Entropy-driven polymerization of protein from the E66 strain of tobacco mosaic virus

Protein of the tobacco mosaic virus mutant E66 has lysine replacing asparagine of the type strain, vulgare, at position 140. Thus, E66 protein should have one more positive or one less net negative charge than vulgare at pH 6 to 7. To investigate the effect of charge, a comparative study of the polymerization of E66 and vulgare proteins at pH 6.0, 6.2, 6.4, 6.6, and 6.8 at ionic strengths 0.15, 0.10, and 0.05 was made by turbidimetry. Polymerization of E66 protein always proceeded at a lower temperature than vulgare. However, the extent of polymerization was much lower in E66, especially at the higher ionic strengths. Sedimentation velocity results paralleled those from turbidity measurements in that E66 protein polymerizes at lower temperatures than vulgare; the 20 S component is more abundant in E66 protein. Osmotic pressure measurements also show that E66 protein is more polymerized than vulgare, especially at lower pH values. Hydrogen ion titrations of E66 protein were carried out from pH 8 to 5 and back to pH 8 in 0.10 m KCl at three temperatures, 4, 10, and 15 °C. These titrations were reversible when carried out slowly. The isoionic point is near pH 5; thus the charge at pH 7.5 is ?3. The reversible titration results were correlated with the aggregates present at the various pH values and temperatures, determined from the areas under the schlieren peaks in sedimentation velocity experiments. It is found that hydrogen ion binding at the three pH values is correlated with the disappearance of the smallest aggregates and is independent of the type of higher polymer formed. To investigate the effect of ionic strength and pH on the characteristic temperature corresponding to an optical density increment of 0.01 by the method used previously for vulgare, two sets of turbidity measurements were carried out. In the first one the ionic strength was changed from 0.025 to 0.15 in increments of 0.025 at pH 6.0 and 6.4. In the other set, the ionic strength was kept constant at 0.10 and the pH changed from 5.9 to 6.7 in increments of 0.1 pH units. When the analysis of these data was carried out, $\text{Δ}\text{H}{}^1\text{= 30}\text{kcal/mol}$ was obtained. For the salting out constant a value of 1.7 was found, compared to 2.2 for vulgare, a result consistent with the fact that E66 should be less hydrophobic than vulgare. The electrical work term ΔW_el also turns out to be about one-half that for vulgare, which is expected from the lower net negative charge on E66 protein.     

Bee visits and the nectar of Echium vulgare L. and Sinapis alba L.

Abstract. 1. The time-course of anthesis of Echium vulgare is described.
2. Diel changes in the sugar concentration of the nectar, the quantity of nectar and the quantity of sugar per flower are illustrated for E.vulgare and for Sinapis alba.
3. These changes are interpreted in terms of (a) the periodicity of secretion and (b) the influence of microclimate and insect visits on post-secretory changes in the composition and volume of nectar.
4. There was hour-to-hour and day-to-day variation in the species composition and the proportion of workers taking nectar rather than nectar plus pollen among the social bees visiting E.vulgare.
5. Honeybee visits to E.vulgare were more numerous in humid weather, when there was enough nectar per flower for their relatively short tongues to reach, and in an area where the corollas grew shorter than they did elsewhere.
6. The significance of changes in the caloric content, volume, concentration, viscosity and sugar composition is discussed from the points of view of insects and ecologists.     

The first cytochrome P450 in ferns. Evidence for its involvement in phytoecdysteroid biosynthesis in Polypodium vulgare

The fern Polypodium vulgare is a phytoecdysteroid (PE)-producing plant. Cultures of P. vulgare prothalus produce PE, whereas prothalus-derived callus cultures do not. However, this callus line can transform topically applied ecdysone (E) to 20-hydroxyecdysone (20E), which is the last step in the biosynthetic pathway of the main plant PE. This hydroxylation is catalysed by a cytochrome P450 enzyme. E treatment of the callus line results in an increased amount of P450, showing a linear correspondence between the amount of P450 and in vivo E 20-hydroxylation activity, estimated by measuring the bioconversion of E to 20E. This activity can be inhibited by molecules that bind to the P450-heme group. E shows a P450-substrate-binding spectrum with microsomes that overexpress the P450 protein. Finally, a P450 protein was purified from E-treated calli, this being the first P450 to be described in the pterydophyte group.     

酮古龙酸菌细胞色素c的表达、成熟及活性分析

目的:从酮古龙酸菌Y25中扩增细胞色素c基因,在大肠杆菌中表达、成熟并进行生物活性分析。方法:从酮古龙酸菌Y25基因组中PCR扩增细胞色素c基因,构建pET22b表达载体;从大肠杆菌BL21(DE3)中扩增细胞色素c成熟基因簇ccmABCDEFGH,连接到带有山梨糖脱氢酶组成型启动子的pBBR1MCS2-P200载体中;将构建的2个质粒共转化大肠杆菌BL21(DE3),经IPTG诱导表达后进行血红素染色检测;通过氧化还原光谱法对Ni柱亲和纯化的重组蛋白进行活性分析。结果:扩增得到1404 bp的细胞色素c基因及6481 bp的ccmABCDEFGH基因簇;重组菌株经IPTG诱导表达后行SDS-PAGE分析,可见相对分子质量为50×103的表达条带;血红素染色显示重组蛋白结合有血红素;经氧化还原光谱扫描,显示亲和层析纯化得到的目的蛋白有细胞色素c特征吸收峰。结论:从酮古龙酸菌Y25中扩增得到了细胞色素c基因,在大肠杆菌中进行了表达和成熟,表达蛋白具有细胞色素c生物活性。     

酮古龙酸菌细胞色素c基因的克隆及表达

目的:从酮古龙酸菌SCB329中分离细胞色素c(Cytc)相关基因,在大肠杆菌中进行表达并验证。方法:根据酮古龙酸菌SCB329基因组序列设计引物,通过PCR从SCB329基因组中扩增cytc基因,酶切后连接pET22b表达载体,转化至大肠杆菌DH5α后提取质粒,经PCR、质粒双酶切及测序鉴定后,转入大肠杆菌BL21(DE3),并对表达条件进行考察;用Chelating Sepharose珠粒对可溶性的Cytc-His融合蛋白进行纯化;经光谱扫描和血红素染色等方法对表达蛋白定性分析。结果:PCR扩增的cytc基因长513 bp;重组菌在IPTG浓度为0.025 mmol/L的条件下,于28℃诱导10 h后,SDS-PAGE分析可见表达条带,相对分子质量约为18×103;Ni柱亲和层析纯化得到目的蛋白,纯化蛋白经光谱扫描呈现Cytc特征峰,血红素染色呈现阳性结果。结论:从酮古龙酸菌SCB329中分离得到一种cytc基因,并表达纯化了融合蛋白Cytc-His,纯化蛋白呈现Cytc特性,为研究酮古龙酸菌中产酸关键酶的电子传递机制奠定了基础。     

酮古龙酸菌山梨醇脱氢酶的原核表达及活性检测

目的：克隆酮古龙酸菌Y25的山梨醇脱氢酶基因sldh,在大肠杆菌中进行表达并检测表达产物的活性。方法：以酮古龙酸菌Y25基因组DNA为模板,PCR扩增sldh基因,连接到表达载体pTIG,转入大肠杆菌BL21（DE3）,IPTG诱导表达;取表达菌体、菌体裂解上清和沉淀进行SDS-PAGE分析;以山梨醇为底物,通过活性电泳、体外转化及休止细胞转化进行sldh基因表达产物的活性检测。结果：扩增得到1740 bp的山梨醇脱氢酶基因,构建了表达质粒pTIG-sldh并在大肠杆菌中获得表达,SDS-PAGE结果显示表达产物为可溶性形式,相对分子质量约58×10^3;活性电泳结果说明表达产物在以山梨醇为底物时表现出脱氢酶活性,而经体外转化和休止细胞转化后薄层层析检测出转化产物山梨糖的存在。结论：在大肠杆菌中实现了酮古龙酸菌山梨醇脱氢酶的可溶性表达,且表达的重组脱氢酶能将山梨醇脱氢生成山梨糖。     

Biotransformations of putative phytoecdysteroid biosynthetic precursors in tissue cultures of Polypodium vulgare.

Incubation of calli and prothalli of Polypodium vulgare with different tritium-labelled ecdysteroids has led to modification of some previous assumptions about the biosynthesis of ecdysteroids in plants. Thus, 25-deoxy-20-hydroxyecdysone was transformed efficiently in both tissues into 20-hydroxyecdysone (20E), but no 25-deoxyecdysteroids such as pterosterone and inokosterone were formed. Likewise, incubation of 2-deoxyecdysone (2dE) produced exclusively ecdysone (E) and 20E, indicating a high 2-hydroxylase activity in both tissues, despite calli not producing phytoecdysteroids. This 2-hydroxylation was also evident in the transformation of 2,22-dideoxyecdysone (2,22dE) into 22-deoxyecdysone (22dE). Different ecdysteroids that do not occur in P. vulgare were formed in the incubation of 3-dehydro-2,22,25-trideoxyecdysone (3D2,22,25dE) by 3alpha-reduction and 3beta-reduction and 25-hydroxylation processes. The fact that 22,25-dideoxyecdysone and 22dE were the only 2-hydroxylated products formed in this case suggests that only compounds bearing a 3beta-hydroxyl group are substrates for the 2-hydroxylase. Surprisingly, 22-hydroxylation was never observed with either 2,22dE or 3D2,22,25dE, raising the possibility that it could occur at an early step in the biosynthetic pathway. In this respect, labelled 22R-hydroxycholesterol was efficiently converted into E and 20E, whereas 22S-hydroxycholesterol was not transformed into ecdysteroids, because of its unsuitable configuration at C22. Finally, the conversion of 25-hydroxycholesterol into E and 20E was greatly enhanced after thermal treatment of prothalli which induces the release of previously stored ecdysteroids. Thus, P. vulgare prothalli and calli appear to be particularly suitable models for the study of ecdysteroid biosynthesis and its regulation in plants.     

Advanced backcross QTL analysis in barley (Hordeum vulgare L.)

This paper reports on the first advanced backcross-QTL (quantitative trait locus) project which utilizes spring barley as a model. A BC(2)F(2) population was derived from the initial cross Apex ( Hordeum vulgare ssp. vulgare, hereafter abbreviated with Hv) x ISR101-23 ( H. v. ssp. spontaneum, hereafter abbreviated with Hsp). Altogether 136 BC(2)F(2) individuals were genotyped with 45 SSR (simple sequence repeat) markers. Subsequently, field data for 136 BC(2)F(2) families were collected for 13 quantitative traits measured in a maximum of six environments. QTLs were detected by means of a two-factorial ANOVA with a significance level of P < 0.01 for a marker main effect and a marker x environment (M x E) interaction, respectively. Among 585 marker x trait combinations tested, 86 putative QTLs were identified. At 64 putative QTLs, the marker main effect and at 27 putative QTLs, the M x E interaction were significant. In five cases, both effects were significant. Among the putative QTLs, 29 (34%) favorable effects were identified from the exotic parent. At these marker loci the homozygous Hsp genotype was associated with an improvement of the trait compared to the homozygous Hv genotype. In one case, the Hsp allele was associated with a yield increase of 7.7% averaged across the six environments tested. A yield QTL in the same chromosomal region was already reported in earlier barley QTL studies.     

Typification of Cerastium fontanum ssp. vulgare (Caryophyllaceae)

The basionym of Cerustium fontanum ssp. vulgare (Hartman) Greuter and Burdet, C. vulgare Hartman, is neotypified, based on a specimen in UPS. Flora Nordica notes No. 14.     

Potential for Integrated Control of Horehound Marrubium vulgare L. by the Plume Moth Wheeleria spilodactylus (Curtis) and 2,4-D

Marrubium vulgare is an introduced perennial herbaceous weed of pastures and natural ecosystems in southern Australia. The moth Wheeleria spilodactylus has recently been established as a biocontrol agent and is spreading widely; its larvae feed on shoots and later leaves of M. vulgare. Glasshouse experiments were carried out to examine how eggs, larvae and adult W. spilodactylus might be affected by herbicide treatment of M. vulgare and thus to determine whether the two control techniques were compatible. Survival and development of larvae was generally little affected by herbicide treatments that were sufficient to damage the weed severely. Adult moths showed large reductions in longevity and fecundity in response to loss of M. vulgare flowers as a food source. Female moths offered a choice of treated or untreated plants as oviposition sites strongly avoided herbicide-treated M. vulgare. The results justify further investigation of the combined effects of herbicide and W. spilodactylus in the management of M. vulgare infestations.     

酮古龙酸菌醇醛脱氢酶基因的克隆及表达

目的：从酮古龙酸菌SCB329株中分离山梨糖生物氧化相关酶的基因并进行表达验证。方法：根据酮古龙酸菌SCB329株基因组序列设计引物,通过PCR从SCB329株基因组中扩增醇醛脱氢酶基因aadh;构建载体pBMP3-aadh并在大肠杆菌中表达,经活性染色、体外转化反应等方法考察表达产物的活性。结果：目的产物能够催化山梨糖、葡萄糖、果糖、木糖等多种含羟基及羰基化合物脱氢,并能将L-山梨糖直接转化为2-酮基-L-古龙酸。结论：从酮古龙酸菌SCB329株中分离到一种醇醛脱氢酶基因,可为该菌株糖酸转化机制的研究提供帮助。     

酮古龙酸菌细胞色素c氧化酶亚基Ⅱ的融合表达及纯化

目的:利用大肠杆菌融合表达酮古龙酸菌细胞色素c氧化酶亚基Ⅱ(CcoⅡ)与谷胱甘肽S-转移酶(GST)并纯化。方法:根据酮古龙酸菌Y25基因组序列设计引物,通过PCR扩增CcoⅡ基因,酶切后连接pGEX-KG表达载体,转化至大肠杆菌获得重组菌,经IPTG诱导表达融合蛋白GST-CcoⅡ,用谷胱甘肽-Sepharose 4B树脂亲和纯化融合蛋白,并利用Western印迹及质谱对表达蛋白进行鉴定。结果:扩增得到867 bp的CcoⅡ基因,构建了pGEX-KG-CcoⅡ融合表达载体,重组菌经0.4 mmol/L IPTG于20℃诱导16 h,SDS-PAGE分析显示有可溶性表达条带,相对分子质量约为59×103;Western印迹及质谱分析表明,利用亲和层析方法纯化到了目的蛋白。结论:表达并纯化了GST-CcoⅡ融合蛋白,为酮古龙酸菌电子传递链的研究奠定了基础。     

Contemporary seasonal and altitudinal variations of leaf structural features in oregano (Origanum vulgare L.)

The effects of elevation (200, 950 and 1760 m) and season (April-October) on leaf morphological, anatomical, ultrastructural, morphometrical and photosynthetic parameters were studied in Origanum vulgare plants. Observations aimed at the determination of the alterations in leaf structure and function associated with differential growth and adaptation of plants. Raising elevation results in a progressive decrease of plant height. During the growing period, summer plants are taller than spring and autumn plants at all elevations examined. In high-altitude populations (O. vulgare ssp. vulgare), the blade size becomes reduced in June leaves as compared with October leaves, while it does not change remarkably in low-altitude populations (O. vulgare ssp. hirtum). Leaf thickness remains more or less stable during the growing period. Expanded leaves in June and October at 200 m elevation contain dark phenolics only in their epidermis, whereas leaves of August are densely filled with phenolics in all of their tissues. In June at 1760 m elevation, leaves are devoid of phenolics, which, however, occur in the epidermis of the leaves in August and October. At higher altitudes, larger mesophyll chloroplasts with more starch grains are present in June leaves, whereas in August and October leaves chloroplasts are smaller with fewer starch grains. Leaf stomata and non-glandular hairs increase in number from the lowland to the upland habitats, whereas glandular hairs decrease in number. During the growing season, the density of stomata and of glandular and non-glandular hairs progressively increases. In the low- and mid-altitude oregano populations, leaf chlorophyll a content and PSII activity significantly increase in October, whereas they simultaneously decrease in the high-altitude population, suggesting a phenomenon of chilling-induced photoinhibition. The highest photochemical efficiency of PSII appears in the mid-altitude population (having characteristics intermediate between those of O. vulgare ssp. hirtum and ssp. vulgare) where environmental conditions are more favourable. This conclusion is also confirmed by the observation that the 950 m O. vulgare population has larger and thicker leaves with highly developed palisade and spongy parenchymas.     

醇醛脱氢酶的分离纯化及其基因文库的构建和筛选

在维生素C的发酵生产过程中,普通生酮基古龙酸菌S2(Ketogulonigenium vulgare)能产生醇醛脱氢酶,将L-山梨糖转化为VC的前体2-酮基-L-古龙酸(2-KLG)。通过超声波破碎菌体、硫酸铵分级沉淀、DEAE Sepharose Fast Flow阴离子交换层析,QSepharose High Performance柱层析等过程,从普通生酮基古龙酸菌S2发酵液中分离纯化了醇醛脱氢酶,并用该纯化酶免疫新西兰兔制备出了合格抗血清。同时,普通生酮基古龙酸菌S2基因组DNA经Sau3AⅠ部分酶切后,与黏粒载体pKC505连接,用包装蛋白进行包装,转染大肠杆菌DH5浕,构建了基因组文库。最后应用免疫酶斑点技术(Dot-ELISA)从12000个克隆子中筛选得到一个阳性克隆K719#。通过检测该基因工程菌的活性,表明K719#具有使L-山梨糖转化为2-KLG的功能,从而使醇醛脱氢酶在大肠杆菌中获得了高效表达,这为简化VC的生产工艺奠定了基础。     

Comparative high resolution map of the six-rowed spike locus 1 (vrs1) in several populations of barley, Hordeum vulgare L

Multiple alleles at the vrs1 locus control the development and fertility of the lateral spikelets of barley (Hordeum vulgare L.), which is a key character in the study of yield, utilization and domestication. In this study, six linkage maps of the vrs1 locus were constructed, using different mapping populations developed from nine different barley cultivars (H. vulgare subsp. vulgare) or mutant and wild barley (H. vulgare subsp. spontaneum). A total of 8387 chromosomes (gametes) were sampled for analysis based on a hypothesis that orders of marker loci were the same over the different parental lines. The results showed that four markers and the vrs1 locus in all cases were arranged in the same order, which was in a good agreement with the hypothesis. This makes the linkage maps suitable for the positional cloning of the alleles at the vrs1 locus.     

A Review of: “Centrifugation in Biology and Medical Science,P. Sheeler,Wiley/Interscience,New York, 1981; hardbound, 269 pages, $35.00”

ABSTRACT

Peroxidases (E.C. 1.11.1.7., hydrogen donor oxidoreductase) utilize hydrogen peroxide or substituted peroxides for the oxidation of a large number of substrates. Peroxidases are widely distributed and have been isolated from many higher plants (1). The wide distribution of the enzyme suggests that it could be of great biological importance, but the physiological functions and metabolic control of these enzymes are still poorly understood. The simultaneous presence of amine oxidase and peroxidase in cell walls suggests that the peroxide generated on oxidation of the amines could be utilized by the peroxidase (2,3). Recently we have purified an amine oxidase from Hordeum vulgare (4) and we have attempted to purify the peroxidase in order to study in vitro the reconstituted coupled system. β-glucosidase (β-D-glucoside glucohydrolase E.C. 3.2.1.21.) is capable of transforming glucosides in glucose and the corresponding aglycone or disaccarides as cellobiose, sophorose, gentiobiose. This enzyme is widely distributed in plants, fungi, bacteria, yeasts and animals (5, 6). In the homogenate of Hordeum vulgare seedlings we also found β-glucosidase activity and also attempted to purify β- glucosidase. This enzyme copurified whit peroxidase up to the last step. We report here the isolation of peroxidase and β-glucosidase from Hordeum vulgare see-dlings: some molecular and kinetic properties are given.     

桂东、桂东南晚泥盆世一个弗拉斯期放射虫动物群

本文系统描述了广西玉林、贺县等地晚泥盆世榴江组硅质岩中的放射虫化石4属14种,其中有1新属2新种。这个放射虫动物群的多数分子曾见于苏联南乌拉尔地区和澳大利亚的弗拉斯阶。     

EPIDERMAL FEATURES AND SILICA DEPOSITION IN LEMMAS AND AWNS OF ZIZANIA (GRAMINEAE)

In four species of Zizania (Gramineae: Oryzeae) epidermal features of pistillate and staminate lemmas, paleas, and awns were studied by scanning electron microscopy (SEM) and energy dispersive X-ray analysis. Features observed were silica bodies, siliceous papillae, pitted siliceous papillae, stomata, microhairs, and prickle hairs. Staminate lemmas have all of these features. Pistillate lemmas have silica bodies and prickle hairs, lack stomata, and differ among species in occurrences of microhairs and siliceous papillae and pitted siliceous papillae. Awns of pistillate lemmas have silica bodies, prickle hairs, microhairs, and stomata; therefore, they possess a more complete set of features than their attached lemmas. Shapes of silica bodies on pistillate lemmas differ among species. A taxonomic key based on SEM observation of pistillate lemmas separates the four species by the shapes of silica bodies, arrangement of prickle hairs, and occurrences of microhairs and siliceous papillae. The main silica-containing structures are silica bodies, siliceous papillae, pitted siliceous papillae, and to a lesser extent prickle hairs. Pitted siliceous papillae with circular raised rims are formed by collapse or exfoliation of the tops of siliceous papillae; these have not been previously described in grasses. Comparison of epidermal features in the lemmas and leaves of Zizania shows that the former lack three kinds of nonsilicified papillae and epicuticular wax that are present on the latter but the lemmas have siliceous papillae and pits that are absent in leaves.     

Cation regulation by the terrestrial isopod Armadillidium vulgare (Crustacea: Isopoda: Oniscidea) during dehydration in air

Many terrestrial arthropods display tight osmotic and ionic regulation of the hemolymph during dehydration. In this study, we sought to quantify the level of regulation of the major hemolymph cations in the terrestrial isopod Armadillidium vulgare (Isopoda, Oniscidea). Inulin space measurements showed that the hemolymph comprises 52 ± 2.2% of the hydrated water content but contributes 71 ± 9.8% of water losses during desiccation. Hemolymph concentrations of Na+, K+ and Ca2+ were measured in variably dehydrated animals using ion-selective microelectrodes and compared with predicted concentrations assuming no regulation. Na+ and Ca2+ are quite tightly regulated, showing respective concentration increases of 20.8% and 7.1% following a 50% reduction in hemolymph volume, but K+ showed no measurable regulation. The excreted cation fraction during desiccation is negligible. Sites of ion sequestration were examined by injecting 22Na and ??Ca into the hemolymph of hydrated animals and assaying tissue-specific activities following dehydration. Na+ is apparently sequestered non-specifically by an unknown mechanism. Ca2+ accumulates in the dorsal somatic tissues, possibly in the calcium pool of the cuticle. How A. vulgare avoids significant disruptions of E(m) and neuromuscular function in the absence of K+ regulation, and how it sequesters Na+, both pose intriguing challenges for future work.     

ON THE HOST RANGE AND SYSTEMATIC POSITION OF THE BACTERIA RESPONSIBLE FOR THE YELLOW SLIME DISEASES OF WHEAT (TRITICUM VULGARE VILL.) AND COCKSFOOT GRASS (DACTYLIS GLOMERATA L.)

Inoculations with Corynebacterium rathayi (E. F. Sm.) Dowson, alone or combined with the eelworm Anguina tritici into the leaves and shoots of cocksfoot grass ( Dactylis glomerata L.) and wheat ( Triticum vulgare Vill.), fail to cause infection. Inoculations made through the soil with Corynebactmim rathuyi and C. tritici (Hutchinson) Burkh. plua Anguina tritici cause infection in wheat but not in cocksfoot grass. The symptoms produced on wheat resemble those of yellow slime disease of cocksfoot grass. Successful infection of wheat with Corynebacterium rathay. or C. tritici is dependent on the presence, in the soil surrounding the plants, of Anguina tritici together with one of these bacterial species, which are considered to be so closely related as to suggest that Corynebacterium tritici is but a geographical variety of C. rathayi.