Structural Evidence for α-Synuclein Fibrils Using in Situ Atomic Force Microscopy

Human α-synuclein is a presynaptic terminal protein and can form insoluble fibrils that are believed to play an important role in the pathogenesis of several neurodegenerative diseases such as Parkinson‘s disease, dementia with Lewy bodies and Lewy body variant of Alzheimer‘s disease. In this paper, in situ atomic force microscopy has been used to study the structural properties of α-synuclein fibrils in solution using two different atomic force microscopy imaging modes: tapping mode and contact mode. In the in situ contact mode atomic force microscopy experiments α-synuclein fibrils quickly broke into fragments, and a similar phenomenon was found using tapping mode atomic force microscopy in which α-synuclein fibrils were incubated with guanidine hydrochloride (0.6 M). The α-synuclein fibrils kept their original filamentous topography for over 1h in the in situ tapping mode atomic force microscopy experiments. The present results provide indirect evidence on how 13-sheets assemble into α-synuclein fibrils on a nanometer scale.     

Accumulation of cadmium and its effects on growth, development and hemolymph biochemical compositions in Boettcherisca peregrina larvae （Diptera： Sarcophagidae）

Newly emerged larvae of the fleshfly, Boettcherisca peregrina were exposed to two different CdCl2 concentrations of 100μg/g and 400 mg/g diet fresh weight （DFW）. They were administered in the diets until the end of larval stage. Cd-exposed larvae accumulated significant amounts of Cd and this accumulation increased with the exposure dose and time. The body weights were lightened and lengths of larvae were shortened considerably after Cd exposure, especially at the higher Cd concentration. The total larval duration was also extremely affected due to Cd exposure. The average duration was prolonged significantly by 14 h at the lower Cd concentration, while it was increased by 33.7 h over controls at the higher Cd concentration. A significant decrease in contents of either soluble proteins, total lipids or caloric values in the hemolymph occurred due to Cd exposure throughout the entire tested period but after 120 h of Cd exposure. In contrast, when exposed to Cd with its higher concentration, total sugar contents in the hemolymph were increased strikingly over the whole tested time, except after 96 h of Cd exposure, while they were not apparently altered except after 24 h of Cd exposure at the lower concentration. Thus, it is suggested that Cd exposure shows significant adverse impact on the growth and development, as well as metabolism, in larvae of this fleshfly, depending on its exposure time and dose.     

Use of a dietary exposure system for screening of insecticidal compounds for their toxicity to the planthopper Laodelphax striatellus

We developed a dietary exposure assay for screening insecticidal compounds for their toxicity and for assessing the side effects of insecticidal proteins produced by genetically engineered （GE） plants on the planthopper Laodelphax striatellus Fallen. The fitness bioassay confirmed that the diet fulfills the requirements to be used in the dietary exposure system. To validate the efficacy of the dietary exposure system, nymphs of L. striatellus were fed diets treated with different concentrations of an inorganic stomach poison, potassium arsenate （PA）, or a cysteine protease inhibitor, E-64. The results showed that with increasing concentrations of E-64, the larval development time was prolonged, the adult weight was reduced and the survival rate of L. striatellus was decreased. Similarly the survival rates of L. striatellus consistently decreased with increasing PA content in the diet. The data indicate that the dietary exposure assay is able to detect the effects of insecticidal compounds on L. striatellus. Subsequently, this assay was successfully used for assessing the potential toxicity of Cry2Aa. The results showed that L. striatellus larvae were not negatively affected when fed the artificial diet containing purified Cry2Aa at 300 μg/g diet. In the assay, the stability and bioactivity of crystal （Cry） proteins in the food sources were confirmed by enzyme-linked immunosorbent assay and sensitive-insect bioassays. These results show that L. striatellus is not sensitive to Cry2Aa. We conclude that the dietary exposure system is valid and useful for assessing the toxicity of insecticidal compounds produced by GE plants on planthoppers.     

Aluminum ions induce DNA synthesis but not cell proliferation in human fibroblasts in vitro

The effect in vitro of aluminum (Al) ions on DNA synthesis and human dermal fibroblast proliferation using [Al] concentrations from 1.85 to 74 μM and incubation periods of 1, 2, 3, 4, and 5 d was assessed. The lowest concentration of Al that exerted a slight positive, although not significant, effect on DNA synthesis was 1.85 μM, after d 3 or 5 of incubation. The stimulating action of Al was more evident and statistically significant from concentrations of 3.7 μM and 2 d exposure onward. This Al-induced effect on [³H] thymidine incorporation into DNA increased in a time-dependent manner as [Al] in the culture medium rose, provoking increments of up to 322% above the control at [Al] 74 μM and 5 d incubation. In contrast, Al salts moderately increased fibroblast division in a continuous manner only from 7.4 to 74 μM after 3 d of incubation. Although significant overall, the minimal and inconstant mitogenic activity of Al differs greatly from and is not parallel to DNA synthesis, which is not clearly related to exposure times or Al concentrations. Abnormalities in Al-induced cellular metabolic processes described herein and their influence on the cell cycle may constitute a toxicity mechanism for human tissues, leading to disease development. Further studies are required to determine whether these findings can be extrapolated to in vivo situations.     

Inhibitory effect of parvovirus H—1 on the formation of colonies of human hepatoma cell line in vitro and its tumors in nude mice

The inhibitory effect of parvovirus H-1 on the colonyforming ability.in vitro of QGY-7703,a cultured human hepatoma cell line,and on the formation and growth of its tumors in nude mice was studied.With higher multiplicity of infection(MOI) of H-1 given,survival of the QGY-7703 cells was found to be decreased.H-1 DNA amplification level at 30h postinfection(p.i.) was detected to be 7.4 times higher than that at 2h by dispersed cells assay,while the cells were delayed to enter into S phase.Plaques were formed in the indicator cells(new-born human kidney cell line,NBK) by progeny H-1 virus particles released from the infected QGY-7703 cells by infectious cell center assay.The formation of tumors in nude mice by QGY-7703 cells which were injected s c at 2h postinfection was observed to by prevented in 2 proups with given MOI 25 and 50.The tumor growth of MOI 10 group occurred at a lower exponential rate than that of control,after a 20d latent period.It was evident that parvovirus H-1 exhibited a direct inhibitory effect on the formation and growth of human hepatoma cells in vivo as well as in vitro.     

cDNA clone and expression analysis of α-Tropomyosin during Japanese flounder(Paralichthys olivaceus) metamorphosis

Tropomyosin(TM) plays a critical role in skeletal and cardiac muscle development and function. To assess the functional significance of α-TM in Japanese flounder(Paralichthys olivaceus) development and metamorphosis, cDNA from Japanese flounder was cloned and α-TM mRNA measured during development and metamorphosis. The full-length cDNA is 1 191 bp, including a 5'-untranslated region of 114 bp, a 3'-UTR of 222 bp, and an open reading frame of 855 bp encoding a polypeptide of 284 amino acids. Real-time quantitative PCR revealed that α-TM mRNA is initially expressed in unfertilized ovum, indicating the α-TM gene is maternal. Relatively low mRNA levels were observed in different embryonic stages. A higher level of α-TM mRNA was detected 3 days post hatching(dph), while the highest level was measured at 29 dph(metamorphic climax) after which it declined towards the end of metamorphosis. The expression of α-TM mRNA was up-regulated in thyroid hormone-treated larvae at 36 dph, but there was no marked difference at other stages when compared to control animals. After thiourea treatment, the expression of α-TM mRNA declined slightly. These data provide basic information that can be utilized in further studies into the role of α-TM in P. olivaceus development and metamorphosis.     

1,8-二羟基蒽醌对大型溞的毒性效应

The study on the acute,sublethal and chronic toxicity of 1,8-dihydroxyanthraquinone (1,8-dihAQ) to Daphnia magna showed that the 48 h LC₅₀ was 0.37 mg稬^-1,and the feeding behavior of Daphnia magna was severely affected by the compound.When exposed to 0.2 mg稬^-1 of 1,8-dihAQ for 5 h,the filtration and ingestion rate of Daphnia magna was inhibited by 97%.Chronic toxicity test results indicated that the reproduction ability decreased dramatically after exposing to sublethal concentration of 1,8-dihAQ.It could be inferred that reproduction parameters and intrinsic rate of natural increase were the sensitive parameters in characterizing sublethal toxicity.The NOEC and LOEC values for reproduction parameters were also given.     

Monovalent cation transporters; establishing a link between bioinformatics and physiology

Toxic aluminum (Al) ion is a major constraint to plant growth in acid soils. Aluminum tolerance in wheat (Triticum aestivum L.) is strongly related to the Al-triggered efflux of malate from root apices. A role of the secreted malate has been postulated to be in chelating Al and thus excluding it from root apices (malate hypothesis), but the actual process has yet to be fully elucidated. We measured Al content and root growth during and after Al exposure using seedlings of near-isogenic lines [ET8 (Al tolerant) and ES8 (Al sensitive)] differing in the capacity to induce Al-triggered malate efflux. Aluminum doses that caused 50% root growth inhibition during 24-h exposure to Al in calcium (Ca) solution (0.5 mM CaCl₂, pH 4.5) were 50 μM in ET8 and 5 μM in ES8. Under such conditions, the amount of Al accumulated in root apices was approximately 2-fold higher in ET8 than ES8. Al-treated seedlings were then transferred to the Al-free Ca solution for 24 h. Compared to control roots (no Al pretreatment), root regrowth of Al-treated roots was about 100% in ET8 and about 25% in ES8. The impaired regrowth in ES8 was observed even after 24-h exposure to 2.5 μM Al which had caused only 20% root growth inhibition. The addition of malate (100 μM) during exposure to 50 μM Al in ES8 enhanced root growth 1.6 times and regrowth in Al-free solution 7 times, resulting in similar root growth and regrowth as in ET8. Short-term Al treatments of ES8 for up to 5 h indicated that the Al-caused inhibition of root regrowth started after 1-h exposure to Al. The stimulating effect of malate on root regrowth was observed when malate was present during Al exposure, but not when roots previously exposed to Al were rinsed with malate, although Al accumulation in root apices was similar under these malate treatments. We conclude that the malate secreted from root apices under Al exposure is essential for the apices to commence regrowth in Al-free medium, the trait that is not related to the exclusion of Al from the apices.     

Effects of Phosphorus Nutrient on the Hydraulic Conductivity of Sorghum （Sorghum vulgare Pers.） Seedling Roots Under Water Deficiency

Hydroponic experiments were conducted in a growth chamber and changes in the hydraulic conductivity of sorghum (Sorghum vulgare Pers.) roots (Lpr) at the three-leaf stage were measured using the pressure chamber method. Water deficiency was imposed with polyethylene glycol (PEG) 6000 and the phosphorus (P) levels were controlled by complete Hoagland solution with and without P nutrient. The objective of this study was to investigate the effect of P nutrition on root Lpr under water deficiency. The results showed that the Lpr in P deficiency treatments decreased markedly, but the Lpr recovered to the same value as that of control when sufficient P was supplied for 4-24 h. Water deficiency decreased Lpr, but the hydraulic conductivity of the roots with sufficient P supply was still higher than that of plants without P supply. When resuming water supply, the Lpr of the water-deficient plants under P supply recovered faster than that of plants without P supply, which indicates that plants with sufficient P nutrient are more drought tolerant and have a greater ability to recover after drought. The treatment of HgCl2 indicated that P nutrient could regulate the Lpr by affecting the activity and the expression levels of aquaporins.     

Isolation and characterization of insecticidal activity of （Z）- asarone from Acorus calamus L.

The insecticidal activity of Acorus calamus L. rhizome-derived material against adults of Sitophilus zeamais Motschulsky was examined by using repellency method and contact toxicity. The biologically active constituent of the A. calamus rhizome was separated and identified. The results showed that the ethanol extract of A. calamus had strong repellency and contact effect to S. zeamais and the active constituent of the A. calamus was characterized as （Z）-asarone by spectroscopic analysis. Responses from the tests varied with exposure times and doses. In the repellency test, ethanol extract of A. calamus had 93.92% repellency at 629.08 μg/cm^2 but only 71.38% at 157.27 μg/cm^2 12 h after treatment. As a contrast, （Z）-asarone showed 84.50% repellency at 314.54μg/cm^2 and 77.02% at 78.63 μg/cm^2 12 h after treatment. In the filter paper diffusion test, ethanol extract of A. calamus caused 95.56% and 17.78% mortality to S. zeamais at 314.54 μg/cm^2 and 78.63 μg/ cm^2 4 days after treatment, while （Z）-asarone brought about 100.00% and 15.56% mortality at 40.89 μg/cm^2 and 15.73 μg/cm^2 respectively. These results indicate that the insecticidal activity of the A. calamus extract may be due to （Z）-asarone.     

Mutation of Residue Arginine^18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the α-subunit of cytochrome bsss （Cyt bsss） except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem （PS） Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSll complexes. In the present study, two arginine18 （R18） mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid （E） and glycine （G）. The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type （WT） levels and that, after irradiation at high light intensity, oxygen evolution for the PSll of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSll （Fv/Fm） of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the α-subunit of Cyt bsss and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt bss9 maintain the structure of the PSll complex and its activity, although it is not directly bound to the heme group.     

Interactive effects of α-NAA and UV-B radiation on the endogenous hormone contents and growth of Trichosanthes kirilowii Maxim seedlings

The impact of UV-B radiation on endogenous hormones in plants has recently drawn attention from researchers. The mechanism for reduced stem elongation by UV-B might be due to changes in the phytohormone levels, especially IAA, which plays a role in stem elongation. In this study, effects of UV-B radiation on Trichosanthes kirilowii Maxim (T. kirilowii) seedlings in greenhouse-grown plants were investigated. The results indicated that: (1) In comparison to controls, exposure to 0.029 Jm^?2 s^?1. UV-B radiation led to accumulation of endogenous abscisic acid (ABA) and zeatinriboside (ZR) in the plant contents, and decreased contents of endogenous indole-3-acetic acid (IAA) and gibberellic acid (GA_1/3). Exposure to UV-B radiation reduced the height and leaf area of plants. As a result, total biomass (plant dry weight) was lower. (2) In comparison to controls, addition of 2 mg l^?1 α-naphthaleneacetic acid (α-NAA) slightly increased the contents of IAA, GA_1/3 and ZR, and decreased the content of ABA in leaves. This addition of α-NAA significantly increased plant height and leaf area, but only slightly increased total biomass. (3) Addition of α-NAA to UV-B-exposed plants: increased the content of endogenous IAA, GA_1/3 and ZR; decreased accumulation of endogenous ABA; and increased plant height and leaf area in comparison to plants that only were exposed to UV-B. Moreover, total biomass increased slightly. This suggests that addition of α-NAA may compensate to a certain extent for the lack of IAA resulting from UV-B radiation; it also increases the content of GA_1/3 and ZR, decreases the accumulation of ABA, and promotes the growth of plants.     

Interactive effects of α-NAA and UV-B radiation on the endogenous hormone contents and growth of Trichosanthes kirilowii Maxim seedlings

The impact of UV-B radiation on endogenous hormones in plants has recently drawn attention from researchers. The mechanism for reduced stem elongation by UV-B might be due to changes in the phytohormone levels, especially IAA, which plays a role in stem elongation. In this study, effects of UV-B radiation on Trichosanthes kirilowii Maxim (T. kirilowii) seedlings in greenhouse-grown plants were investigated. The results indicated that: (1) In comparison to controls, exposure to 0.029 Jm^?2 s^?1. UV-B radiation led to accumulation of endogenous abscisic acid (ABA) and zeatinriboside (ZR) in the plant contents, and decreased contents of endogenous indole-3-acetic acid (IAA) and gibberellic acid (GA_1/3). Exposure to UV-B radiation reduced the height and leaf area of plants. As a result, total biomass (plant dry weight) was lower. (2) In comparison to controls, addition of 2 mg l^?1 α-naphthaleneacetic acid (α-NAA) slightly increased the contents of IAA, GA_1/3 and ZR, and decreased the content of ABA in leaves. This addition of α-NAA significantly increased plant height and leaf area, but only slightly increased total biomass. (3) Addition of α-NAA to UV-B-exposed plants: increased the content of endogenous IAA, GA_1/3 and ZR; decreased accumulation of endogenous ABA; and increased plant height and leaf area in comparison to plants that only were exposed to UV-B. Moreover, total biomass increased slightly. This suggests that addition of α-NAA may compensate to a certain extent for the lack of IAA resulting from UV-B radiation; it also increases the content of GA_1/3 and ZR, decreases the accumulation of ABA, and promotes the growth of plants.     

Distribution of α-D-mannose residue on zona pellucida and its role(s) in fertilization in pigs

The present study aims to identify the distribution of α-D-mannose residues on zona pellucida (ZP) and their role(s) in fertilization in pigs. In experiment 1, in vitro matured pig oocytes were freed from cu- mulus cells and treated with fluorescein isothiocyanate-labelled Lens culinaris (FITC-LCA), a D-mannose specific binding lectin. After 30 min of treatment, LCA bound evenly throughout the ZP with strong fluorescence. In experiment 2, when LCA-treated oocytes were used for in vitro fertilization, the number of sperm bound to ZP was significantly decreased, and sperm penetration was almost com- pletely blocked. In experiment 3, polysaccharide mannan was added to the in vitro fertilization medium as a competitive inhibitor. Both the number of sperm bound to ZP and the rate of fertilized oocytes were significantly reduced in the mannan-treated group compared with the control group. In experiment 4, spermatozoa were incubated with mannan in vitro. The number of acrosome-reacted spermatozoa was evidently increased in a time-dependent manner during the incubation. These results suggest that α-D-mannose residues presenting on pig ZP might be an important component of sperm receptor and might induce sperm acrosome reaction and thus facilitate the sperm penetration into the ZP.     

Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）, the partially purified MoFe protein （Avl） preparation was obtained from Azotobacter vinelandii Lipmann OP by chroma- tography on DEAE-cellulose （DE52） and Sephacryl S-200 columns and analyzed by PAGE and matrix- assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Avl band, the preparation was shown to have three other main bands that migrated slower than Av 1. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase （PGI）. In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av 1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaC1 concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.     

Effects of Exogenous Spermidine on Antioxidant System Responses of Typha latifolia L. Under Cd^2＋ Stress

The effects of foliar spraying with spermidine (Spd), ranging in concentration from 0.25 to 0.50 mmol/L, on the antioxidant system under Cd^2 stress (range 0.1- 0.2 mmol/L Cd^2 ) in Typha latifolia L. grown hydroponically were investigated in order to offer a referenced evidence for an understanding of the mechanism by which polyamines (PAs) relieve the damage to plants by heavy metal and improve the phytoremediation efficiency of heavy metal-contaminated water. The results showed that Cd^2 stress induced oxidative injury, as evidenced by an increase in the generation of superoxide anion (O2), as well as the hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents in both leaves and caudices. With the exception of superoxide dismutase (SOD) activity in the leaves, an increase in the activities of catalase (CAT), guaiacol peroxidase (GPX), and glutathione reductase (GR) was observed in both leaves and caudices, SOD activity was increased in caudices, and ascorbate peroxidase (APX) activity was increased in leaves following Cd^2 treatment. The reduced glutathione (GSH) content in both leaves and caudices and the reductive ascorbate content in leaves was obviously increased, which were prompted by the application of exogenous Spd. Spraying with Spd increased the activity of GR and APX in both leaves and caudices, whereas the activity of SOD, CAT, and GPX was increased only in caudices following spraying with Spd. The generation of O2 and the H2O2 and MDA content in both leaves and caudices decreased after spraying with Spd. The decrease in MDA was more obvious following the application of 0.25 than 0.50 mmol/L Spd. It is supposed that exogenous Spd elevated the tolerance of T. latifolia under Cd^2 stress primarily by increasing GR activity and the GSH level.     

Identification of Contaminations Hiding Beneath the α- and β-Subunits of Partially Purified Nitrogenase MoFe Protein on the Sodium Dodecyl Sulfate Gel

To identify the unknown proteins that would contaminate the α- and β-subunits of nitrogenase MoFe protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the partially purified MoFe protein (Av 1) preparation was obtained from Azotobacter vinelandii Lipmann OP by chromatography on DEAE-cellulose (DE52) and Sephacryl S-200 columns and analyzed by PAGE and matrixassisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The Av 1 preparation was shown to have two main bands at the position of the α- and β-subunits of crystalline Avl on the SDS gel. However, on the anoxic native PAGE, in addition to the Ay 1 band, the preparation was shown to have three other main bands that migrated slower than Avl. Of these three main bands, the protein with the fastest migration was identified as bacterioferritin elsewhere. The proteins on the other two bands, termed Upper and Middle, were suggested to be two different homopolymers with the same apparent subunit electrophoretic mobilities as the α- and β-subunits of Avl, respectively. By analysis of MALDI-TOF mass spectrometry, the Upper was identified as GroEL, which belongs to the heat shock protein 60 family, and the Middle was identified as glucose-6-phosphate isomerase (PGI). In our preparation, anoxic native electrophoresis indicated that GroEL was composed of 14 identical subunits and that PGI was composed of 10 identical subunits. This is the first report of PGI, with so many subunits. The contaminating proteins in the Av 1 preparation, mainly GroEL and PGI, could be totally or partially removed from Av1 if the shoulders and center of the elution peak were collected separately from the Sephacryl S-200 column and the center fraction was purified further by Q-Sepharose developed with an NaCl concentration gradient. Thus, Avl with more than 90% purity was obtained. Obviously, this modified method is useful for the purification of mutant MoFe proteins with a high purity.