Purification and properties of Pinus taeda arginase from germinated seedlings

In germinated loblolly pine (Pinus taeda L.) seeds arginine accumulates in the seedling during its growth immediately following germination. The enzyme arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is responsible for hydrolyzing this arginine into ornithine and urea. Loblolly pine arginase was purified to homogeneity from seedling cotyledons by chromatographic separation on DE-52 cellulose, Matrex Green and arginine-linked Sepharose 4B. The enzyme was purified 148-fold and a single polypeptide band was identified as arginase. The molecular mass was determined to be 140 kDa by FPLC, while the subunit size was shown to be 37 kDa by SDS-PAGE, predicting a homotetramer holoprotein. Removal of manganese from the enzyme abolishes catalytic activity, which can be restored by incubating the protein with Mn²⁺. Antibodies, raised against the arginase subunit, are able to immunotitrate arginase activity and are monospecific for arginase on immunoblots.     

Broad-specificity quinate (shikimate) dehydrogenasefrom Pinus taeda needles

Two proteins having quinate dehydrogenase (QDH, quinate:NAD(P)⁺-oxidoreductase, EC 1.1.1.24) and shikimate dehydrogenase (SDH, shikimate:NADP⁺-oxidoreductase, EC 1.1.1.25) activities were purified about 3 000-fold from young loblolly pine (Pinus taeda L.) needles. A combination of ammonium sulfate solubilization, and chromatographies on DEAE-cellulose, 2′, 5′ ADP-Sepharose and Mono-Q was used. Throughout all purification steps, the QDH activity consistently co-purified with the activity of the first of three forms of SDH, and the ratio of QDH/SDH was constant (variation from 1.63 to 1.89). These data indicate that both QDH and SDH activities are catalyzed by a single broad-specificity quinate (shikimate) dehydrogenase. Gel chromatography on Superdex 75 was used to estimate the native molecular mass of two forms of the enzyme as 35 and 53 kDa.     

Annual and seasonal changes in fine root biomass of a Quercus ilex L. forest

The biomass, production and mortality of fine roots (roots with diameter <2.5 mm) were studied in a typical Mediterranean holm oak (Quercus ilex L.) forest in NE Spain using the minirhizotron methodology. A total of 1212 roots were monitored between June of 1994 and March of 1997. Mean annual fine root biomass in the holm oak forest of Prades was 71±8 g m^–2 yr^–1. Mean annual production for the period analysed was 260+11 g m^–2 yr^–1. Mortality was similar to production, with a mean value of 253±3 g m^–2 yr^–1. Seasonal fine root biomass presented a cyclic behaviour, with higher values in autumn and winter and lower in spring and summer. Production was highest in winter, and mortality in spring. In summer, production and mortality values were the lowest for the year. Production values in autumn and spring were very similar. The vertical distribution of fine root biomass decreased with increasing depth except for the top 10–20 cm, where values were lower than immediately below. Production and mortality values were similar between 10 and 50 cm depth. In the 0–10 cm and the 50–60 cm depth intervals, both production and mortality were lower.     

林分密度对思茅松人工林根系生物量空间分布的影响

以云南省普洱市思茅松人工林中14a龄林为研究对象,对5种造林密度(1m×1m、1.5m×1.5m、2m×1m、2m×2m、2m×3m)的思茅松人工林的林分特征及其根系生物量空间分布进行调查分析,以明确林分密度对思茅松人工林细根生物量空间分布的影响,为思茅松人工林经营管理提供理论依据。结果表明:(1)树高和胸径随着林分密度的减少呈增大趋势,而生物量呈减少趋势。(2)14a思茅松人工林的细根生物量随林分密度的增大而减少,单株细根生物量随林分密度的减小而增大,但粗根与死根生物量在5个林分密度之间无显著差异。(3)细根生物量主要分布在土壤上层,其中40.21%~54.73%的细根集中在0~10cm土壤深度,不同林分密度的细根生物量随土层深度的增加而减少的趋势较为明显,随着林分密度的减小,土壤上层的细根生物量比例呈先增加后减少的趋势。(4)林分密度和土壤深度对细根生物量有显著影响,而且乔木个体大小差异与细根生物量具有极显著的负相关关系,而单株细根生物量与林分密度、乔木个体大小差异、地下生物量及胸高断面积之间呈显著或极显著的负相关关系,而与树高和胸径之间呈显著正相关关系。     

The role of M cells in Salmonella infection

Intestinal M cells, the specialised antigen-sampling cells of the mucosal immune system, are exploited by Salmonella and other pathogens as a route of invasion. Salmonella entry into M cells and colonisation of Peyer’s patches involve mechanisms critical for infection of cultured cells as well as factors not accurately modelled in vitro.     

黄土区不同林龄刺槐人工林细根的衰老生理特征

以黄土高原刺槐人工林为研究对象,采用手工挖掘法,配合完整土块法获取根系样品,分析幼龄(11a),中龄(22a),成熟(34a)刺槐人工林细根活力、可溶性糖含量、可溶性蛋白含量和细胞膜透性等细根衰老生理指标的差异,为深入了解刺槐细根的生长和衰老机制提供参考。结果表明:(1)在生长季节,刺槐细根活力表现为,幼龄林成熟林中龄林,可溶性糖含量、可溶性蛋白含量随着林龄增大而增加,而细胞膜透性则随林龄的增加而减小。(2)随着根序增加,根活力和可溶性糖含量增加,而可溶性蛋白含量和细胞膜透性则呈波动式降低。这表明,在生长季节幼龄林细根较中龄林和成熟林更容易出现衰老,刺槐不同根序衰老具有顺序性,衰老先从1级根开始,然后是2级根和3级根。     

Degradation and Utilization of Xylans by the Rumen AnaerobePrevotella bryantii(formerlyP. ruminicolasubsp.brevis) B14

Freshly harvested whole cells from cultures ofP. bryantiiB₁4 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantiiB₁4 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation withP. bryantiishowed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobeRuminococcus flavefaciensgave little increase in overall pentose utilization suggesting that externalP. bryantiixylanases are as effective as the clonedR. flavefaciensenzymes in releasing products that can be utilised byP. bryantiicells. The xylanase system ofP. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.     

卡西亚松家系幼林的生长节律

为了解卡西亚松(Pinus kesiya)生长与气象因子间的关系,对17个卡西亚松家系幼林的周年生长性状进行研究。结果表明,17个卡西亚松家系的胸径、树高和抽梢次数存在显著差异(P0.05),可分为速生型(Ⅰ)和慢生型(Ⅱ),其中7、25、61、104、41家系属于速生型,其胸径和树高年均生长量分别为2.03 cm和1.46 m。两类型家系的生长趋势一致,胸径一年内出现2次生长高峰(2和8月),树高出现3次生长高峰(2-3、6和8-10月)。有3次抽梢高峰,分别在5月(春梢)、8-9月(夏梢)和11月(秋梢),一年抽1～4次新梢,以抽2次新梢的植株最多(47%),且在春(71.78%)夏(77.33%)两季抽梢的比例较高。月平均气温对卡西亚松树高的生长影响最大,在1月出现短暂的生长停滞。因此,卡西亚松幼林抚育管理宜在胸径和树高生长高峰之前进行,即5-6和12-翌年1月,1月进行幼林修枝抚育。     

Secretion of different listeriolysin cognates by recombinant attenuated Salmonella typhimurium: superior efficacy of haemolytic over non-haemolytic constructs after oral vaccination

Viable antigen (Ag) delivery systems expressing defined pathogen-derived proteins represent powerful candidates for future vaccination strategies. Here, recombinant (r)Salmonella typhimurium aroA strains secreting listeriolysin (Hly) of Listeria monocytogenes in haemolytic or non-haemolytic form were constructed to direct these carriers into cytosolic or phagosomal host cell compartments, respectively. Oral and intravenous (i.v.) vaccination of mice with either construct induced ‘transporter associated with antigen processing’-dependent protection against the intracellular bacterial pathogen L. monocytogenes. Comparison of oral immunization with both rSalmonella constructs revealed superior vaccine efficacy of the haemolytic rS. typhimurium Hlys construct as compared to the non-haemolytic rSalmonella Hlys₄₉₂ strain. In contrast, efficacy of i.v. vaccination with either rSalmonella strain did not significantly differ. Therefore, rSalmonella strains secreting biologically active Hly represent valuable delivery systems for heterologous rAg or DNA which should be exploited for future mucosal vaccination strategies.     

Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae

Streptococcus pneumoniae is a major pathogen causing potentially life-threatening community-acquired diseases in both the developed and developing world. Since 1967, there has been a dramatic increase in the incidence of penicillin-resistant and multiply antibiotic-resistant pneumococci worldwide. Prevention of access of the antibiotic to the target, inactivation of the antibiotic and alteration of the target are mechanisms that S. pneumoniae has developed to resist antibiotics. Recent studies on antibiotic-tolerant pneumococcal mutants permitted development of a novel model for the control of bacterial cell death.     

Seed germination and seedling fitness in Mesua ferrea L. in relation to fruit size and seed number per fruit

Effect of fruit size and seediness (seed number per fruit) was examined on germination and early growth of seedlings in Mesua ferrea L. Fruiting incidence (number of fruited trees in a population) and fruit loading (number of fruits per tree) vary from one year to the other, and were greater in 1997 than in 1998. Seeds from large fruits (> 40 g) are preferred for forestry plantations and those from small fruits (< 40 g) are discarded, despite a greater proportion of small fruits (63.2 %) than large fruits (36.8 %). A fruit, large or small in size, may contain one, two, three or four seeds. The germination percentage of seeds increased from 1-seeded through 4-seeded fruits both in laboratory and greenhouse conditions, and both in case of large and small fruits. Conversely, the mean seed weight and germination time decreased along this gradient, i.e. seeds from 1-seeded fruits were the heaviest and required maximum time for germination, and the seeds from 4-seeded fruits were the lightest and required minimum time for germination. The seeds from small fruits were lighter in weight, achieved lower germination percentages and required greater germination time than the seeds from large fruits in all four seeded categories. Seedlings from seeds from 1-seeded fruits survived better and with stronger vigour after 1 year of growth than seedlings from 2-, 3- and 4-seeded fruits. Further, seedling survival and vigour were greater for seeds from large rather than small fruits.     

Evaluation of the enzymes involved in primary nitrogen metabolism in Tilia platyphyllos-Tuber borchii ectomycorrhizae

No information is available about Tuber borchii Vittad. ammonium metabolism during its life cycle, which involves the succession of three distinct phases. In this direction, the levels of glutamine synthetase (GS; EC 6.3.1.2), glutamate synthase (GOGAT; EC 1.4.1.13-14) and glutamate dehydrogenase (GDH; EC 1.4.1.2-4) were evaluated in Tilia platyphyllos Scop.-Tuber borchii Vittad. ectomycorrhizae, free living mycelium and non-inoculated roots. In the plant roots, GS shows high specific activity and only NADH-GDH (EC 1.4.1.2) is detectable; on the other hand, in free living mycelium GS and NADPH-GDH (EC 1.4.1.4) can be detected. Ectomycorrhizal metabolism was found to be deeply influenced by the two symbiotic partners. In fact, GS and both forms of GDH are present and their specific activities are higher than those found in the plant root and in the mycelial cells.     

Frequency and effort of reproduction in female Vipera aspis from a southern population

The frequency of reproduction of the asp viper (Vipera aspis, Viperidae) was studied in a population living along the coasts of central Italy. An annual reproductive cycle seemed to be the rule during the 5-year study period. Annual reproduction, high average mass of reproductive females, and large size of neonates, compared with other northern or continental populations, are presumably due to the particularly suitable climatic conditions of the area, as in most coastal habitats of the Mediterranean region. Such a scenario should influence the extent of the feeding period, which allows females, within a few months after parturition, to regain their previous body condition and reproduce again the following year.     

武夷山不同海拔黄山松细根碳、氮、磷化学计量特征对土壤养分的适应

细根的生态化学计量特征承载着植物生存环境的变化信息,从而为探索全球变化对植物内在机制的影响提供理论依据。以江西武夷山国家级自然保护区内五个不同海拔梯度(1200、1400、1600、1800、2000 m)的黄山松为对象,运用挖掘法采样后测定细根C、N、P含量及化学计量比特征,研究不同的海拔下细根对土壤养分变化的适应规律。结果表明:(1)黄山松细根C含量年平均值为(486.27±64.32)mg/g,海拔对其没有显著的影响,与土壤养分之间不存在显著的相关关系。(2)细根N含量年平均值为(9.26±2.09)mg/g,海拔对其没有显著的影响,但与土壤C含量存在显著的正相关关系。(3)细根P含量年平均值为(0.39±0.13)mg/g,与海拔梯度及土壤P含量均存在极显著正相关关系,而与土壤碳氮比呈显著负相关关系。(4)细根氮磷比为26.94±12.51,与海拔梯度、土壤P含量及土壤碳氮比均显著负相关。因此,黄山松细根吸收N是以消耗C为代价;细根P主要受土壤P供应量的限制;武夷山地区N沉降将进一步增加植物的氮磷比,加剧黄山松生长的P限制。     

武夷山不同海拔黄山松细根性状季节变化

细根作为植物吸收养分和水分的主要器官,其功能性状对森林生态系统功能具有重要影响。以武夷山黄山松为研究对象,通过对不同季节(春季、夏季、秋季和冬季)和不同海拔(1200、1400、1600、1800 m和2000 m)的黄山松细根的功能性状的测定,分析其细根性状特征随海拔和季节变化的规律。结果表明:(1)黄山松细根比根长(SRL),比根面积(SRA)均随海拔先升高后降低,其均值分别为(9.32±0.35) cm/g与(276.41±68.10) cm~2/g;根组织密度(RTD)随海拔先降低后升高,均值为(0.16±0.05) g/cm~3。根平均直径(AvgDiam)随海拔增加变化不显著,均值为(0.097±0.004) mm。SRL和SRA在海拔1600 m处达到最大,而RTD和AvgDiam的最大值出现在海拔1800 m或2000 m处。(2)SRL和SRA在夏季或秋季达到最大,RTD和AvgDiam最大值则出现在冬季或春季。季节和海拔对各细根性状都有显著影响(P0.01),但季节与海拔对根性状并没有产生显著的交互作用(P0.05)。(3)SRL与SRA间的异速生长指数是1.25,显著大于1.0(P0.01);SRL与RTD存在负等速生长关系,而与AvgDiam存在显著负异速生长关系(P0.01);SRA与RTD,以及RTD与AvgDiam间均存在显著负异速生长关系(P0.01),但SRA与AvgDiam之间不存在异速生长关系。黄山松的细根性状在1600 m处倾向于增加比根长和比根面积,而在海拔1800 m或2000 m处则倾向于增加组织密度与根直径,这与黄山松细根性状从夏秋到冬春的季节变化规律相类似。同时,相对于比根面积来说,黄山松的细根在海拔1600 m处和夏秋季节更倾向于投资比根长来增加养分的吸收。     

Molecular and biochemical comparison of two different apyrases from Arabidopsis thaliana

Recent advances in the Arabidopsis sequencing project has elucidated the presence of two genes Atb561-A and Atb561-B that show limited homology to the DNA sequence encoding for the mammalian chromaffin granule cytochrome b-561 (cyt b-561). Detailed analysis of the structural features and conserved residues reveals, however, that the structural homology between the presumptive Arabidopsis proteins and the animal proteins is very high. All proteins are hydrophobic and show highly conserved transmembrane helices. The presumably heme-binding histidine residues in the plant and animal sequences as well as the suggested binding site for the electron acceptor, monodehydroascorbate, are strictly conserved. In contrast, the suggested electron donor (ascorbate) binding site is not very well conserved between the plant and animal sequences questioning the function of this motif. Sequence analysis of the Atb561-B gene demonstrates a different splicing than that initially predicted in silico resulting in a protein with nine extra amino acids and a significantly higher homology to the other cyt b-561 sequences. The homology between the plant and animal sequences is further supported by the strong similarity between a number of biochemical properties of the chromaffin cyt b-561 and the cyt b-561 isolated from bean hook plasma membranes. Since the mammalian cyt b-561 is considered specific to neuroadrenergic tissues, the identification of a closely related homologue in an aneural organism demonstrates that these proteins constitute a new class of widely occurring membrane proteins. Both the plant and animal cyt b-561 are involved in transmembrane electron transport using ascorbate as an electron donor. The similarity between these proteins therefore suggests, for the first time, that this transport supports a number of different cell physiological processes. An evolutionary relationship between the plant and animal proteins is presented.     

Storage reserve mobilization during low temperature germination and early seedling growth in Brassica napus

In western Canada, oilseed rape (Brassica napus L. var. oleifera cv. Westar) is seeded during the early months of spring, when ambient temperatures are well below the optimum. This can result in poor seedling emergence. The objectives of the present study were to determine which developmental stages are sensitive to low temperature and whether the effects are thermal or developmental in nature. Seed was germinated at 22, 10 and 6 °C. Fresh weight changes and seedling growth were assessed on the basis of equal accumulated heat units, and the mobilization of storage reserves was assessed by employing antibodies against isocitrate lyase (ICL; EC 4.1.3.1), oleosin and cruciferin. Additionally, de novo protein synthesis was determined by quantifying the incorporation of methionine via in vivo labelling. Low temperature resulted in poor germination and early seedling growth with phase II of germination being most sensitive. At 10 °C, there was a temporal delay in germination that did not affect the overall success of germination. This was a thermal effect as seed at the lower temperatures required the equivalent of 16–24 degree days before germination occurred. Also, seedling growth at 10 °C was lower in comparison to seedlings grown at 22 °C. Seed at 6 °C displayed slow and incomplete germination and poor seedling growth as a result of both thermal and developmental effects.     

Differential effects of light and nitrogen on production of hypericins and leaf glands in Hypericum perforatum

The effect of light intensity and root nitrogen supply on the levels of leaf hypericins was examined for St. John’s wort (Hypericum perforatum L.) grown in a sand culture system with artificial lighting. Increasing the light intensity illuminating St. John’s wort plants from 106 to 402 μmol·m^–2·s^–1 resulted in a continuous increase in the level of leaf hypericins. Using a leaf dissection approach, the association of hypericins with the dark glands on the leaves was shown, and it was found that increasing light intensity resulted in a parallel increase in the number of dark glands. In this respect, a linear relationship was observed between leaf gland number and the level of leaf hypericins (R = 0.901). While a decrease in nitrogen supply to St. John’s wort plants also yielded an increase in the level of leaf hypericins, this response occurred in a discontinuous manner over the range of nitrogen levels tested and no significant effect upon the number of dark leaf glands was observed. Overall, these effects of increased light intensity and decreased nitrogen supply on leaf hypericins appear to be independent and additive, and may reflect differences in the sites and processes where these environmental parameters impact production of these phytochemicals.     

海南岛北部木麻黄林凋落物与土壤养分、细根生物量的关系

通过对海口市桂林洋海岸木麻黄防护林地表凋落物进行持续清除处理,与保留凋落物层的对照样地进行比较实验,研究防护林凋落物-土壤养分-细根生物量三者之间的关系。结果表明:(1)处理样地与对照样地的年凋落物量分别是6.0162和5.9505t·hm-2,但无显著性差异。两种样地一年内凋落物量变化的时间格局较一致。凋落物组成均为小枝占比例最高,且小枝凋落物量月动态与凋落物总量月动态相似,杂物占比例次之,皮+枝占的比例最小;(2)N、P、K元素归还量的月动态变化在两样地中均较一致。两样地中小枝养分归还量的大小顺序为N>K>P;(3)处理样地在短时间无新鲜凋落物输入情况下,两样地的土壤养分含量无显著性差异;(4)两样地的细根生物量季节动态均呈现"单峰"特征,细根生物量在实验期间无显著性差异,但处理样地的细根生物量有随时间延长低于对照样地的趋势,清除凋落物的处理可能会引起细根生物量的减少。     

广西不同林龄杉木、马尾松人工林根系生物量及碳储量特征

为了解不同林龄杉木、马尾松人工林地地下根系生物量及碳储量特征,以广西杉木、马尾松主产区5个不同林龄阶段(幼龄林、中龄林、近熟林、成熟林、过熟林)的人工林为研究对象,采用全根挖掘法和土钻法获取标准木根系生物量、灌草根系生物量和林分细根生物量,并测定其碳含量,分析其不同林龄阶段地下根系生物量和碳储量分配特征。结果表明:杉木、马尾松林地下根系总生物量分别在9.06—31.40Mg/hm~2和7.91—53.40Mg/hm~2之间,各林龄阶段根系总生物量总体上呈现随林龄增加而增加的趋势,杉木林细根生物量随林龄的增加呈现出先减后增的趋势,马尾松呈现出逐渐减小的趋势;林分各层次根系碳含量表现为乔木灌木草本、细根;杉木、马尾松地下根系碳储量变化趋势与生物量变化趋势相同,杉木、马尾松林不同林龄阶段各层次根系和土壤细根总碳储量分别在7.56—21.97Mg/hm~2和8.86—29.95Mg/hm~2之间;地下根系碳储量总体上以乔木根系占优势,且随林龄的增大其比例呈增加的趋势。