Transfer ribonucleic acid from Scenedesmus obliquus D3: General properties

Transfer RNA has been isolated from the green alga Scenedesmus obliquus by phenol extraction at 60°. The proportions of major bases and some of t     

Transfer ribonucleic acid from Scenedesmus obliquus D3: Purification of the major formylatable methionine-accepting species

The major formylatable methionine tRNA of Scenedesmus obliquus has been purified using two column chromatographic steps: (1) chromatography on     

The 520 nm absorbance changes in Scenedesmus obliquus and its relation to Photosystem I

The kinetics (region of seconds) of the light-induced 520 nm absorbance change and its dark reversal have been studied in detail in the wild type and in some pigment and photosynthetic mutants of Scenedesmus obliquus. The following 5 lines of evidence led us to conclude that the signal is entirely due to the photosystem I reaction modified by electron flow from Photosystem II.Gradual blocking of the electron transport with 3(3,4-dichlorophenyl)-1,1-dimethylurea resulted in diminution and ultimate elimination of the biphasic nature of the signal without reducing the extent of the absorbance change or of the dark kinetics. On the contrary, blocking electron flow at the oxidizing side of plastoquinone with 2, $\text{5-}\text{dibromo}\text{-3-}\text{methyl}\text{-6-}\text{isoprophyl}\text{-p-}\text{benzoquinone}$ or inactivating the plastocyanin with KCN, prolonged the dark reversal of the absorbance change apart from abolishing the biphasic nature of the signal.Action spectra clearly indicate that the main signal (I) is due to electron flow in Photosystem I and that its modification (Signal II) is due to the action of Photosystem II.Signal I is pH independent, whereas Signal II demonstrates a strong pH dependence, parallel to the O₂-evolving capacity of the cells.Chloroplast particles isolated from the wild type Scenedesmus cells demonstrated in the absence of any added artificial electron donor or acceptor and also under non-phosphorylation conditions the 520 nm absorbance change with approximately the same magnitude as whole cells. The dark kinetics of the particles were comparatively slower. Removal of plastocyanin and other electron carriers by washing with Triton X-100 slowed down the kinetics of the dark reversal reaction to a greater extent. A similar positive absorbance change at 520 nm and slow dark reversal was also observed in the Photosystem I particles prepared by the Triton method.Mutant C-6E, which contains neither carotenoids nor chlorophyll $\text{b}$ and lacks Photosystem II activity, demonstrates a normal signal I of the 520 nm absorbance change. This latter result contradicts the postulate that carotenoids are the possible cause of the 520 nm absorbance change.     

光、温限制后铜绿微囊藻和斜生栅藻的超补偿生长与竞争效应

研究了铜绿微囊藻(Microcystis aeruginosa)和斜生栅藻(Scenedesmus obliquus)低温和低光照限制后的超补偿效应,以及共培养条件下的竞争效应。结果表明,低温和低光照均显著抑制微藻的生长发育,但低温对铜绿微囊藻的抑制效应更强,而斜生栅藻则对低光胁迫更敏感。经过低光和低温培养后,铜绿微囊藻和斜生栅藻在恢复正常培养时藻细胞密度短期内都表现出超补偿增长效应,但不同藻类超补偿模式不同,斜生栅藻补偿生长时间不超过1周,而铜绿微囊藻的补偿效应可以持续10天;此外,统计结果表明铜绿微囊藻细胞密度对低温限制解除表现出更显著的补偿生长,斜生栅藻则在低光解除后表现出更强的超补偿效应。微藻叶绿素a指标在光恢复条件下都表现出显著的补偿效应,但温度恢复过程中叶绿素a含量与藻密度增长不同步,低温胁迫对恢复正常培养后微藻叶绿素a的形成产生了一定的负效应;铜绿微囊藻产毒株(912)在两种恢复模式下脱氢酶活性显著高于对照,产毒株(912)脱氢酶活性的补偿响应明显高于其它两种材料。共培养实验结果表明斜生栅藻同铜绿微囊藻产毒株(912)相比处于竞争劣势,而在同无毒株(469)的共培实验中,尽管连续正常培养情况下两者竞争能力差异不显著,但在恢复培养条件下斜生栅藻竞争能力显著高于后者。因此产毒型铜绿微囊藻低温和低光后的补偿生长效应以及对斜生栅藻的竞争优势可能是蓝藻爆发的内源性机制之一。     

A comparative approach towards thylakoid membrane proteome analysis of unicellular green alga Scenedesmus obliquus

The chlorophyll (Chl)-containing membrane protein complexes from the green alga Scenedesmus obliquus have been isolated from the thylakoid membranes by solubilization with dodecyl-β-maltoside and fractionation using a sucrose density gradient. The Chl-containing protein fractions were characterized by absorption spectroscopy, tricine SDS PAGE, BN-PAGE, and dynamic light scattering (DLS). BN-PAGE showed the presence of seven protein complexes with molecular weights in the range of 68, 118, 157, 320, 494, 828 and 955 kDa, respectively. Furthermore, light scattering reveals the simultaneous presence of particles of different sizes in the 3-4 nm and 6.0-7.5 nm range, respectively. The smaller size is related to the hydrodynamic radius of the trimer Light Harvesting Complex (LHCII), whereas the larger size is associated with the presence of photosystem I and photosystem II reaction centers. Additionally, functional information regarding protein-protein interactions was deconvoluted using coupling 2-D BN-PAGE, MALDI-TOF MS and a detailed mapping of S. obliquus photosynthetic proteome of the solubilized thylakoid membranes is therefore presented.     

铜绿微囊藻和斜生栅藻非稳态营养盐限制条件下的生长竞争特性

采用“脉冲”添加方法进行了非稳态条件下铜绿微囊藻（M.）和斜生栅藻（S.）分别在氮磷单营养盐和双营养盐限制时的共培养试验。试验结果显示：当添加频率为1d时,无论何种营养盐限制,M.均成为优势藻种。氮限制条件下,氮时均浓度范围在0.3—2.4 mg/L时,M.始终具有竞争优势。磷限制条件下,磷浓度范围在0.018—0.035 mg/L时,S.只在生长初期阶段占优。氮磷双营养盐限制条件下,添加液的氮磷质量比为35:1（设定为最优比）,添加频率为8 d时,两种藻表现出共生特征;而偏离最优比时（N:P=70:1,17:1）,在不同的添加频率下均未出现共生现象,且氮的时均浓度为0.6—4.8 mg/L时（70:1）,M.具有竞争优势,而降低为0.15—0.3 mg/L时（17:1）,S.占优。随着添加频率的变化,两种藻的细胞大小也会随之改变,S.随着营养盐浓度的降低而增大,且在双营养盐限制条件下变化更显著。上述试验结果分析表明：两种藻竞争能力与添加频率相关,在藻种浓度的变化上,按照‘拾遗－机会’交替竞争理论,M.表现出机会主义者特征,而S.则表现出拾遗者的特征,两者的共生特征也符合‘中度干扰’假说。藻细胞大小变化表明,两种藻均可以改变大小实现最大限度争夺受限营养盐。在低浓度时,S.细胞大小的变化同样也变现出了“拾遗者”的特征。     

A mutant strain of Scenedesmus obliquus deficient in ribulose diphosphate carboxylase,cytochrome f and Photosystem II activity

The partial reactions of photosynthesis shown by strain F208, a non-photosynthetic mutant strain of Scenedesmus obliquus, have been compared with those performed by other mutant strains which lacked; Photosystem II activity (strains 11 and F131), cytochrome f (strain 50), P-700 and cytochrome f (strain F119), and P-700 (strains F139 and 199). In this respect the properties of strain F208 were those that would be expected if Photosystem II activity and cytochrome f were not present in this strain. Examination of the composition of strain F208 has shown the absence of cytochrome f in both the soluble and the membrane-bound form. The considerably lower level of plastoquinone compared to that found in the wild type is characteristic of the strains which lack Photosystem II activities.Fraction 1 protein could not be detected in extracts of strain F208 by sedimentation velocity experiments in the ultracentrifuge, and only 7% of the wild type ribulose diphosphate carboxylase activity was found after chromatography of these extracts on DEAE-cellulose.The properties of strain F208 are compared with those of the ac-20 and cr-1 strains of Chlamydomonas rheinhardi, both of which have a deficiency of ribulose diphosphate carboxylase which is considered to result from a deficiency of chloroplast ribosomes. Strain F208 resembles these strains in its abnormal chloroplast ultrastructure and its decreased levels of the RNA forms derived from the chloroplast ribosomes when compared with the wild type.Chloroplast fragments isolated from strains of S. obliquus which lacked cytochrome f (strains 50 and F208) were able to use diaminodurene and ascorbate as an electron donor to Photosystem I. Since this reaction was inhibited by mercuric salts it would appear that plastocyanin, but not cytochrome f, was involved in this electron transfer.     

PSII-efficiency,polysaccharide production,and phenotypic plasticity of Scenedesmus obliquus in response to changes in metabolic carbon flux

To investigate the response of Scenedesmus obliquus to changes in metabolic carbon flux, S. obliquus was cultured in medium with different concentrations of glyoxylate over 9 days. Results showed that growth rates were not affected in the lower concentration glyoxylate (0.25 and 0.5 mM). However, growth rate of S. obliquus was inhibited in the higher concentration glyoxylate (0.85 and 1.25 mM) during the early phase before recovering at higher densities. Changes in growth rates in different glyoxylate concentrations were in line with changes in F_v/F_m and Φ_PSII. Colony formation was observed in S. obliquus in the four glyoxylate treatments. As a consequence, the mean number of cells per particle of S. obliquus in the glyoxylate treatments were significantly higher than those in the control. The total polysaccharide content of S. obliquus cells increased with increased glyoxylate concentrations. The increased glyoxylate-stimulated polysaccharide levels were directly correlated with colony size of S. obliquus.     

Polyethyleneimine-induced flocculation and flotation of cyanobacterium Anabaena flos-aquae for gas vesicle production

Gas vesicles are hollow, proteinaceous structures found in some strains of cyanobacteria. They have been used to increase the oxygen supply and improve the cultivation of shear-sensitive mammalian cells. However, the production and, especially, collection of cells and gas vesicles were laborious and ineffective. In this study we examined the use of the cationic polymer, polyethyleneimine (PEI), for improving the cell harvesting by flocculation and flotation. PEI was examined to determine the appropriate molecular weight, pH range, and dosage. The dose of 20–30 mg/l of PEI with molecular weight of 25,000 in the pH range of 6.0–8.5 was found to provide effective and efficient cell flocculation and flotation. As the PEI dose increased, the rate of flotation increased but the clearance (collection) efficacy declined slightly. The culture samples used in this study were taken from light-limiting continuous culture systems at different dilution rates (0.05–0.24 h⁻¹). Without PEI addition, the cells at dilution rates lower than 0.1 h did not float while those at higher dilution rates would float slowly. With PEI addition, the flocculated cells at the dilution rate of 0.05 h⁻¹ sank and those of higher dilution rates would float and the flotation rate increased with increasing specific growth rate. Nonetheless, PEI flocculation and flotation (or sedimentation) could be used to harvest cells at a wide range of growth states.     

Depletion of urate in human nasal lavage following in vitro ozone exposure

Ozone, a strong oxidant present in summer smog, is thought to primarily react with antioxidant molecules found in the epithelial lining fluid of the respiratory tract. In humans, as much as 40% of inhaled ozone can be removed in the nasal cavity where the major extracellular antioxidant has been identified as uric acid. The present study was undertaken to examine urate/oxidant interactions in human nasal lavage fluid following in vitro exposure to ozone at concentrations relevant to the U.K. Lavage fluid was collected from 8 volunteers using a modified Foley catheter which permits prolonged contact of isotonic saline with the anterior nasal cavity. Nasal lavage samples in multiwell plates were exposed to ozone at concentrations of 50, 100 and 250 ppb. Samples were removed at intervals from 15 to 240 min following exposure and assayed for uric acid depletion. Uric acid concentrations in the nasal lavage were found to fall from 8.52 (time zero) to 3.99 μM, 0.05 and 0.07 μM after 240 min at 50, 100 and 250 ppb ozone respectively. At a non-environmentally relevant ozone concentration of 1000 ppb, uric acid was completely depleted after 60 min. Regression analysis showed a linear correlation between rate of loss of urate and ozone concentration (R² = 0.97). A novel, non-invasive technique is described to investigate antioxidant compromise and its importance in individual subjects. We conclude that uric acid in nasal lavage samples is scavenged by ozone in a dose and time dependant manner.     

Aging of Utricularia traps and variability of microorganisms associated with that microhabitat

Various authors have described algae in aquatic Utricularia traps as commensals, as stress factors or as prey. This study examined the diversity and abundance of organisms (prey, algae, protozoa and bacteria) in the traps of aquatic Utricularia reflexa in relation to prey occurrence and trap age. The number of organisms increased with the trap age. In both young and old traps, phytoplankton dominated of all organisms found. In young traps, Scenedesmus spp. and Characiopsis sp. were the most abundant algae, while Scenedesmus spp. and the palmelloidal form of Euglena spp. dominated in the old traps. Most of the algal species found stayed alive in the trap environment. The number of living algae and ciliates inside the traps increased with the increasing trap age, too. As the number of Paramecium bursaria inside traps consistently increased with the trap age and number of bacteria, which serve as a food for them, ciliates can be regarded as commensals, but not as prey for the plant. The predominant organisms in the traps were those that can be considered either commensals or intruders, exceeding captured macroscopic prey.     

A collagenase assay using [3H-methyl]collagen

A new assay procedure for collagenase is presented. Highly radioactive substrate is prepared by methylation of native collagen. The ³H-labelled protein is readily attacked by bacterial as well as by mammalian collagenase and resistant to other proteinases. The sensitivity of this assay is higher than that of the enzymic methods hitherto available.     

NMR-based metabonomic analysis on effect of light on production of antioxidant phenolic compounds in submerged cultures of Inonotus obliquus

This study was designed to investigate the light effect on biosynthesis of antioxidant phenolic compounds by Inonotus obliquus grown in submerged cultures using ¹H NMR spectroscopy combining multivariate pattern recognition strategies. I. obliquus were exposed to a range of light conditions and resultant data were compared to those from field-grown sclerotia and the mycelia grown in daylight. Daylight illumination inhibited biosynthesis of davallialactone and phelligridins and other hispidin analogs. Continuous darkness enhanced the formation of phelligridins, davallialactone and inoscavins. Phelligridins and davallialactone also occurred in the mycelia grown in blue and red light with levels lower than those found in darkness. In addition, polyphenols synthesized under daylight conditions showed less potential antioxidant activity than those determined with other light regimes. These findings demonstrate that light regulates biosynthesis of polyphenols in I. obliquus and their subsequent antioxidant activities, and ¹H NMR-based metabolic profiling is a cost-effective approach for evaluating light effects on fungal metabolisms.     

Primary metabolites of Phaseolus vulgaris nodules

More ethanol soluble material (carbohydrate and amino nitrogen) was found in both host cell and bacteroid components of Phaseolus vulgaris nodules from plants grown at 28 W/m² than from plants grown at 7 W/m². The range of compounds identified was similar at the two irradiances. On feeding ¹⁴CO₂ to the plant tops at either irradiance the labelling patterns of carbohydrates and organic acids in the nodule host cells and bacteroids suggested that any or all of the following substances could be donated by the host to the bacteroids for general metabolism: sucrose, fructose, glucose, an unidentified carbohydrate, malic acid and an organic acid co-chromatographing with 6-phosphogluconate. Distribution and labelling patterns of nodule amino compounds were consistent with the hypothesis that ammonia is the primary product of nitrogen fixation within bacteroids, and that this ammonia is transported to host cells for assimilation, initially into glutamine and glutamate.     

Ectopic expression of FaesAP3, a Fagopyrum esculentum (Polygonaceae) AP3 orthologous gene rescues stamen development in an Arabidopsis ap3 mutant

Arabidopsis thaliana APETALA3 (AP3) and Antirrhinum majus DEFICIENS (DEF) MADS box genes are required to specify petal and stamen identity. AP3 and DEF are members of the euAP3 lineage, which arose by gene duplication coincident with radiation of the core eudicots. In order to investigate the molecular mechanisms underlying organ development in early diverging clades of core eudicots, we isolated and identified an AP3 homolog, FaesAP3, from Fagopyrum esculentum (buckwheat, Polygonaceae), a multi-food-use pseudocereal with healing benefits. Protein sequence alignment and phylogenetic analyses revealed that FaesAP3 grouped into the euAP3 lineage. Expression analysis showed that FaesAP3 was transcribed only in developing stamens, and differed from AP3 and DEF, which expressed in developing petals and stamens. Moreover, ectopic expression of FaesAP3 rescued stamen development without complementation of petal development in an Arabidopsis ap3 mutant. Our results suggest that FaesAP3 is involved in the development of stamens in buckwheat. These results also suggest that FaesAP3 holds some potential for biotechnical engineering to create a male sterile line of F. esculentum.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Corynebacterium glutamicum using propionate as a precursor

Lipopolysaccharides free P[3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)] production was achieved using recombinant Corynebacterium glutamicum harboring polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha. Cells grown on glucose with feeding of propionate as a precursor of 3HV unit accumulated 8-47 wt% of P(3HB-co-3HV). The 3HV fraction in the copolymer was varied from 0 to 28 mol% depending on the propionate concentrations.