Leaf Developmental Age Controls Expression of Genes Encoding Enzymes of Chlorophyll and Heme Biosynthesis in Pea (Pisum sativum L.)

The effects of leaf developmental age on the expression of three nuclear gene families in pea (Pisum sativum L.) coding for enzymes of chlorophyll and heme biosynthesis have been examined. The steady-state levels of mRNAs encoding aminolevulinic acid (ALA) dehydratase, porphobilinogen (PBG) deaminase, and NADPH:protochlorophyllide reductase were measured by RNA gel blot and quantitative slot-blot analyses in the foliar leaves of embryos that had imbibed for 12 to 18 h and leaves of developing seedlings grown either in total darkness or under continuous white light for up to 14 d after imbibition. Both ALA dehydratase and PBG deaminase mRNAs were detectable in embryonic leaves, whereas mRNA encoding the NADPH:protochlorophyllide reductase was not observed at this early developmental stage. All three gene products were found to increase to approximately the same extent in the primary leaves of pea seedlings during the first 6 to 8 d after imbibition (postgermination) regardless of whether the plants were grown in darkness or under continuous white-light illumination. In the leaves of dark-grown seedlings, the highest levels of message accumulation were observed at approximately 8 to 10 d postgermination, and, thereafter, a steady decline in mRNA levels was observed. In the leaves of light-grown seedlings, steady-state levels of mRNA encoding the three chlorophyll biosynthetic enzymes were inversely correlated with leaf age, with youngest, rapidly expanding leaves containing the highest message levels. A corresponding increase in the three enzyme protein levels was also found during the early stages of development in the light or darkness; however, maximal accumulation of protein was delayed relative to peak levels of mRNA accumulation. We also found that although protochlorophyllide was detectable in the leaves immediately after imbibition, the time course of accumulation of the phototransformable form of the molecule coincided with NADPH:protochlorophyllide reductase expression. In studies in which dark-grown seedlings of various ages were subsequently transferred to light for 24 and 48 h, the effect of light on changes in steady-state mRNA levels was found to be more pronounced at later developmental stages. These results suggest that the expression of these three genes and likely those genes encoding other chlorophyll biosynthetic pathway enzymes are under the control of a common regulatory mechanism. Furthermore, it appears that not light, but rather as yet unidentified endogenous factors, are the primary regulatory factors controlling gene expression early in leaf development.     

Ion-translocating ATPases in tendrils ofBryonia dioica Jacq.

Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils ofBryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg²⁺ATP-driven transport of⁴⁵Ca²⁺. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca²⁺-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca²⁺-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K⁺, Mg²⁺-ATPase and the Ca²⁺-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.Abbreviations CAM calmodulin - CCCP carbonylcyanidem-chlorophenylhydrazone - IC₅₀ concentration giving 50% inhibition - PM plasma membrane - rER rough endoplasmic reticulum - sER smooth endoplasmic reticulum - FC fusicoccin - U₃+U₃ the two PM-rich upper phases obtained after phase partitioning of microsomal membranes The authors wish to thank the Deutsche Forschungsgemeinschaft, Bonn, Germany, and the Fonds der Chemischen Industrie, Frankfurt, Germany (literature provision) for financial support.     

Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants

Immunocytochemical localization of polygalacturonic acid (pectin) and methyl-esterified pectin in the walls of pollen tubes of 20 species of flowering plants grown in vitro was investigated by using monoclonal antibodies (MAbs) JIM5 and JIM7 and by means of confocal laser scanning microscopy (CLSM). In general, periodic annular deposits of pectins were found coating the tube wall in species possessing solid styles, and a more uniform pectin sheath in tube walls in species having hollow styles or no styles. We hypothesize that the periodic ring-like structure of the pectin sheath reinforces pollen tubes for passing through the transmitting tract in the style. Esterified pectin which prevents Ca²⁺-induced gelification of pectate is located predominantly at the apex. This implies that pectin esterification is related to tip wall loosening that is required for cell wall expansion during tip growth of pollen tubes. The occurrence of unesterified pectins in other areas of pollen tube walls suggests that de-esterification of pectin following tip expansion leads to a more rigid form of pectin that contributes to the construction of the pollen tube wall.     

Regeneration of catalytic activity of glutamine synthetase mutants by chemical activation: exploration of the role of arginines 339 and 359 in activity.

In order to understand the nature of ATP and L-glutamate binding to glutamine synthetase, and the involvement of Arg 339 and Arg 359 in catalysis, these amino acids were changed to cysteine via site-directed mutagenesis. Individual mutations (Arg-->Cys) at positions 339 and 359 led to a sharp drop in catalytic activity. Additionally, the Km values for the substrates ATP and glutamate were elevated substantially above the values for wild-type (WT) enzyme. Each cysteine was in turn chemically modified to an arginine "analog" to attempt to "rescue" catalytic activity by covalent modification; 2-chloroacetamidine (CA) (producing a thioether) and 2,2'-dithiobis (acetamidine)(DTBA) (producing a disulfide) were the reagents used to effect these chemical transformations. Upon reaction with CA, both R339C and R359C mutants showed a significant regain of catalytic activity (50% and 70% of WT, respectively) and a drop in Km value for ATP close to that for WT enzyme. With DTBA, chemically modified R339C had a greater kcat than WT glutamine synthetase, but chemically modified R359C only regained a small amount of activity. Modification with DTBA was quantitative for each mutant and each modified enzyme had similar Km values for both ATP and glutamate. The high catalytic activity of DTBA-modified R339C could be reversed to that of unmodified R339C by treatment with dithiothreitol, as expected for a modified enzyme containing a disulfide bond. Modification of each cysteine-containing mutant to a lysine "analog" was accomplished using 3-bromopropylamine (BPA).(ABSTRACT TRUNCATED AT 250 WORDS)     

Two conformational states of Candida rugosa lipase.

The structure of Candida rugosa lipase in a new crystal form has been determined and refined at 2.1 A resolution. The lipase molecule was found in an inactive conformation, with the active site shielded from the solvent by a part of the polypeptide chain-the flap. Comparison of this structure with the previously determined "open" form of this lipase, in which the active site is accessible to the solvent and presumably the substrate, shows that the transition between these 2 states requires only movement of the flap. The backbone NH groups forming the putative oxyanion hole do not change position during this rearrangement, indicating that this feature is preformed in the inactive state. The 2 lipase conformations probably correspond to states at opposite ends of the pathway of interfacial activation. Quantitative analysis indicates a large increase of the hydrophobic surface in the vicinity of the active site. The flap undergoes a flexible rearrangement during which some of its secondary structure refolds. The interactions of the flap with the rest of the protein change from mostly hydrophobic in the inactive form to largely hydrophilic in the "open" conformation. Although the flap movement cannot be described as a rigid body motion, it has very definite hinge points at Glu 66 and at Pro 92. The rearrangement is accompanied by a cis-trans isomerization of this proline, which likely increases the energy required for the transition between the 2 states, and may play a role in the stabilization of the active conformation at the water/lipid interface. Carbohydrate attached at Asn 351 also provides stabilization for the open conformation of the flap.     

Changes and Relationship of PAF and TNF in Rats with Myocardial Ischaemia and Reperfusion Injury

IN THIS STUDY IT IS REPORTED THAT: (1) the levels of blood platelet-activating factor and serum tumour necrosis factor significantly increased after coronary ligation and reperfusion, compared with sham-ligated controls, in an anaesthetized rat model; (2) compared with vehicle controls, pretreatment with the PAF antagonist BN 50739 (10 mg/kg, i.v.) produced significant decreases in infarct size (from 29.6 +/- 4.0% to 22.4 +/- 2.1%, p < 0.05 after 3 h ligation, and from 28.5 +/- 9.5% to 10.5 +/- 4.5%, p < 0.01 after 4 h reperfusion) and the level of serum TNF (from 10.4 +/- 7.7 U/ml to 3.9 +/- 4.8 U/ml, p < 0.05); and (3) a significan positive correlation was found between the level of blood PAF or serum TNF and infarct size. The present results indicate that PAF and TNF may be important mediators involved in myocardial ischaemia and reperfusion injury, and that PAF antagonists may exert a protective effect on ischaemic or reperfused myocardium by inhibiting the interaction of PAF and TNF.     

华南淡水鱼类单殖吸虫的研究──XVI．锚首虫属一新种（指环虫目：锚首虫科）

从广西荔蒲河所产的美丽小条鳅Ｍｉｃｒｏｎｏｅｍａｃｈｅｉｌｕｓｐｕｌｃｈｅｒ（ＮｉｃｈｏｌｓｅｔＰｏｐｅ）鳃上采得单殖吸虫一种,经鉴定为锚首虫属一新种──小脊锚首虫,Ａｎｃｙｒｏｃｅｐｈａｌｕｓｌｉｒｅｌｌａｔｕｓｓｐ．ｎｏｖ。新种与Ａ．ｂｏｔｉａｅ．Ｍａｅｔａｌ．１９８１后吸器几了质结构相似,但雄性交接器,阴道结构显著不同。     

Structure—activity studies leading to potent chloride channel blockers: 5e-tert-butyl-2-[4-(substituted-ethynyl)phenyl]-1,3-dithianes

5e-tert-Butyl-2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes with selected functional groups (R) on the ethynyl moiety are potent blockers of the GABA-gated chloride channel measured as inhibitor concentration (IC₅₀) for 4-n-[³H]propyl-1-(4-ethynylphenyl)-2, 6,7-trioxabicyclo[2.2.2]octanebinding to bovine brain membranes. The terminal R substituents were introduced by coupling 5e-tert-butyl-2e-(4-iodophenyl)-1,3-dithiane with HC ≡ CR or 5e-tert-butyl-2e-(4-ethynylphenyl)-1,3-dithiane with XR. The potency of the parent compound (R=H) with an IC₅₀ of 21 μM is equaled or exceeded by up to 7-fold (i.e. IC₅₀ = 3–21 μM) by several carboxylic acids [R = (CH₂)_nCO₂H (n = 0–3), (CH_2nOCH₂CO₂H (n = 1–3) and CH₂SCH₂ CO₂H] and their esters and two phosphonic acids (CH₂CH₂PO₃H₂ and CH₂OCH₂PO₃H₂) but not their esters. These carboxyl and phosphonic acids (and their salts) include the most potent water-soluble chloride channel blockers known. Conversion to the monosulfones increases activity of the R = H and CH₂OH analogs by 1.2- to 3-fold but decreases that of the R = CH₂CH₂CO₂R′ (R′ = H or CH₃) derivatives by 3- to 13-fold. Quantitative structure-activity analyses for 44 2-[4-(substituted-ethynyl)phenyl]-dithianes suggests that the principal feature of the R substituent for high activity is its polarizable volume modeled as molecular refractivity, i.e. this substituent is not a well-defined pharmacophore and undergoes a structurally non-specific interaction with the receptor. These observations lay the background for preparing candidate affinity probes.     

从水稻根部悬浮培养细胞分离原生质体及植株再生

以长香稻(Oryza sativa L．)(糯稻)的幼嫩种子根为材料,在Ms和N6培养基上诱导产生结构松软而分散的愈伤组织,经过AA液体培养基振荡悬浮培养,悬浮培养细胞经酶解后得到了活性较高的原生质体,试验结果表明这是分离原生质体较理想的材料。在附加2,4-D lmg／L(以下单位同)、KT(激动素)0．2、各氨酰胺876、天门冬酰胺266的Ms琼脂糖培养基中,诱导出了大量愈伤组织,在含KT2、NAA(α-萘乙酸)0．2、zT(玉米素)0．2、cH(水解酪蛋白)1000和4％椰子汁的N6培养基中成功地诱导出了由原生质体再生的植株。当代原生质体再生植株能正常开花、结实、产生种子;染色体数均为二倍性(2n＝24),最显著的特点是结实率低,穗粒数减步、生育期延迟。     

抗鞘翅目δ－内毒素 及毒素基因文库的构建

研究了对鞘翅目昆虫有毒效的5个苏云金芽孢杆菌新菌株YM-03、Sph04-04、YK14-01、SH11-05、Sph16-01伴孢晶体的蛋白质组成,以柳蓝叶甲为供试虫测定了它们的LC_(50)值,其中YM-03毒力最高。测定了YM-03晶体蛋白N-末端部分氨基酸序列。用琼脂糖凝胶电泳快速检测了5株菌的质粒组成,证明其质粒图型各不相同。以广谱的cosmid质粒pLAFRI为载体,通过限制酶EcoRI部分酶解获得“目的”DNA片段,构建了菌株YM-03的总DNA文库,对17个抗性克隆的质粒进行酶切分析表明,其中含有外源片段的克隆占总数的76％,超过要求的理论值。以人工合成的杀鞘翅目基因的18bp保守序列片段为探针,筛选了近1200个抗性克隆,获得了3个阳性克隆,LE392(PBYM2)、LE392(pBYM3)和LE392(pBYM4),毒力测定试验表明LE392(pBYM3)和LE392(pBYM4)有一定表达,表明其携带有δ-内毒素基因。