植物激素IBA与6-BA对摇瓶分批流加异养培养小球藻的生长及化学组成的影响

研究了植物激素IBA与6-BA对摇瓶分批流加异养培养小球藻的生长及化学组成的影响。结果表明,IBA与6-BA处理摇瓶分批流加异养培养的小球藻,促进了小球藻的生长,提高了小球藻的生物量(15．0％)与对糖转化率(14．8％),同时也提高了小球藻的蛋白质含量(16．3％)。氨基酸分析结果表明,IBA与6-BA能提高摇瓶分批流加异养培养的小球藻的α-氨基酸含量(含量达31．2％,而对照的仅26．0％),增加必需氨基酸的含量及比例,尤其两种重要α-氨基酸Arg和Lys的含量分别提高达22．9％和30．1％,强化了异养小球藻的营养价值。IBA与6-BA能提高摇瓶分批流加异养培养的小球藻叶绿素及类胡萝卜素含量。培养工艺研究与化学组成分析将为植物激素应用于小球藻的异养培养奠定基础。     

几种化学物质对小球藻生长和蛋白含量的效应

研究了不同有机和无机碳源及生长素对小球藻生长量及叶绿素和蛋白质含量的效应。葡萄糖、蔗糖、乙酸钠和碳酸氢钠及萘乙酸均能提高小球藻的生长量和叶绿素含量,对小球藻具有明显的促生效应。培养基加入乙酸钠和萘乙酸小球藻细胞蛋白质含量由对照的43．4％提高到46．9％和52．5％。     

利用芽孢菌发酵废水培养小球藻的研究

研究表明,在25℃、光照12h／d（4500lx）条件下,芽孢茵发酵废水对小球藻的生长有促进作用,且促进作用随废水浓度的升高而增强。浓度为10％Ya发酵废水对小球藻的促进增殖作用与对照组相近,纯发酵废水培养的小球藻最高密度为对照组的7．4倍。研究同时表明,小球藻对芽孢菌发酵废水有较强的降解能力,通过10d的培养,发酵废液中的COD下降95．53％,氨态氮下降81．27％,亚硝酸盐氮下降88．33％。     

普通小球藻对嗪草酮、骠马和甲草胺的敏感性研究

通过96h的毒性实验,研究了普通小球藻对3种不同作用机制农田常用除草剂嗪草酮、骠马和甲草胺的敏感性．结果表明。在实验条件下,嗪草酮、甲草胺对普通小球藻的毒性随时间的推移有加重趋势。并呈现很好的剂量效应关系;最高抑制生长浓度(嗪草酮0．24mg·L^-1,甲草胺12．8mg·L^-1)处理组的最大比增长率分别为对照组的12．38％和31．58％;骠马低浓度对普通小球藻的抑制作用不明显,并呈一定的生长促进效应,0．08mg·L^-1浓度组普通小球藻最大比增长率为对照组的111．44％,而高浓度则具有明显的生长抑制作用,并随时间推移,毒性逐渐减弱．嗪草酮、骠马和甲草胺的96hEC50分别为0．021、0．937和5．54mg·L^-1．普通小球藻对嗪草酮最敏感。其次为骠马和甲草胺．3种除草剂在实验条件下对普通小球藻叶绿素a含量的影响和对普通小球藻生长的影响相似。表现出较好的剂量．效应关系．     

酵母及与藻类搭配对萼花臂尾轮虫饵料效果的研究

研究了２种酵母单独投喂萼花臂尾轮虫的最适密度、饵料效果及与藻类搭配的投喂效果．结果表明,在５种密度下,面包酵母和啤酒酵母的最适密度分别为１０和５×１０６个·ｍｌ－１．用面包酵母和啤酒酵母培养轮虫所得到的种群密度和瞬时增长率分别是用蛋白核小球藻培养的４０．２％、２６．０％和８５．５％、７６．６％．用面包酵母和蛋白核小球藻适当比例搭配投喂轮虫,其效果接近或超过单一用小球藻在最适密度下的培养效果．     

几种绿藻对西草净的抗性与活性氧防御物质

４种绿藻对除莠剂西草净（Ｓｉｍｅｔｒｙｎ）的反应有很大差异,小球藻、羊角月芽藻、衣藻和四鞭藻的ＥＣ＿（５０）（影响浓度）值分别为７００、２５、２７和１１μｇ／Ｌ不同浓度西草净都能影响绿藻的光合作用,浓度为２００ｐｐｍ６时,羊角月牙藻和四鞭藻的光合抑制率分别为９５．３％和９４．０％,但对小球藻几乎无影响,当浓度增至２０００ｐｐｍ时,对小球藻的光合抑制率为８７％。小球藻中活性氧防御物（ＳＯＤ、ＡｓＡ－ＰＯＤ、ＧＲ等）的活性均比衣藻、羊角月牙藻等高几倍到几十倍,显示小球藻对西草净的抗性和忍耐性可能与它细胞内所含的活性氧防御物活性有关,它们的高活性使小球藻在西草净污染后,对体内生成的活性氧（Ｏ＿２、Ｈ＿２Ｏ＿２、Ｏ＿２等）伤害有解毒作用。     

小球藻诱变选育及池塘水体净化效能研究

采用N＋束注入、甲基磺酸乙酯二步诱导法对蛋白核小球藻分步进行诱变,筛选出使蛋白核小球藻定向变异藻株,得到了最佳培养条件。常规生化分析结果表明,诱变后得到的小球藻新品种的蛋白质含量最高提高27．23％;     

小球藻RubisCO的纯化及其在异养向自养转变过程中的含量变化

经硫酸铵分部沉淀、ＳｅｐｈａｃｒｙｌＳ－３００和ＤＥＡＥ－纤维素柱层析纯化了小球藻ＲｕｂｉｓＣＯ,得率为１５％,比活力达１．２３２μｍｏｌＣＯ２ｍｓ－１ｍｉｎ－１,分子量是５００ｋＤ,它和菠菜叶片ＲｕｂｉｓＣＯ在分子量、亚基组成和免疫特性等方面相似,反映ＲｕｂｉｓＣＯ在高等和低等植物中有较高的同源性。自养小球藻ＲｕｂｉｓＣＯ占细胞可溶性蛋白质的２４％。而异养转变后的小球藻细胞内不含ＲｕｂｉｓＣＯ。异养小球藻向自养生长转变过程中,２０ｈ后细胞内叶绿素含量逐渐增加,２４ｈ时细胞内出现ＲｕｂｉｓＣＯ,２４ｈ后大量增加,至４１ｈ时含量达最高峰;标志着小球藻细胞光合作用能力的恢复和加强。     

小球藻的优化培养及胞内多糖的提取

小球藻细胞中所含有的多糖和糖蛋白等活性物质具有明显的抗肿瘤,抗病毒感染及增强机体免疫力等作用。首先考察了几个重要的环境因素对小球藻生长及胞内多糖含量的影响。确定了优化的环境条件为：光照17h,培养温度15℃,营养盐KNO3浓度为2mmol/L。在此优化条件下,大批量培养小球藻至平衡期,离心收集藻细胞,再经过冻融与超声波破碎,三氯乙酸沉淀蛋白质,最后通过SephadexG-75凝胶柱分离得到了两种分子量不同的多糖,并结合醋酸纤维素膜电泳证明了同样的结论。     

自养条件下小球藻净化氮磷能力的研究

研究了在自养条件下,小球藻净化氮、磷的能力。实验结果表明,小球藻对氮、磷的吸收在开始的前12h内速度比较快,利用率达70％和60％左右;吸收氮、磷2d,利用率可分别达到75％和62％。低浓度的氮、磷组合有利于小球藻对氮的吸收,在磷浓度50～100mg／l范围内可以有效地吸收磷,吸收率接近60％。升高温度有利于小球藻对磷、氮的吸收。     

Rapid purification of a high molecular weight human brain protein by chromatography on controlled pore glass.

Controlled pore glass (CPG) chromatography was employed to simply (one pass through column) and rapidly (60 minutes) purify a human brain specific protein having a high molecular weight (approximately 250,000 mol. wt.) from a crude brain extract containing proteins of varying molecular weights. This method, either exactly as described herein or by adjusting the pore size of the CPG, should be adaptable to other purification problems.     

A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics

Antibodies are highly specific recognition molecules which are increasingly being applied to target therapy in patients. One type of developmental antibody-based therapy is antibody directed enzyme prodrug therapy (ADEPT) for the treatment of cancer. In ADEPT, an antibody specific to a tumor marker protein delivers a drug-activating enzyme to the cancer. Subsequent intravenous administration of an inactive prodrug results in drug activation and cytotoxicity only within the locale of the tumor. Pilot clinical trials with chemical conjugates of the prodrug activating enzyme carboxypeptidase G2 (CPG2) chemically conjugated with an antibody to and carcinoembryonic antigen (CEA), have shown that CPG2-mediated ADEPT is effective but limited by formation of human antibodies to CPG2 (HACA). We have developed a recombinant fusion protein (termed MFE-CP) of CPG2 with an anti-CEA single chain Fv antibody fragment and we have developed methods to address the immunogenicity of this therapeutic. A HACA-reactive discontinuous epitope on MFE-CP was identified using the crystal structure of CPG2, filamentous phage technology and surface enhanced laser desorption/ionization affinity mass spectrometry. This information was used to create a functional mutant of MFE-CP with a significant reduction (range 19.2 to 62.5%, median 38.5%) in reactivity with the sera of 11 patients with post-therapy HACA. The techniques described here are valuable tools for identifying and adapting undesirable immunogenic sites on protein therapeutics.     

Rapid activity-dependent delivery of the neurotrophic protein CPG15 to the axon surface of neurons in intact Xenopus tadpoles

CPG15 (aka neuritin) is an activity-induced GPI-anchored axonal protein that promotes dendritic and axonal growth, and accelerates synaptic maturation in vivo. Here we show that CPG15 is distributed inside axons and on the axon surface. CPG15 is trafficked to and from the axonal surface by membrane depolarization. To assess CPG15 trafficking in vivo, we expressed an ecliptic pHluorin (EP)-CPG15 fusion protein in optic tectal explants and in retinal ganglion cells of intact Xenopus tadpoles. Depolarization by KCl increased EP-CPG15 fluorescence on axons. Intraocular kainic acid (KA) injection rapidly increased cell-surface EP-CPG15 in retinotectal axons, but coinjection of TTX and KA did not. Consistent with this, we find that intracellular CPG15 is localized to vesicles and endosomes in presynaptic terminals and colocalizes with synaptic vesicle proteins. The results indicate that the delivery of the neurotrophic protein CPG15 to the axon surface can be regulated on a rapid time scale by activity-dependent mechanisms in vivo.     

Effect of cyclopentaneglycine on metabolism in Salmonella typhimurium

Cyclopentaneglycine (CPG) inhibited the growth of wild-type Salmonella typhimurium. The inhibition was overcome by isoleucine or any isoleucine precursor formed after threonine. CPG appeared to mimic isoleucine as a strong inhibitor of the activity of l-threonine deaminase. The analogue was a poor inhibitor of isoleucyl-transfer ribonucleic acid synthetase. CPG did not appear to be incorporated into protein nor did it replace isoleucine in repression. Cells that had recovered from growth inhibition by CPG had derepressed levels of the isoleucine-valine biosynthetic enzymes.     

CPG70 is a novel basic metallocarboxypeptidase with C-terminal polycystic kidney disease domains from Porphyromonas gingivalis

In a search for a basic carboxypeptidase that might work in concert with the major virulence factors, the Arg- and Lys-specific cysteine endoproteinases of Porphyromonas gingivalis, a novel 69.8-kDa metallocarboxypeptidase CPG70 was purified to apparent homogeneity from the culture fluid of P. gingivalis HG66. Carboxypeptidase activity was measured by matrix-assisted laser desorption ionization-mass spectrometry using peptide substrates derived from a tryptic digest of hemoglobin. CPG70 exhibited activity with peptides containing C-terminal Lys and Arg residues. The k(cat)/K(m) values for the hydrolysis of the synthetic dipeptides FA-Ala-Lys and FA-Ala-Arg by CPG70 were 99 and 56 mm(-1)s(-1), respectively. The enzyme activity was strongly inhibited by the Arg analog (2-guanidinoethylmercapto)succinic acid and 1,10-phenanthroline. High resolution inductively coupled plasma-mass spectrometry demonstrated that 1 mol of CPG70 was associated with 0.6 mol of zinc, 0.2 mol of nickel, and 0.2 mol of copper. A search of the P. gingivalis W83 genomic data base (TIGR) with the N-terminal amino acid sequence determined for CPG70 revealed that the enzyme is an N- and C-terminally truncated form of a predicted 91.5-kDa protein (PG0232). Analysis of the deduced amino acid sequence of the full-length protein revealed an N-terminal signal sequence followed by a pro-segment, a metallocarboxypeptidase catalytic domain, three tandem polycystic kidney disease domains, and an 88-residue C-terminal segment. The catalytic domain exhibited the highest sequence identity with the duck metallocarboxypeptidase D domain II. Insertional inactivation of the gene encoding CPG70 resulted in a P. gingivalis isogenic mutant that was avirulent in the murine lesion model under the conditions tested.     

Age-dependent Occurrence of the Intestinal Ciliate Balantidium coli in Pigs at a Danish Research Farm

A cross sectional study of the prevalence and intensity of Balantidium coli in pigs was carried out on a Danish research farm. The prevalence of B. coli infection increased from 57% in suckling piglets to 100% in most pig groups ≥4 weeks old. The mean number of cysts per gram faeces (CPG) of pigs aged 12 weeks and younger were ≤206, whereas pigs aged 28 weeks and >52 weeks had significantly higher counts of ≥865 CPG. Although some lactating sows had very high CPG’s, no significant differences in CPG could be detected between the intensities of pregnant sows, lactating sows and empty and dry sows. No human cases of B. coli infection have been published in Denmark though it is zoonotic.     

NDRG2 and TLR7 as novel DNA methylation prognostic signatures for acute myelocytic leukemia

Acute myelocytic leukemia (AML) is an aggressive malignant tumor and typically fatal without treatment. Identification and development of novel biomarkers could be beneficial for the diagnosis and prognosis of AML patients. Here, we aimed to identify the accurate DNA methylation prognostic signatures for AML patients. The DNA methylation data of AML patients and corresponding clinical information were retrieved from The Cancer Genome Atlas database. CPG sites that correlates closely with the survival of the AML patients were identified and further combined into CPG sites pairs to screen the survival-related pairs. The prognostic signatures were identified by the C-index and forward search algorithms and validated by the verification group. Finally, the functional enrichment analysis was performed on these CPG sites. As a result, a total of 498 CPG sites associated with the overall survival of AML patients was obtained. A prognostic signature composed of 10 CPG sites pairs was obtained and validated. The functional enrichment analysis showed prognostic genes were mainly enriched in tumor protein processing, cell differentiation, blood leukocyte immunity, and platelet growth factor pathways. In summary, we identified two accurate prognostic methylation signatures (NDRG2 and TLR7), which would be served as a novel therapy target for AML.     

Molecular Cloning, Sequencing and Phylogenetic Analysis of Coat Protein Gene of a Biologically Distinct Citrus Tristeza Virus Isolate Occurring in Central India

Citrus tristeza virus (CTV) culture, collected from a fifteen-year-old wilted and declined mosambi sweet orange [Citrus sinensis (L) Osb] plant, was maintained under green house into acid lime [C. aurantifolia Swing] and mosambi seedlings. It gave positive reaction in ELISA, both with CTV specific polyclonal antibodies (G604) and monoclonal antibody MCA13, which specifically detects severe CTV strains. Coat protein gene (CPG) of the virus was amplified by RT PCR using CPG specific primers yielding an amplicon of 672 bp. Sequence analysis of the CPG amplicon showed 97% nucleotide sequence homology with Chinese isolate CTV-0002 (Acc. No. AJ518841) and isolate S4 (Acc. No. EF063109). In phylogenetic analysis, the present CTV isolate was displayed in different clade than other reported Indian CTV isolates, but it shared a separate clade with isolates from China, Israel, Jordan and New Zealand.     

Dehydration of crude protein from Ginkgo biloba L. by microwave freeze drying

The paper optimized the parameters of microwave freeze drying (MFD) of crude Ginkgo biloba protein (CPG) using response surface methodology (RSM) based on the analysis of its proximate composition. The results showed that coefficients of determination, R(2) values for drying time and protein solubility were greater than 0.9500. The drying time and protein solubility of CPG varied curvilinearly with increase of microwave power and pre-freeze temperature, and drying time varied linearly with material thickness. The optimum MFD condition was microwave power of 408-421 W, material thickness of 15 mm and pre-freeze temperature of -20°C to -21°C, respectively.     

Extraction of Wheat Flour Proteins with Sodium Dodecyl Sulfate and Their Molecular Weight Distribution

By extraction of wheat flour with sodium dodecyl sulfate (SDS) solution at pH 6.8, about 76% of the total flour nitrogen solubilized into clear supernatant. This solvent was more effective for extraction of wheat protein than 0.01 m acetic acid, aluminium lactate-lactic acid buffer (pH 3.1), AUC-solvent (0.1 m acetic acid, 3 m urea and 0.01 m cetyltrimethyl-ammomum bromide) and 3,5-diiodosalicylic acid lithium salt etc. The molecular weight distribution of the SDS-soluble proteins was studied by SDS-polyacrylamide gel electrophoresis and by molecular sieve chromatography on controlled pore glass (CPG–10–500) without prior reduction of disulfide linkages of the proteins. Most of the SDS-soluble proteins had molecular weight of less than 75,000, suggesting single-chained proteins. A small amount of relatively high molecular weight proteins which contained intermolecular disulfide linkages was also detected in the gel of electrophoresis, while high molecular weight protein which did not migrate into gel matrix during electrophoresis without prior reduction of disulfide linkages existed in trace amount in the SDS-soluble fraction.

The SDS-insoluble proteins were almost completely extracted by further extraction with SDS in combination with 2-mercaptoethanol or with mercuric chloride.