Coimmobilization of D-amino acid oxidase and catalase by entrapment ofTrigonopsis variabilis in radiation polymerised Polyacrylamide beads

Trigonopsis variabilis induced for D-amino acid oxidase and catalase was immobilized by entrapment in Polyacrylamide beads obtained by radiation polymerisation. Permeabilization of the cells was found to be essential for optimal activity of the enzymes in free cells. However, the process of entrapment itself was found to eliminate the permeability barrier of cells immobilized in Polyacrylamide. The two enzymes exhibited a differential response on Polyacrylamide entrapment. Thus, D-amino acid oxidase activity was stabilized to heat inactivation whereas catalase in the same cells showed a destabilization on entrapment in Polyacrylamide. The coimmobilized enzyme preparation showed an operational half life of 7–9 days after which the D-amino acid oxidase activity remained stable at a value 35–40% of that of the initial activity for a study period of 3 weeks. Coimmobilization of MnO₂ was not effective in enhancing the operational life of the enzyme preparation.     

Size selection of latex beads by blackfly larvae (Diptera: Simuliidae) in the laboratory

In laboratory experiments, blackfly larvae collected from a lake outlet, a woodland and a meadow stream were tested for size selection of latex beads of < Ito > 100 μm diameters. 3 suspensions of varying proportions for each size category were supplied to these blackfly larvae in common experiments. Comparisons between the size frequency distributions of particles supplied and the particle compositions in the larval guts showed intra- and interspecific differences and were quantified by calculating Jacobs' electivity index. In all species selection of larger particles increased with the larger larval instars. Although there was a positive selectivity of small particles in some cases, the ingested proportion of large particles increases volumes and biomasses of gut content and may be more important for larval growth than small particles.     

Studies on NK cell purification by negative selection in human peripheral blood

We have attempted to improve negative selection procedures for the large scale purification of human CD _in3 ^– CD56⁺ NK cells. In a series of experiments, purifications of NK cells from 10⁸ PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3⁺ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 10⁸ cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 10⁹ PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads.     

云南羯布罗香树脂的化学成分

从云南产羯布罗香(Dipterocarpus tubinatus Gaertn. f.)树脂中分离得到6个三萜化合物,鉴定为羟基达玛烯酮—Ⅱ(hydroxydammarenone-Ⅱ,达玛烯二醇—Ⅱ(dammarenediol—Ⅱ),白桦脂酸(betulonk acid),亚细亚酸(asiatic acid),3,23-O-异丙叉亚细亚酸(3,23-O-isopropylidene asiatic acid)和崩大碗酸(madasiatic acid)。其中崩大碗酸系首次从龙脑香科树脂中分离得到。     

31P NMR analysis of intracellular pH of Swiss mouse 3T3 cells: Effects of extracellular Na+ and K+ and mitogenic stimulation

Summary Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional³¹P and¹⁹F probes of intracellular pH (pH _c ) were found to be impracticable. Cells were therefore superfused with 1 to 4mm 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na⁺ with choline or K⁺ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pH _c on external Na⁺ concentration (c _Na ^o ). pH _c also depended on intracellular Na⁺ concentration (c _Na ^o ). Increasingc _Na ^c by withdrawing external K⁺ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducingc _Na ^o produced a larger acid shift in pH _c than with external K⁺ present. Comparison of separate preparations indicated that pH _c was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pH _c of Swiss mouse 3T3 cells using³¹P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.     

油松茎次生结构中树脂道的结构分布和发育的研究

油松茎的次生结构中树脂道存在于次生维管组织中。其中,次生木质部内具有水平的和垂直的两类树脂道,而次生韧皮部内则仅有水平的树脂道。两类树脂道都由上皮细胞和鞘细胞包围着胞间道构成,其中木质部内的树脂道具有死鞘细胞,而韧皮部中的则都系生活细胞。在心材中,垂直树脂道形成拟侵填体。在次生木质部内,垂直树脂道常分布于早材的外部区域和最初形成的晚材中,它们与水平树脂道连接,腔道贯通,从而形成二维网状结构。垂直树脂道来源于纺锤状原始细胞的衍生细胞,而水平树脂道来源于射线原始细胞,两者都以裂生方式发生。     

中国鲎凝集素的分离纯化及性质研究

 经N-乙酰氨基葡萄糖交联琼脂糖亲和层析及以交联琼脂糖介质的高效液相分子筛层析,从中国鲎细胞溶解物中分离纯化了一种凝集素,其活性比原料鲎试剂提高128倍。鲎凝集素SDS电泳时表现出分子量为69000,和72000的二个亚基。N-乙酰氨基葡萄糖、D-半乳糖,D-甘露糖及岩藻糖等对鲎凝集素凝集鸡红细胞的活性有显著抑制作用,加热60℃,10分钟可使凝集素活性基本丧失。CaCl_2为凝集素活性所必需。鲎凝集素与肺炎球菌C多糖有沉淀反应。     

High-throughput RNA extraction from plant samples based on homogenisation by reciprocal shaking in the presence of a mixture of sand and glass beads

We describe here a reliable high-throughput method for extraction of RNA from fresh or frozen plant tissue that obviates laborious and time-consuming homogenisation by mortar and pestle. The method is based on homogenisation by high-speed reciprocal shaking in presence of a mixture of inexpensive abrasive materials; i.e., quartz sand and glass beads. After homogenisation, the method follows a standard procedure for RNA extraction by phenol/LiCl. Yield and quality of RNA obtained by homogenisation with the sand/glass bead mix are identical to those obtained by mortar and pestle.     

湿地松木质部和针叶松脂合成基因分析

为挖掘湿地松(Pinus elliottii)松脂合成相关的基因,对不同采脂期的木质部和针叶进行高通量转录组测序,与火炬松(Pinus taeda)参考基因组进行比对,共获得了68 211条unigenes,546 356 450条clean reads,平均比对率达90.21%。将不同时期木质部、木质部与针叶间进行两两对比,以P＜0.05,|log₂FoldChange|＞1.0为标准来筛选差异基因,并进行GO和KEGG富集分析。结果表明,参与萜类物质合成的差异基因有133个,其中大部分富集在MEP途径,从差异基因中挑选8个产脂相关的候选基因进行RT-qPCR验证,确定HMGR、DXS、TPS、ABC转运蛋白基因与产脂存在关联性。通过转录组测序与分析,挖掘出133个参与松脂萜类物质合成相关的差异基因,其中萜烯合酶基因(TPS)和ABC转运基因在正调控萜类物质合成中发挥关键作用。     

Hybridoma perfusion systems: a comparison study

The efficiency of lgM production by hybridoma cells (1) cultured in suspension; (2) entrapped in alginate beads; or (3) packed in hollowfiber cartridge bioreactors, were compared in long-term perfusion cultures. The results showed that steady-state cell concentration and antibody production, per liter of perfused medium per day, were similar when cells were either entrapped in alginate beads of maintained in suspension. These values were also similar whether cells were maintained at high density in a hollowfiber cartridge bioreactro, or at low density in suspension. This work points out that cell behavior and antibody yield are comparable overall in the various perfusion systems currently used. However, a significant reduction of antibody production appeared whenever a part of the viable cells was lost in the filtrate. The reduction was due both to a decrease of viable cell yield and a decline of lgM productivity on a percell basis. This result is well in agreement with the previously presented model of "grow or die" cell cycle system of hybridoma, which proposes that the ratio of arrested to proliferating cells in perfusion cultures, should be increased in proportion to cell retention in the bioreactor, with a concomitant increase of lgM productivity.