Abscisic Acid-Induced Membrane Potential Changes in Barley Aleurone Protoplasts: a Possible Relevance for the Regulation of rab Gene Expression

The effect of ABA on the membrane potential of barley (Hordeumvulgare cv. Himalaya) aleurone protoplasts was studied by measuringthe distribution of the lipophilic cation tetraphenylphosphonium(TPP⁺). The resting membrane potential (E_m) according to ourmeasurements with TPP⁺ is about –53 mV and is in agreementwith membrane potential values as measured with intracellularmicroelectrodes (about –55 mV). The TPP⁺-measurementscould demonstrate a clear dependence of the resting E_m on theexternal pH (pH_e). Stimulation of the protoplasts with ABA induced a transienthyperpolarization of the membrane to –62 mV as measuredwith TPP⁺. The hyperpolarization was ABA-concentration dependent. Inhibition of the H⁺-ATPases with the specific proton pump inhibitorsdiethylstilbestrol (DES) or Micanozole effectively preventedhyperpolarization. This indicates that the hyperpolarizationis consistent with the activation of plasma membrane H⁺-ATPases.The K⁺-inward rectifier inhibitor BaCl₂ was able to prolongthe hyperpolarization. This result suggests that the hyperpolarizationcauses the opening of K⁺-channels. The ABA-induced proton-pump activation may be involved in ABA-inducedgene-expression, as DES was able to inhibit this gene expression.BaCl₂ did only show a slight inhibitory effect on ABA-inducedgene-expression. (Received January 4, 1994; Accepted April 12, 1994)     

Purification and characterization of α-glucosidase from Aspergillus carbonarious

Summary An -glucosidase was purified from Aspergillus carbonarious CCRC 30414 over 20 fold with 37 % recovery. Its molecular mass was estimated to be 328 kDa by gel filtration with an optimum pH from 4.2 to 5.0, and pI=5.0. The optimum temperature is at 60°C over 40 min. The enzyme was partially inhibited by 5 mM Ag⁺, Hg²⁺, Ba²⁺, Pb²⁺, and Aso₄ ⁺.     

Characterization of inhibitory effects of NH2OH and its N-methyl derivatives on the O2-evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca²⁺ and Cl^-) are essential for oxidation of H₂O to O₂ by Photosystem II. The Mn reductants NH₂OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H₂O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O₂ evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl^-, F^- and SO₄ ^2-) is able to slow NH₂OH and CH₃NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl^- is capable of attenuating CH₃NHOH and (CH₃)₂NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl^- fails to protect against NH₂OH inhibition in salt-washed membranes. These results indicate that the attack by NH₂OH and its N-methyl derivatives on Mn occurs at different sites in the O₂-evolving complex. The small reductant NH₂OH acts at a Cl^--insensitive site whereas the inhibitions by CH₃NHOH and (CH₃)₂NOH involve a site that is Cl^- sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl^- sensitivity of their binding to Mn in the O₂-evolving complex.Abbreviation MES 4-morpholinoethanesulfonic acid - PS II Photosystem II     

人肝刺激因子对大鼠实验性慢性肝损伤的保护作用

从健康孕妇水囊引产４─６个月龄的胎儿取肝,采用ＬａＢｒｅｃｑｕｅ方法提取人肝刺激因子（ｈＨＳＳ）。经３Ｈ－胸腺嘧啶核苷参入肝ＤＮＡ法测定其生物活性。表明此ｈＨＳＳ可刺激肝细胞ＤＮＡ合成。采用皮下注射ＣＣｌ４和饮用１０％乙醇来制备慢性肝损伤动物模型,观察了ｈＨＳＳ的保护肝脏作用。结果表明：ｈＨＳＳ可使ＣＣｌ４－乙醇所致慢性肝损伤大鼠的死亡率、血清谷丙转氨酶水平、肝组织中羟脯氨酸含量的升高以及肝组织中丙二醛的含量降低。肝组织切片表明：ｈＨＳＳ能减轻肝组织的损伤程度,促进肝细胞再生,并能明显防止肝纤维化的形成和发展。可见,ｈＨＳＳ对ＣＣｌ４－乙醇所致的慢性肝损伤大鼠有明显的保护作用,其机制可能与促进肝细胞再生及抑制肝细胞膜的脂质过氧化有关。     

四氯化碳损伤成年大鼠肝细胞后原癌基因（c－fos／c－jun）表达的观察

用四氯化碳（ＣＣｌ４）损伤正常大鼠后,采用Ｗｅｓｔｅｒｎ印迹法和免疫组化法观察肝细胞原癌基因（ｃ－ｆｏｓ／ｃ－ｊｕｎ）的表达。Ｗｅｓｔｅｒｎ印迹法表明,当成年大鼠的静息期肝细胞受到ＣＣｌ４损伤性刺激后,ｃ－ｆｏｓ／ｃ－ｊｕｎ产物（Ｆｏｓ和Ｊｕｎ）水平升高,在ＣＣｌ４处理后３０ｍｉｎ开始升高,在４ｈ时消失。８ｈ后Ｆｏｓ／Ｊｕｎ再度出现,并持续２４ｈ以上。ＩＣＣ法表明,Ｊｕｎ阳性细胞为靠近肝中央静脉区的肝实质细胞。根据上述资料推测,肝受ＣＣｌ４损伤后肝细胞的原癌基因ｃ－ｆｏｓ／ｃ－ｊｕｎ出现即时的与滞后的两次表达,这与肝细胞进入细胞周期有关,这种基因表达也许可作为肝再生过程中识别特殊体液因子的标志。     

单纯疱疹病毒通用及Ⅱ型特异性DNA探针的制备及应用研究

单纯疱疹病毒（ＨＳＶ）Ⅰ型及Ⅱ型之间有很多共同抗原,能引起血清学交叉反应,鉴别诊断比较困难。本实验利用重组ＤＮＡ技术,将部分ＨＳＶ－２ＤＮＡ的ＰｓｔＩ片段克隆到载体质粒ＰＳＫ中,并筛选出两个重组质粒（Ｐ和Ｐ）只与ＨＳＶ－２反应,与ＨＳＶ－１不反应,这两个重组质粒中所含的ＨＳＶ－２ＤＮＡ片段大小分别是３．１和４．３ｋｂ,另外,还筛选了一个重组质粒（ＰＨＳＶ２－１,含５．８ｋｂＨＳＶ－２ＤＮＡ片段）与ＨＳＶ－１和ＨＳＶ－２均反应。将４．３ｋｂ的片段用光生物素标记后作为探针检测了１５９份人阴道拭子,其中２３份样品呈阳性反应,其余均为阴性,从２３份阳性样品中挑选１２价涂片用间接荧光抗体法检测也都呈阳性反应,随机挑选的几份杂交反应阴性样品在间接荧光试验中也是阴性。本实验制备的ＨＳＶ通用及ＨＳＶ－２型特异性探针将比常规的血清学方法诊断和鉴别ＨＳＶ－１和ＨＳＶ－２感染更为可靠。     

大鼠肝癌中α_2-巨球蛋白等几种分泌性蛋白基因表达的变化

用Northern blot方法对二乙基亚硝胺所诱发的大鼠肝癌中内源性蛋白酶抑制因子α_2-巨球蛋白(α_2-M)、非特异性免疫抑制剂α_1-酸性糖蛋白(α_1-AGP)及雄性激素正调控的α-2u球蛋白(α-2u)三种分泌性蛋白基因表达情况进行了分析。结果表明在大部分(14/16)肝癌样品中α_2-M RNA水平显著降低;而α_1-AGP RNA水平显著高于正常对照水平;α-2u RNA水平明显下降,但在某些雄性大鼠肝癌样品中该基因却有一定程度的表达。这些结果说明,一些肿瘤宿主血浆中α_2-M水平的显著下降及α_1-AGP水平的明显升高分别是由于基因表达活性的下降及升高所致。α-2u基因表达的异常提示,在癌变过程中机体的内分泌功能发生了某些变化。     

Accelerated organic matter decomposition in thermokarst lakes upon carbon and phosphorus inputs

Mineralization of dissolved organic matter (DOM) in thermokarst lakes plays a non-negligible role in the permafrost carbon (C) cycle, but remains poorly understood due to its complex interactions with external C and nutrient inputs (i.e., aquatic priming and nutrient effects). Based on large-scale lake sampling and laboratory incubations, in combination with ¹³C-stable-isotope labeling, optical spectroscopy, and high-throughput sequencing, we examined large-scale patterns and dominant drivers of priming and nutrient effects of DOM biodegradation across 30 thermokarst lakes along a 1100-km transect on the Tibetan Plateau. We observed that labile C and phosphorus (P) rather than nitrogen (N) inputs stimulated DOM biodegradation, with the priming and P effects being 172% and 451% over unamended control, respectively. We also detected significant interactive effects of labile C and nutrient supply on DOM biodegradation, with the combined labile C and nutrient additions inducing stronger microbial mineralization than C or nutrient treatment alone, illustrating that microbial activity in alpine thermokarst lakes is co-limited by both C and nutrients. We further found that the aquatic priming was mainly driven by DOM quality, with the priming intensity increasing with DOM recalcitrance, reflecting the limitation of external C as energy sources for microbial activity. Greater priming intensity was also associated with higher community-level ribosomal RNA gene operon (rrn) copy number and bacterial diversity as well as increased background soluble reactive P concentration. In contrast, the P effect decreased with DOM recalcitrance as well as with background soluble reactive P and ammonium concentrations, revealing the declining importance of P availability in mediating DOM biodegradation with enhanced C limitation but reduced nutrient limitation. Overall, the stimulation of external C and P inputs on DOM biodegradation in thermokarst lakes would amplify C-climate feedback in this alpine permafrost region.     

不同密度格木幼林的土壤理化与林下植被特征

为探索适合格木(Erythrophleum fordii)人工林在幼龄阶段的种植密度,在不同林分密度(2 m×1 m、2 m×2 m、2 m×3 m、3 m×3 m)的6 a生格木人工林下设置标准样地,采用土壤质量评价和灰色关联度等方法,探究不同密度下格木幼林的土壤理化与林下植被特征。结果表明,密度2 m×3 m下的林木胸径、树高最优,较最低水平高16.7%、27.9%;土壤总孔隙度最大,全N、硝态N、铵态N含量最高,灌木草本多样性最高。相关性分析表明土壤化学性质对灌木草本的多样性影响最大。不同林分密度下格木幼林土壤理化性质及林下植物多样性有显著差异,因此,选择合适的林分密度对人工林土壤肥力的可持续利用及林分的经营培育至关重要。