Expression of Anti-Lipopolysaccharide Factor Isoform 3 in Chlamydomonas reinhardtii Showing High Antimicrobial Activity

Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene (ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.     

小鼠H-Y单克隆抗体ELISA检测方法的建立

以纯系 BAL B/ c雄性小鼠脾细胞腹腔注射免疫同系雌性小鼠 9次 ,获得的抗血清经雌、雄鼠脾细胞吸收后用于精子细胞毒性试验 ,测得 H- Y抗血清效价为 1/ 16 0。选取免疫应答最好的雌鼠脾细胞与 SP2 / 0骨髓瘤细胞融合 ,用精子细胞毒性方法筛选效价较高的细胞株制备腹水 ,建立优化的 EL ISA反应条件。优化后的 EL ISA反应条件为 :抗原4℃过夜 ,加 H- Y抗血清 37℃反应 12 0 m in,加 HRP- Ig G 37℃反应 30 min,加 TMB 2 5℃ 30 min。优化后的 EL ISA与常规 EL ISA比较 ,可以明显降低阴性吸光值 ,检测灵敏度从 1/ 32 0上升为 1/ 12 80     

复方吡喹酮注射液防治羊脑多头蚴病的试验研究

为了获得复方吡喹酮注射液治疗羊脑多头蚴病的最佳治疗剂量,本试验应用复方吡喹酮注射液30、50、80 mg/kg BW三个剂量组对患羊脑多头蚴病的绵羊进行治疗,设80 mg/kg BW剂量的丙硫咪唑作为药物对照组.试验结果表明,30 mg/kg BW剂量组治愈率较低,为68.97%,不能达到治疗目的;50和80 mg/kg BW剂量对羊脑多头蚴均有很强的驱杀作用,对病羊的治愈率分别为91.84%和92.85%,但两组间差异不显著(P＞0.05);高、中剂量组(80、50 mg/kg BW)与低剂量组、药物对照组比较差异极显著(P＜0.01).50 mg/kg BW剂量为防治羊脑多头蚴病的最佳治疗剂量.     

健骨注射液硬膜外腔注射治疗腰腿痛52例的疗效观察

用中草药健骨注射液行硬膜外腔注射治疗急慢性腰腿痛52例,结果症状消失与功能恢复者占40.7％,总有效率为92.3％,其疗效不亚于用普鲁卡因加强的松龙注射液,且未见有何副作用,使用无禁忌症。     

浊度法测定维生素C注射液细菌内毒素含量

应用BET-32型细菌内毒素测定仪定量测定维生素C注射液的细菌内毒素含量，干扰试验表明，维生素C注射液在400倍稀释时，测定内毒素的干扰最小。7个不同批号的维生素C注射液测定结果显示，7批的回收率均符合测定要求，其中4个批号的人用维生素C注射液内毒素含量符合药典标准，兽用3个批号产品的内毒素含量偏高。     

Expression of a recombinant crustacean hyperglycemic hormone of the kuruma prawn Penaeus japonicus in methylotrophic yeast Pichia pastoris

ABSTRACT:   Until now, six crustacean hyperglycemic hormones (CHH) designated Pej-SGP-I, -II, -III, -V, -VI and -VII have been characterized in the kuruma prawn Penaeus japonicus . All CHH consist of 72 amino acid residues and have an amidated carboxyl (C)-terminus. In the present study, we expressed Pej-SGP-III in methylotrophic yeast Pichia pastoris in order to obtain a large quantity of recombinant CHH possessing biological activity. A cDNA encoding Pej-SGP-III that had been previously cloned was processed by polymerase chain reaction (PCR) and the resulting product was ligated into an expression vector. Pichia pastoris was transformed with this vector after which a recombinant Pej-SGP-III was expressed having an additional amino acid residue (glycine) at the C-terminus (rPej-SGP-III-Gly), a form considered to be a putative precursor of this hormone. rPej-SGP-III-Gly secreted into the culture medium was purified by reversed-phase HPLC, and amidated using a peptidylglycine alpha-amidating enzyme. The amidated rPej-SGP-III (rPej-SGP-III) showed hyperglycemic activity in in vivo bioassay almost comparable to that of the natural Pej-SGP-III. rPej-SGP-III thus obtained will be a useful tool not only for its physiological study but also for the determination of its 3-D structure.     

不同海拔下甲醇替代率和主喷正时对RCCI发动机性能的影响

为探究不同海拔条件下甲醇/柴油反应活性控制压燃（reactivity controlled compression ignition, RCCI）发动机的运行特性,该研究基于甲醇/柴油双燃料发动机试验台架,试验研究1 800r/min、100%负荷和3 200r/min、100%负荷下不同甲醇替代率、柴油喷射正时对发动机燃烧与排放性能的影响规律。结果表明：不同海拔条件下随着甲醇替代率的增加,缸压和瞬时放热率峰值逐渐升高,燃烧始点和燃烧中心前移,当量有效燃油消耗率（equivalent brake specific fuel consumption, ESFC）降低,有效热效率升高,NO_x和碳烟排放大幅降低,THC（total hydrocarbons）和CO排放增加。1 800 r/min、100%负荷工况下,甲醇替代率由0增至20%,0、1 000、2 000m海拔下最大缸压平均增加1.72MPa,瞬时放热率峰值平均升高25.08J/(°),ESFC平均降低4.67%,有效热效率平均升高4.90%,NO_x和碳烟排放分别平均降低16.63%和50%,THC和CO排放量分别平均增加142.03、388.18 mg/kg。3 200 r/min下甲醇替代率由0增至7%,不同海拔高度下ESFC平均降低1.76%,有效热效率平均升高1.79%,NO_x和碳烟排放量分别平均降低8.17%和20.70%。海拔高度由0升至2 000m,1 800r/min、20%甲醇替代率与3 200r/min、7%甲醇替代率下,瞬时放热率峰值分别降低4.80和8.08J/(°),燃烧中心分别推迟1.44°和1.43°,有效热效率分别降低0.82%和0.68%,ESFC分别升高2.10%和1.99%,NO_x排放量分别减少10.61%和7.35%,碳烟排放分别增加26.54%和32.12%,THC排放分别升高29.88%和15.45%,CO排放量分别增加22.42%和18.15%。固定甲醇替代率后,随着柴油主喷正时提前,不同海拔条件下缸压和放热率峰值逐渐升高,燃烧中心向上止点靠近,ESFC逐渐降低,有效热效率升高,碳烟排放减少,NO_x、THC和CO排放增加。1 800 r/min、15%甲醇替代率下,主喷正时从-1.5°提前至-7.5°,不同海拔高度下ESFC平均降低8.27%,有效热效率平均升高9.08%,碳烟排放平均减少90.94%。为提升高海拔条件下甲醇/柴油RCCI发动机的热效率和燃油经济性,可以适当增大柴油主喷正时。研究结果可为不同海拔环境下甲醇/柴油RCCI发动机燃烧与污染物排放控制优化提供参考。     

A review of antimicrobial peptides: defensins and related cationic peptides

Cationic antimicrobial peptides are present throughout the plant and animal kingdoms and bear striking structural and functional similarities across species lines. They provide primitive, nonspecific means of combating a variety of bacteria, fungi, enveloped viruses, and protozoa. Some are also cytotoxic against host cells, including neoplastic cells. Cationic antimicrobial peptides may play various roles in inflammation and tissue repair. Antimicrobial peptides are found in epithelial tissues regularly exposed to microbial attack as well as in cells whose primary function is defense against potential pathogens. They constitute an important part of the nonoxidative antimicrobial arsenal of leukocytes. They are preformed and/or readily synthesized when the cells are stimulated by exposure to pathogens. They exert their effects directly by inserting into membranes of target cells and forming ion channels which increase membrane permeability; however, antimicrobial peptides can also act as opsonins to facilitate phagocytosis. Resistance to defensins is a virulence factor for organisms such as Salmonella sp. The study of cationic antimicrobial peptides is increasing our understanding of innate immunity, inflammation, and the pathogenesis of genetic diseases such as specific granule disease in humans. Therapeutic applications of antimicrobial peptides are currently under investigation.     

Evaluation of a radioimmunoassay for type III procollagen peptide in the dog

Type III procollagen peptide (P-3-P) is a serum marker for hepatic fibrosis in humans. The utility of a commercially available radioimmunoassay for P-3-P was evaluated in the dog. The specificity of the assay was assessed by polyacrylamide gel electrophoresis (PAGE) of canine serum and purified bovine P-3-P, followed by Western immunoblotting with rabbit aniti-P-3-P serum. The sensitivity was assessed by performing the radioimmunoassay on dilutions of sera from 22 dogs. Polyacrylamide gel electrophoresis of purified bovine P-3-P and sera from two dogs suspected of having elevated P-3-P concentrations revealed no homologous bands of staining. Western immunoblotting showed marked cross-reactivity of the high antisera concentrations with several components of the serum proteins, but none corresponding to the purified P-3-P. All tested sera from dogs had minimal competitive binding with radiolabeled P-3-P in the radioimmunoassay. Dilution curves of dog sera did not parallel either the standard curve or the dilution curve of a known test human serum. There were no statistically different P-3-P concentrations in any of the groups of dogs studied. It was concluded that currently available radioimmunoassay kits for the measurement of P-3-P in the human are not applicable in the dog. Seemingly, the structure or metabolism of canine P-3-P may vary significantly from that of the bovine or human, limiting the sensitivity and specificity of this assay in the dog.     

In ovo embryotoxicity of α-endosulfan adversely influences liver and brain metabolism and the immune system in chickens

There is a lack of laboratory-based embryonic chicken toxicity studies with the ecologically relevant low dose/s of endosulfan that utilizes a more practical approach such as the chorioallontoic membrane (CAM) injection. In this investigation, 2μg AR grade α-endosulfan/egg (40% of LD₅₀ for embryos) was injected through the CAM in 12-day-old chicken embryos and the activities of glucose-6-phosphatase (G6Pase, EC 3.1.3.9), fructose 1,6-diphosphatase (FDPase, EC 3.1.3.11), adenosine triphosphatase (ATPase, EC 3.6.1.3) and succinic dehydrogenase (SDH, EC 1.3.99.1) and DNA and RNA content in liver and brain tissues and acetyl cholinesterase (AChE, EC 3.1.1.7) in the latter were determined at 24, 48, and 72 h post-exposure. The wet weight of the embryos did not differ between groups. Following endosulfan exposure, except increase in the hepatic ATPase activity (P < 0.01), there was a significant decrease in the following parameters: G6Pase activity in both the liver and brain (P < 0.01), SDH activity in the brain (P < 0.01), brain overall DNA and RNA concentration (P < 0.05), brain AChE activity (P < 0.01). Exposure of 18-day-old embryos to 2-μg endosulfan for 24 h caused decrease (P < 0.01) in the lymphocyte count and IgG content. Histopathology of thymus and bursa of Fabricius revealed a reduction in the population of thymic follicles, smaller thymocytes with the clear vacuoles in cytoplasm and fewer bursocytes accompanied by infiltration of erythrocytes in lymphoid follicles of the endosulfan-treated embryos. It was inferred that in ovo injection of 0.041 μg/g egg weight of α-endosulfan suppress gluconeogenesis (main energy source in embryonic life), nerve transmission, and immunity.