指标的影响。 以辐照后的幼苗为材料研究了低能N+注入对过氧化氢酶（CAT）、 过氧化物酶（POD）、 超氧化物歧化酶（SOD）活性及还原型谷胱甘肽（GSH）含量的影响。 研究结果表明: N+注入后种子活力先升高后降低, 苗高在5×1017 N+/cm2时显著低于对照; 60Co和紫外线辐照对种子活力有较显著的影响, 辐照后的根长和苗高均显著低于对照; N+束辐照后的幼苗CAT酶活随剂量的变化规律性不明显, POD和SOD酶活总体趋势随剂量的增加先升高后降低, GSH含量随剂量的增大先降低后升高。 由此可知, 辐照诱变可以诱导小麦一系列的生理变化。60Co-γ射线和紫外线UV C对小麦胚根的伤害较大, 导致幼苗在后期大量死亡。N+注入对小麦的损伤效应较60Co和紫外线γ射线和紫外线UV-C小, 一定注量的N+注入处理可促进小麦生长。 In this paper, the biological effects,such as germination percentage, germination index and the length of seedlings and roots were investigated by using low energy N+, 60Co- γ rays and ultraviolet UV C to irradiate the embryos of wheat seeds （Xiaoyan81）. The anti oxidative enzyme system (CAT,POD, SOD) and GSH content were studied as well. The results show that the vitality of seed increased and then reduced with increasing the dosage of N+ ion implantation. The length of roots and seedlings were significantly higher than control. The seed vigor was significant effected after irradiated by 60Co-γ rays and ultraviolet UV-C, but the length of roots and seedlings were significantly lower than control. After N+ implantation, the changing pattern of CAT was not obviously. The enzymatic activity of POD and SOD were increased at low dosage and reduced at high dosage. The content of GSH was reduced and then raised. The results proved that the damage induced by γ ray and UV C to the radicle of wheat was severe that result in a large number of seedling died. However the damage induced by N+ implantation was lower than that by rays and UV C irradiation. Certain dosage of ion implantation can promote the growth of wheat.     

Study of cell killing effect on S180 by ultrasound activating protoporphyrin IX

The present study was initiated to investigate the potential biological mechanism of cell killing effect on isolate sarcoma 180 (S180) cells induced by ultrasound activating protoporphyrin IX (PPIX). S180 cells were exposed to ultrasound for 30 s duration, at a frequency of 2.2 MHz and an acoustic power of 3 W/cm² in the presence of 120 μM PPIX. The viability of cells was evaluated using trypan blue staining. The generation of oxygen free radicals in cell suspensions was detected immediately after treatment using a reactive oxygen detection kit. A copper reagent colorimetry method was used to measure the level of FFAs released into cell suspensions by the process of cell damage induced by ultrasound and PPIX treatment. Oxidative stress was assessed by measuring the activities of key antioxidant enzymes (i.e., SOD, CAT, GSH-PX) in S180 tumor cells. Treatment with ultrasound and PPIX together increased the cell damage rate to 50.91%, while treatment with ultrasound alone gave a cell damage rate to 24.24%, and PPIX alone kept this rate unchanged. Colorimetry and enzymatic chemical methods showed that the level of FFAs in cell suspension increased significantly after the treatment, while the activity of all the above enzymes decreased in tumor cells at different levels, and were associated with the generation of oxygen free radicals in cell suspension after treatment. The results indicate that oxygen free radicals may play an important role in improving the membrane lipid peroxidation, degrading membrane phospholipids to release FFAs, and decreasing the activities of the key antioxidant enzymes in cells. This biological mechanism might be involved in mediating the effects on S180 cells and resulting in the cell damage seen with SDT.