Effect of solvents on the enzyme mediated degradation of copolymers

The biodegradation of polycaprolactone (PCL), polylactic acid (PLA), polyglycolide (PGA) and their copolymers, poly (lactide-co-glycolide) and poly (D, L-lactide-co-caprolactone) (PLCL) was investigated. The influence of different solvents on the degradation of these polymers at 37 degrees C in the presence of two different lipases namely Novozym 435 and the free lipase of porcine pancreas was investigated. The rate coefficients for the polymer degradation and enzyme deactivation were determined using continuous distribution kinetics. Among the homopolymers, the degradation of PGA was nearly an order of magnitude lower than that for PCL and PLA. The overall rate coefficients of the copolymers were higher than their respective homopolymers. Thus, PLCL degraded faster than either PCL or PLA. The degradation was highly dependent on the viscosity of the solvent used with the highest degradation observed in acetone. The degradation of the polymers in acetone was nearly twice that observed in dimethyl sulfoxide indicating that the degradation decreases with increase in the solvent viscosity. The degradation of the polymers in water-solvent mixtures indicated an optimal water content of 2.5 wt% of water.

Studies on rabbit reticulocyte ribosomes and polyribosomes and their relation to hemoglobin synthesis

This dissertation is divided into three parts.

The first section is concerned with protein synthesis in cellfree systems from reticulocytes. The sub-cellular reticulocyte fractions, reagents, etc. have been examined for the presence of traces of ribonuclease, using. an assay based upon the loss of infectivity of RNA fran bacteriophage MS2. This assay is sensitive to 5 x 10^-7 γ RNase/ml. In addition, the loss of synthetic capacity of an 80S ribosome on dissociation has been studied, and can be attributed to loss of messenger RNA when the monomer is separated into subunits. The presence of ribonuclease has been shown to be a major cause of polyribosome disintegration during cell-free protein synthesis.

The second section concerns the changes in ribosomes and polyribosomes which occur during the maturation of a reticulocyte into an erythrocyte. With increasing age, the cells lose a large proportion of the ribonucleoprotein, but the percentage of ribosomes present as polyribosomes is only slightly altered. The loss of hemoglobin synthesis on maturation is probably due to both the loss of total ribosomes and to the lessened specific activity of the polyribosomes.

The third section contains analytical ultracentrifugation data on 80S ribosomes, polyribosomes, and ribosomal RNA from reticulocytes. The 60s and 40s subunits, obtained by dissociation of the 80s particle with inorganic pyrophosphate, were also studied. The RNA from reticulocyte ribosomes has been examined under a variety of denaturing conditions, including dimethyl sulfoxide treatment, formaldehyde reaction and thermal denaturation. From these studies we can conclude that the 28S and 16S RNA's are single polynucleotide chains and are not made up of smaller RNA subunits hydrogen-bonded together.

普通烟草与毛曼陀罗族间体细胞杂交的研究

采用PEG／DMSO融合法，从普通烟草(Nicotiana tabacum L.cv.Xanthi nc)叶肉和毛曼陀罗(Datura innoxia Mill.)茎或叶愈伤组织原生质体融合获得I5株花盆中健康生长的族间体细胞杂种植株;其中11株是在没有选择压力的条件下获得，4株则是在有IOA和R6G选择系统存在下获得。同时获得一株试管开花且形态异常的植株和一株花盆中生长的嵌合植株。lOmM IOA和I5μ g／ml R6G均能分别有效地抑制烟草叶肉和毛曼陀罗愈伤组织原生质体的分裂;10% DMSO能显著提高原生质体融合率;PEG种类并不重要，但浓度则很重要;BAP较ZT，KT对植株分化有更好的诱导效果。杂种的形态、细胞、同工酶、Southern杂交，花粉育性分析结果如下：1、l5株杂种较双亲普遍株型矮小，生长缓慢，形态接近烟草但不很正常，根据形态特征可分为两种类型：(1)共有8株，其叶片大小、形状、颜色、开花习惯、花类型（单花）等均与毛曼陀罗接近，但子房败育;(2)共有7株，其株型、叶片形状、颜色、光滑度、花形状、类型（圆锥状花序）、颜色更接近烟草，但少数杂种开单花或先单花后圆锥状花序或先单花后两种花并存，且开花时间不一，部分子房败育。2、杂种染色体数目大都在60～90之间，个别者较少(48条)或较多(125条)，没有一株为双二倍体(2n=96)，并全部为混倍体。3、15株杂种植株均有双亲的细胞色素氧化酶同工酶特征谱带;大部分都有双亲的过氧化物酶同工酶特征谱带，少都仅具烟草的谱带。4、Hae Ⅲ／水稻rDNA的Southern杂交分析表明杂种1较双亲多一条谱带，杂种2较双亲也多一条弱带，其它杂种尚待定。5、花粉活力测定表明毛曼陀罗（种子再生而来）的为99%，烟草（原生质体再生而来）为80～90%，而杂种的为24～61%，育性普遍低于双亲。

果实对酵母拮抗菌和外源水杨酸诱导的抗病性应答机理

用生物和非生物因子来进行采后病害的防治，是一个非常有效的方法。诱导抗性作为控制果蔬采后病害的生物技术，已成为该领域的一个研究热点。然而诱导抗性的机制非常复杂，涉及到寄主、病原菌、激发子之间的相互作用关系。本研究主要利用酵母拮抗菌Pichia membranefaciens和SA处理果实，观察其抗性诱导表达和对采后青霉病菌（Penicillium expansum）的抑制作用，并从蛋白质组学水平上对诱导抗性的机理进行了分析。研究结果表明： 1、酵母拮抗菌P. membranefaciens (5 × 107 cells·ml-1)和SA(0.5 mM)处理采后甜樱桃果实，能够明显地降低病害的发病率和病斑直径。酵母菌和SA处理影响到了果实抗氧化酶的活性，同时还改变了POD同工酶谱和甜樱桃果实的总蛋白含量，并诱导了新的蛋白质条带产生。用光学显微镜和扫描电子显微镜技术观察发现，在in vitro条件下P. membranefaciens能够紧密地结合与病原菌的菌丝，而在in vivo条件下这种结合较为松散。 2、借鉴其它模式植物的方法，我们建立了一整套适用于多汁类植物材料的蛋白质组学研究方法。对于芒果，桃，甜樱桃、苹果以及冬枣等果实，都取得了重复性非常好的2-D图谱。我们应用该技术进一步研究了P. membranefaciens (1 × 108 cells·ml-1)以及SA (0.5 mM)处理对桃果实蛋白质组的诱导影响。结果显示，两种激发子处理都能够诱导桃果实产生抗性，从而减轻青霉病引起的腐烂。在诱导处理1 d以后，酵母拮抗菌和SA分别诱导22和16个蛋白的差异表达。质谱鉴定的蛋白属于6大类：代谢，防御反应，转录，能量途径以及细胞结构。有6个蛋白受到两种激发子的共同调控。其中，4种蛋白(包括glutathione peroxidase, polyphenol oxidase precursor, catalase和methionine sulfoxide reductase) 属于抗氧化蛋白，涉及到活性氧代谢。另2个蛋白（Major allergen Pru av 1和peroxidase）是病程相关蛋白，直接参与植物的防御反应。同时一些磷酸化酶和转录因子也受到两种激发子的调节从而参与果实的抗病反应。酶学测定和Northern杂交的结果表明，拮抗菌与SA处理均能影响过氧化氢酶活性及其基因的表达。 3、采前用较高浓度SA (2 mM) 短时间（10s）处理不同成熟期的甜樱桃果实，能够明显降低果实青霉病的病斑直径，并能减轻较低成熟度果实的发病率。在没有接菌的情况下，SA诱导了33个差异表达的蛋白，其中用质谱鉴定出了26个。而在接种病原菌的情况下，SA诱导了19个差异表达的蛋白，并鉴定出了其中的12个。这些蛋白分别涉及到代谢、防御反应、转录、能量途径、信号转导等过程。在没有接种病原菌的情况下，SA处理诱导了Putative DnaJ heat shock protein, PR1-like protein, Peroxidase, Major allergen Pru av 1 (Pru a 1)和Catalase等与抗病有关的蛋白。而在接种病原菌的情况下，诱导了PR1-like protein, Peroxidase和Catalase蛋白的差异表达。通过酶活性测定以及对细胞学定位的研究，我们发现在没有接种病原菌的情况下，POD的活性受到SA的诱导。但是在接种病原菌以后，诱导效果不明显。

Effects of various extenders and permeating cryoprotectants on cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa

The cryoprotective effects of 11 different extenders, TTE, DM, mDM, LG-DM, G-DM, TCG, TEST, TSM, Test-M, Test-H, and LM, on sperm cryopreservation of cynomolgus monkey (Macaca fascicularis) have been compared with glycerol as cryoprotectant. Sperm motility, plasma membrane, and acrosomal integrity were examined to evaluate frozen-thawed sperm function. The results showed that TTE, DM, mDM, LG-DM, G-DM, and TCG exhibited the best and similar protective efficiencies for cynomolgus monkey sperm cryopreservation in terms of sperm motility and plasma membrane integrity (P > .05). The acrosomal integrity for spermatozoa cryopreserved in TCG was statistically lower than that of TTE, DM, mDM, LG-DM, and G-DM (P < .05) but was significantly higher than that of TEST, TSM, Test-M, Test-H, and LM (P < .05). The postthaw sperm motility for 5 other extenders (TEST, TSM, Test-M, Test-H, and LIVI) did not exceed 30%, and the 3 sperm parameters evaluated for them were significantly lower than that of TTE, DM, mDM, LG-DM, G-DM, and TCG (P < .05). On the basis of these findings, 5 commonly used permeating cryoprotectants, glycerol, ethylene glycol, dimethyl sulfoxide, acetamide and propylene glycol have further been tested for their effectiveness on sperm cryopreservation in extenders of TTE, DM, mDM, LG-DM, G-DM, and TCG. The results showed that the sperm cryoprotective efficiencies of glycerol and ethylene glycol were similar and best among 5 permeating cryoprotectant treatments (P > .05). Dimethyl sulfoxide or acetamide resulted in average cryoprotection for cynomolgus monkey spermatozoa: poorer than glycerol or ethylene glycol but better than that of propylene glycol (P < .05). In addition, the action of permeating cryoprotectant appeared to be independent of extenders. The results in the present study demonstrate that 1) TTE, DM, mDM, LG-DM, G-DM, and TCG are excellent extenders and suitable for cynomolgus monkey sperm cryopreservation; 2) the mechanism of action of permeating cryoprotectants are not affected by extender composition; 3) ethylene glycol has a similar cryoprotective efficacy to glycerol that makes it a successful cryoprotectant for sperm cryopreservation in cynomolgus monkeys.

A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae

Isolation of high neutral lipid-containing microalgae is key to the commercial success of microalgae-based biofuel production. The Nile red fluorescence method has been successfully applied to the determination of lipids in certain microalgae, but has been unsuccessful in many others, particularly those with thick, rigid cell walls that prevent the penetration of the fluorescence dye. The conventional "one sample at a time" method was also time-consuming. In this study, the solvent dimethyl sulfoxide (DMSO) was introduced to microalgal samples as the stain carrier at an elevated temperature. The cellular neutral lipids were determined and quantified using a 96-well plate on a fluorescence spectrophotometer with an excitation wavelength of 530 nm and an emission wavelength of 575 run. An optimized procedure yielded a high correlation coefficient (R-2 = 0.998) with the lipid standard triolein and repeated measurements of replicates. Application of the improved method to several green algal strains gave very reproducible results with relative standard errors of 8.5%, 3.9% and 8.6%, 4.5% for repeatability and reproducibility at two concentration levels (2.0 mu g/mL and 20 mu g/mL), respectively. Moreover, the detection and quantification limits of the improved Nile red staining method were 0.8 mu g/mL and 2.0 mu g/mL for the neutral lipid standard triolein, respectively. The modified method and a conventional gravimetric determination method provided similar results on replicate samples. The 96-well plate-based Nile red method can be used as a high throughput technique for rapid screening of a broader spectrum of naturally-occurring and genetically-modified algal strains and mutants for high neutral lipid/oil production. (C) 2009 Published by Elsevier B.V.

Effect of cryoprotectants on hatching rate of red seabream (Pagrus major) embryos

The objectives were to investigate the effect of cryoprotectants on the hatching rate of red seabream embryos. Heart-beat embryos were immersed in: five permeable cryoprotectants, dimethyl sulfoxide (DMSO), glycerol (Gly), methanol (MeOH), 1,2-propylene glycol (PG), and ethylene glycol (EG). in concentrations of 5-30% for 10, 30, or 60 min; and two non-permeable cryoprotectants: polyvinylpyrrolidone (PVP), and sucrose (in concentrations of 5-20% for 10 or 30 min). The embryos were then washed and incubated in filtered seawater until hatching occurred. The hatching rate of the embryos treated with permeable cryoprotectants decreased (P < 0.05) with increased concentration and duration of exposure. In addition, PG was the least toxic permeable cryoprotectant, followed by DMSO and EG, whereas Gly and MeOH were the most toxic. At a concentration of 15% and 30 min exposure, the hatching rate of the embryos immersed in PG was 93.3 +/- 7.0% (mean +/- S.D.), however. in DMSO. EG, Gly. and MeOH, it was 82.7 +/- 10.4, 22.0 +/- 5.7, 0.0 +/- 0.0, and 0.0 +/- 0.0%, respectively. Hatching rate of embryos treated with PVP decreased (P < 0.05) with the increase of concentration and exposure time, whereas for embryos treated with sucrose, there was no significant decrease in comparison with the control at the concentrations used. (C) 2008 Elsevier Inc. All rights reserved.

辐射诱导的HeLa细胞旁效应信号通路研究

随着肿瘤认识的不断深入，肿瘤外科学、肿瘤放射治疗学、肿瘤化学治疗学构成了现代肿瘤治疗学的三大支柱，而放射治疗学的研究对于肿瘤的治疗有重要的临床意义。本文通过对HeLa进行辐射来观察其产生的旁效应信号通路，从而对临床工作的起到一定的帮助。辐照过的细胞通过释放信号分子引起周围未辐照细胞产生一系列的生物学反应的现象，称之为辐射诱导的旁效应。活性氧（Reactive oxygen species, ROS）和一氧化氮（Nitric oxide, NO）是辐射诱导的旁效应信号通路中的两个重要信号分子。本文研究了这两种重要的信号分子在辐射诱导的HeLa细胞旁效应信号通路中的关系。通过微核实验，我们发现X射线以及12C6+ 辐照过的HeLa细胞及其旁观者细胞的微核形成明显增加，而1%的二甲亚砜（Dimethyl sulfoxide，DMSO，ROS清除剂）预处理X射线辐照的细胞则显著抑制了受辐照细胞及其旁观者细胞的微核形成。1 Gy的X射线辐照能够抑制细胞的增殖速率而0.5%和1%的DMSO预处理则能减少X射线的增殖抑制作用，并且DMSO预处理的效果与浓度有关：1%的DMSO比0.5%的DMSO处理更大程度的恢复了受辐照细胞的增殖速率。另一方面，接受条件培养基（Conditioned medium）的旁观者细胞的增殖速率增加，而DMSO预处理产生条件培养基的受辐照细胞则使旁观者细胞的增殖速率降低，且DMSO预处理的效果同样与其浓度相关：浓度越高，条件培养基的刺激生长作用越小。Western blotting和DAF-FM DA荧光探针检测分别显示了辐照过后细胞的诱导型一氧化氮合酶（Inducible nitric oxide synthase, iNOS）和NO水平均升高，而DMSO预处理则降低其水平。因此，我们推测在X射线辐照的HeLa细胞旁效应信号通路当中ROS是NO的上游信号。另外，我们采用培养基转移后立即加DMSO或BMS－345541（IKK/NF-κB抑制剂）的方法研究了旁观者细胞当中的旁效应信号通路。我们发现DMSO和BMS－345541均显著抑制了旁观者细胞的NO水平。因此，在旁观者细胞当中ROS与NF-κB均为NO的上游信号

Free-Radical Solution Copolymerization of the Ionic Liquid Monomer 1-Vinyl-3-Ethylimidazolium Bromide with Acrylonitrile

Ionic liquid monomer 1-vinyl-3-ethylimidazolium bromide (ViEtIM(+)Br(-)) was first used to copolymerize with acrylonitrile (AN) successfully under various conditions. This was achieved with azobisisobutyronitrile as the initiator and dimethyl sulfoxide as the solvent. The kinetics of this copolymerization were studied. The values of the monomer apparent reactivity ratios were calculated by the Kelen-Tudos method. The apparent reactivity ratios of ViEtIM(+)Br(-) (r(ViEtIM+Br-)) and AN (r(AN)) were similar at polymerization conversions of less than 10%, (r(AN) = 0.954, r(ViEtIM+Br-) = 0.976). The copolymers were obtained with high molecular weights and high hydrophilicides. The copolymers were characterized by H-1-NMR, differential scanning calorimetry, and thermogravimetric analysis. These copolymers may be potentially useful in the preparation of precursor fibers and carbon fibers.

A new class of high T-g and organosoluble polynaphthalimides

A new class of soluble six-membered ring polynaphthalimides (PNIs) was synthesized from asymmetrical fluorinated naphthalenesubstituted monomers. All the resulting PNIs were easily soluble in many organic solvents, such as N-methyl-2-pyrrolidinone (NMP), N,N-dimethylacetamide (DMAc), dimethyl sulfoxide (DMSO). and chloroform. They also showed good thermal stability with glass transition temperature of 340-386 degrees C, 10% weight loss temperature in excess of 529 degrees C. Polyimide 3c could be solution-cast into tough and flexible film. The film had a tensile strength, elongation at break, and Young's modulus of about 117.6 Wa, 23.6%, and 1.77 GPa, respectively. The gas permeation property of the film of 3c was investigated with oxygen permeability coefficient (PO2 = 3.99) and permeability selectivity coefficient of oxygen to nitrogen (P-O2/P-N2 = 5.27). Therefore, these materials are expected to be a good alternative to PIs based on five-membered rings with applications in gas separation membranes.

2-cyanoprop-2-yl dithiobenzoate mediated reversible addition-fragmentation chain transfer polymerization of acrylonitrile targeting a polymer with a higher molecular weight

The reversible addition-fragmentation chain transfer (RAFT) polymerization of acrylonitrile (AN) mediated by 2-cyanoprop-2-yl dithiobenzoate was first applied to synthesize polyacrylonitrile (PAN) with a high molecular weight up to 32,800 and a polydispersity index as low as 1.29. The key to success was ascribed to the optimization of the experimental conditions to increase the fragmentation reaction efficiency of the intermediate radical. In accordance with the atom transfer radical polymerization of AN, ethylene carbonate was also a better solvent candidate for providing higher controlled/living RAFT polymerization behaviors than dimethylformamide and dimethyl sulfoxide. The various experimental parameters, including the temperature, the molar ratio of dithiobenzoate to the initiator, the molar ratio of the monomer to dithiobenzoate, the monomer concentration, and the addition of the comonomer, were varied to improve the control of the molecular weight and polydispersity index. The molecular weights of PANS were validated by gel permeation chromatography along with a universal calibration procedure and intrinsic viscosity measurements. H-1 NMR analysis confirmed the high chain-end functionality of the resultant polymers.

Effect of La3+ on structure and electrochemical reaction of microperoxidase-11 in imitated physiological solution

In this paper, the interaction mechanism between La3+ and microperoxidase-11 (MP-11) in the imitated physiological solution was investigated with the electrochemical and spectroscopic methods. It was found that when the molar ratio of La3+, and MP-11 is low, such as 2, La3+ can coordinate with oxygen in the propionic acid group of the heme group in the MP-11 molecule, forming the La-MP-11 complexes and leading to the increase in the non-planarity of the porphyrin cycle in the heme group and then the increase in the extent of exposure of the electrochemically active center, Fe(I I I) in the porphyrin cycle of the heme group. The increase in the extent of exposure of the electrochemically active center, Fe(III) in the porphyrin cycle of the heme group would increase the reversibility of the electrochemical reaction of the La-MP-11 complexes and its electrocatalytic activity for the reduction of H2O2. The results of the chromatographic analysis demonstrated that the average molar ratio of La3+ and MP-11 in the La-MP-11 complexes is 1.62.When the molar ratio of La3+ and MP-11 is high, such as 3, La3+ would shear some amino acid residues of the peptide of MP-11. Therefore, many La3+ ions can bind to the oxygen- and/or nitrogen-containing groups in the sheared amino acid residues except coordinating with the sheared and non-sheared MP-11 molecules.

Synthesis and characterization of hyperbranched poly(ester-amide)s from commercially available dicarboxylic acids and multihydroxyl primary amines

A new method for syntheses of hyperbranched poly(ester-amide)s from commercially available A(2) and CBx type monomers has been developed on the basis of a series of model reactions. The aliphatic and semiaromatic hyperbranched poly(ester-amide)s with multihydroxyl end groups are prepared by in situ thermal polycondensation of intermediates obtained from dicarboxylic acids (A(2)) and multihydroxyl primary amines (CBx) in N,N-dimethylformamide. Analyses of FTIR, H-1 NMR, and C-13 NMR spectra revealed the structures of the polymers obtained. The MALDI-TOF MS of the polymers indicated that cyclization side reactions occurred during polymerization. The hyperbranched poly(ester-amide) s contain configurational isomers observed by C-13 and DEPT C-13 NMR spectroscopy. The DBs of the polymers were determined to be 0.38-0.62 by H-1 NMR or quantitive C-13 NMR and DEPT 135 spectra. These polymers exhibit moderate molecular weights, with broad distributions determined by size exclusion chromatography ( SEC), and possess excellent solubility in a variety of solvents such as N, N- dimethylacetamide, dimethyl sulfoxide, tetrahydrofuran, and ethanol, and display glass-transition temperatures (T(g)s) between -2.3 and 53.2 degrees C, determined by DSC measurements.

Synthesis and characterization of hyperbranched aromatic poly(ester-imide)s

The synthesis and characterization of hyperbranched aromatic poly(ester-imide)s are described. A variety of AB(2) monomers, N-[3- or 4-bis(4-acetoxyphenyl)toluoyl]-4-carboxyl-phthalimide and N-{3- or 4-[1,1-bis(4-acetooxyphenyl)]ethylphenyl}-4-carboxy phthalimides were prepared starting from condensation of nitrobenzaldehydes or nitroacetophenones with phenol and used for synthesis of hyperbranched poly(ester-imide)s containing terminal acetyl groups by transesterification reaction. These hyperbranched poly(ester-imide)s were produced with weight-average molecular weight of up to 6.87 g/mol. Analysis of H-1 NMR and C-13 NMR spectroscopy revealed the structure of the four hyperbranched poly(ester-imide)s. These hyperbranched poly(ester-imide)s exhibited excellent solubility in a variety of solvents such as N,N-dimethylacetamide, dimethyl sulfoxide, and tetrahydrofuran and showed glass-transition temperatures between 217 and 255 degreesC. The thermogravimetric analytic measurement revealed the decomposition temperature at 10% weight-loss temperature (T-d(10)) ranging from 365 to 416 degreesC in nitrogen.

Promoter effect of La3+ on the electrochemical reaction of microperoxidase-11

The effect of La3+ on the electrochemical behavior and structure of heme undecapeptide-microperoxidase-11 (MP-11)-in the aqueous solution was investigated using cyclic voltammetry, circular dichroism (CD) and UV-vis absorption spectrometry. It was found for the first time that La3+ would promote the electrochemical reaction of MP-11 at the glassy carbon (GC) electrode. This is mainly due to the fact that La3+ would induce more beta-turn and alpha-helical conformations from the random coil conformation of MP-11 and increase the non-planarity of the heme.