属特异性T-RFLP技术用于乳酸杆菌的群落分析

【目的】设计乳酸杆菌属特异性T-RFLP技术(末端限制性片段长度多态性分析)对14株乳酸杆菌进行分型。【方法】采用源于16S-23S rRNA基因间隔区序列的乳酸杆菌属特异性引物LAB-rev,乳酸杆菌的属特异性引物,6-FAM荧光标记后结合16S上游通用引物7f用于乳酸杆菌的PCR扩增。【结果】选取HaeⅢ和HhaⅠ进行限制性酶切,最后对酶切后的产物末端测序得到T-RFLP峰谱图,该图谱能够快速准确地对不同种的乳酸杆菌进行定性、定量的分析。【结论】实验成功搭建T-RFLP技术用于微生态环境中乳酸杆菌检测的平台,对于在功能性食品、乳酸饮料和药物对肠道微生态的影响及菌种鉴定等领域有重大意义。     

ATP-inhibited and Ca2+-dependent K+ channels in the soma membrane of cultured leech Retzius neurons

The properties of one ATP-inhibited and one Ca²⁺-dependent K⁺ channel were investigated by the patch-clamp technique in the soma membrane of leech Retzius neurons in primary culture. Both channels rectify at negative potentials. The ATP-inhibited K⁺ channel with a mean conductance of 112 pS is reversibly blocked by ATP (K _i = 100 m), TEA (K _i =0.8 mm) and 10 mm Ba²⁺ and irreversibly blocked by 10 nm glibenclamide and 10 m tolbutamide. It is Ca²⁺ and voltage independent. Its open state probability (P _o) decreases significantly when the pH at the cytoplasmic face of inside-out patches is altered from physiological to acid pH values. The Ca²⁺-dependent K⁺ channel with a mean conductance of 114 pS shows a bell-shaped Ca²⁺ dependence of P _o with a maximum at pCa 7–8 at the cytoplasmic face of the membrane. The P _o is voltage independent at the physiologically relevant V range. Ba²⁺ (10 mm) reduces the single channel amplitude by around 25% (ATP, TEA, glibenclamide, tolbutamide, and Ba²⁺ were applied to the cytoplasmic face of the membrane).We conclude that the ATP-dependent K⁺ channel may play a role in maintaining the membrane potential constant—independently from the energy state of the cell. The Ca²⁺-dependent K⁺ channel may play a role in generating the resting membrane potential of leech Retzius neurons as it shows maximum activity at the physiological intracellular Ca²⁺ concentration.This study was supported by the Deutsche Forschungsgemeinschaft (W.-R. Schlue) and by a fellowship of the Konrad-Adenauer-Stiftung (G. Frey). We thank Dr. Draeger (Hoechst AG) for the gift of glibenclamide. The data are part of a future Ph.D. thesis of G. Frey.     

The membrane potential of Arabidopsis thaliana guard cells; depolarizations induced by apoplastic acidification

The apoplastic pH of guard cells probably acidifies in response to light, since light induces proton extrusion by both guard cells and epidermal leaf cells. From the data presented here, it is concluded that these apoplastic pH changes will affect K⁺ fluxes in guard cells of Arabidopsis thaliana (L.) Heynh. Guard cells of this species were impaled with double-barrelled microelectrodes, to measure the membrane potential (E_m) and the plasma-membrane conductance. Guard cells were found to exhibit two states with respect to their E_m, a depolarized and a hyperpolarized state. Apoplastic acidification depolarized E_m in both states, though the origin of the depolarization differed for each state. In the depolarized state, the change in E_m was the result of a combined pH effect on instantaneously activating conductances and on the slow outward rectifying K⁺ channel (s-ORC). At a more acidic apoplastic pH, the current through instantaneously activated conductances became more inwardly directed, while the maximum conductance of s-ORC decreased. The effect on s-ORC was accompanied by an acceleration of activation and deactivation of the channel. Experiments with acid loading of guard cells indicated that the effect on s-ORC was due to a lowered intracellular pH, caused by apoplastic acidification. In the hyperpolarized state, the pH-induced depolarization was due to a direct effect of the apoplastic pH on the inward rectifying K⁺ channel. Acidification shifted the threshold potential of the channel to more positive values. This effect was accompanied by a decrease in activation times and an increase of deactivation times, of the channel. From the changes in E_m and membrane conductance, the expected effect of acidification on K⁺ fluxes was calculated. It was concluded that apoplastic acidification will increase the K⁺-efflux in the depolarized state and reduce the K⁺-influx in the hyperpolarized state. Received: 28 April 1997 / Accepted: 10 November 1997     

硝化抑制剂DCD、 DMPP对褐土氮总矿化速率和硝化速率的影响

采用15N库稀释-原位培养法研究了硝化抑制剂DCD、DMPP对华北盐碱性褐土氮总矿化速率和硝化速率的影响.试验在山西省运城市种植玉米的盐碱性土壤上进行,设单施尿素、尿素+DCD、尿素+DMPP 3个处理.结果表明:施肥后2周,DCD、DMPP分别使氮总矿化速率和氮总硝化速率减少了25.5％、7.3％和60.3％、59.1％,DCD对氮总矿化速率的影响显著高于DMPP,两者对氮总硝化速率的影响无显著差异;而在施肥后7周,不同硝化抑制剂对氮总硝化速率的影响存在差异.施肥后2周,3个处理的土壤氮总矿化速率和硝化速率分别是施肥前的7.2 ～10.0倍和5.5 ～21.5倍;NH4+和NO3-消耗速率分别是施肥前的9.1 ～12.2倍和5.1 ～8.4倍,这是由氮肥对土壤的激发效应所致.硝化抑制剂使氮肥更多地以NH4+形式保持在土壤中,减少了NO3-的积累.土壤氮总矿化速率和总硝化速率受硝化抑制剂的抑制是N2O减排的主要原因.     

Monoclonal antibodies reveal changes in predator efficiency with prey spatial pattern

Spatially explicit predator–prey interactions can alter the predatory potential of natural enemies augmented through conservation biological control. To test hypotheses regarding such interactions and predatory efficiency, we used a combination of molecular techniques and mark–release–recapture to study the foraging behaviour of a generalist carabid predator, Poecilus cupreus , in response to spatial patterns of its cereal aphid prey ( Metapolophium dirhodum and Sitobion avenae ). Beetle and aphid numbers were measured across two grids of sampling locations, within which aphid spatial pattern had been manipulated to generate patchy and more homogenous distributions. Aphid consumption was measured by enzyme-linked immunosorbent assays (ELISA) of beetle gut contents, using an aphid-specific monoclonal antibody. Movement and distribution patterns suggest that P. cupreus does not aggregate at, nor instigate prey-taxis within, aphid patches. However, more than two-thirds of the 2169 P. cupreus tested by ELISA had consumed aphids and the proportion of beetles containing aphid proteins was positively related to aphid density. Against expectation, the proportion of predators feeding on aphids was greatest where prey were homogenously distributed, and this was attributed to the loss of partial refuges for prey in aphid patches. The functional value of this type of uniform foraging strategy is ideally suited to early colonization of the crop habitat, when aphid numbers are low, before populations build up and form strong spatial patterns.     

Distinct fucosylation of M cells and epithelial cells by Fut1 and Fut2, respectively, in response to intestinal environmental stress

The intestinal epithelium contains columnar epithelial cells (ECs) and M cells, and fucosylation of the apical surface of ECs and M cells is involved in distinguishing the two populations and in their response to commensal flora and environmental stress. Here, we show that fucosylated ECs (F-ECs) were induced in the mouse small intestine by the pro-inflammatory agents dextran sodium sulfate and indomethacin, in addition to an enteropathogen derived cholera toxin. Although F-ECs showed specificity for the M cell-markers, lectin Ulex europaeus agglutinin-1 and our monoclonal antibody NKM 16-2-4, these cells also retained EC-phenotypes including an affinity for the EC-marker lectin wheat germ agglutinin. Interestingly, fucosylation of Peyer’s patch M cells and F-ECs was distinctly regulated by α(1,2)fucosyltransferase Fut1 and Fut2, respectively. These results indicate that Fut2-mediated F-ECs share M cell-related fucosylated molecules but maintain distinctive EC characteristics, Fut1 is, therefore, a reliable marker for M cells.     

西加毒素的危害及其检测技术

西加毒素是由少数几种海洋底栖微藻产生,具有极强毒性的生物毒素,它能够通过食物链传递而累计在多种珊瑚鱼体内,继而造成人类因食用鱼类而中毒。近年来,随着珊瑚鱼类在世界范围内的广泛贸易,西加毒素中毒已经成为全球性的健康问题。为给预防西加毒素引起的食物中毒提供借鉴,对西加毒素的分子结构、化学性质、生物来源、制毒机理、中毒症状和对人类的危害进行了综述。就目前主要的检测方法进行了技术特性的介绍,并对常用检测方法的优缺点进行了比较分析。     

南极假丝酵母脂肪酶A的表面展示及酶学性质研究

采用a凝集素作为载体蛋白,首次将南极假丝酵母脂肪酶A展示在酿酒酵母细胞表面,通过MD平板筛选获得表面展示型的CALA酵母工程菌株。免疫荧光检测显示CALA被成功展示在酵母细胞壁表面,重组子经诱导后能在三丁酸甘油酯板上形成透明圈,说明展示的CALA具有活性。重组酵母在液体培养基培养72 h,活性达到最高,为80.4 U/g干细胞。酿酒酵母展示的CALA最适温度及pH值为70°C和pH 8.0。经50°C保温2 h,仍含有60%水解酶活力。展示的CALA在pH 7.0和pH 8.0溶液中比较稳定。经DMSO处理2 h,展示的CALA仍保持70%的活性。以上结果表明酵母展示的CALA可作为一种有潜质商业用途的全细胞催化剂。     

DNA condensation by TmHU studied by optical tweezers,AFM and molecular dynamics simulations

The compaction of DNA by the HU protein from Thermotoga maritima (TmHU) is analysed on a single-molecule level by the usage of an optical tweezers-assisted force clamp. The condensation reaction is investigated at forces between 2 and 40 pN applied to the ends of the DNA as well as in dependence on the TmHU concentration. At 2 and 5 pN, the DNA compaction down to 30% of the initial end-to-end distance takes place in two regimes. Increasing the force changes the progression of the reaction until almost nothing is observed at 40 pN. Based on the results of steered molecular dynamics simulations, the first regime of the length reduction is assigned to a primary level of DNA compaction by TmHU. The second one is supposed to correspond to the formation of higher levels of structural organisation. These findings are supported by results obtained by atomic force microscopy.