Intra- and interspecific variations in vacuolar flavonoids among Ficus species from the Budongo Forest,Uganda

Leaves of 14 species of Ficus growing in the Budongo Forest, Uganda, were analysed for vacuolar flavonoids. Three to six accessions were studied for each species to see whether there was intraspecific chemical variation. Thirty-nine phenolic compounds were identified or characterised, including 14 flavonol O-glycosides, six flavone O-glycosides and 15 flavone C-glycosides. In some species the flavonoid glycosides were acylated. Ficus thonningii contained in addition four stilbenes including glycosides. Most of the species could be distinguished from each other on the basis of their flavonoid profiles, apart from Ficus sansibarica and Ficus saussureana, which showed a very strong intraspecific variation. However, on the whole flavonoid profiles were sufficiently distinct to help in future identifications.     

ent-Labdane glycosides from the aquatic plant Potamogeton lucens and analytical evaluation of the lipophilic extract constituents of various Potamogeton species

Two new ent-labdane glycosides, one known furano-ent-labdane and a new hydroxylated fatty acid were isolated from the dichloromethane extract of the freshwater aquatic plant Potamogeton lucens. The new compounds were assigned the structures of beta-d-glucopyranosyl-8(17),13-ent-labdadien-16,15-olid-18-oate, 18-beta-d-glucopyranosyloxy-8(17),13-ent-labdadien-16,15-olide and 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trien-oic acid by spectroscopic means. The algicidal activity of these compounds was tested against Raphidocelis subcapitata. Based on our previous study of Potamogeton pectinatus, other constituents were identified in P. lucens by LC-UV-MS, LC-NMR and GC-MS. The lipophilic extract profiles of both species are presented. Two other species, Potamogeton perfoliatus and P. crispus, were also investigated by analytical comparison of their non-polar extracts. The distribution of ent-labdanes characterized in Potamogeton is summarized.     

Triterpene saponins and flavonoids in the seeds of Trifolium species

Seeds of 57 species of the genus Trifolium have been studied for the occurrence and concentration of soyasapogenol B glycosides and flavonoids. It was shown that all tested species contained soyasaponin I and in some species astragaloside VIII and/or 22-O-glucoside and 22-O-diglucoside of soyasaponin I were also present. Total concentration of saponins ranged from trace amounts up to 10 mg/g(DM). It was suggested that soyasapogenol B glycosides could be recognized as chemotaxonomic character of Fabaceae family. All but three tested species contained flavonoids. The majority of species contained quercetin as a sole flavonoid or in the mixture with a number of unidentified flavonoid components. Concentration of quercetine in some species was at the level of about 3 mg/g(DM). This high quercetin concentration and soyasaponin occurrence makes the seeds of some Trifolium species a potential source of health beneficial phytochemicals, to be used in human nutrition.     

The comparison of mechanically stimulated and spontaneous firings in traps of aquatic carnivorous Utricularia species

Firing and resetting of traps in aquatic Utricularia species are associated with water flows and trap volume changes. In this study, trap thickness was used as a measure of water flow and was monitored automatically using an electronic position sensor. Isolated traps from three aquatic Utricularia species were monitored over the course of 1-2 days to verify spontaneous firings (without any mechanical stimulation) and describe their basic characteristics. Isolated traps of three Utricularia species were initially fired by mechanical stimulation and allowed to naturally reset within a period of 24-48 h. Within this resting period, spontaneous firings were found in the traps of all species and in two trap age categories of U. vulgaris. The timing of spontaneous firings was found to be irregular. Spontaneous firings ranged between 0.29 and 2.4 during the 24-h resting period and the mean time between two spontaneous firings was highly variable within each species (319-891 min). There was no quantitative difference between spontaneous and mechanically stimulated firings of the traps. Spontaneous firings could explain how phytoplankton or detritus enters traps even when no prey species are present.     

Variations of floral traits among different life forms illustrate the evolution of pollination systems in Potamogeton species from China

Four leaf morpha groups (floating-leaved, submerged broad-leaved, submerged linear-leaved and filiform-leaved) and three inflorescences type groups (erect, facultative floating and floating) can be found in the genus Potamogeton. Life forms in the genus correlate with pollination types (anemophily, epihydrophily and hydroautogamy). Floral traits vary greatly among different life forms and pollination types. Both floating and submerged broad-leaved species produce erect inflorescences that are suited for anemophilous pollination. These flowers also have the highest pollen/ovule (P/O) ratio (25,013) and the smallest pollen grain volume (6046 μm³). On the contrary, submerged filiform-leaved species produce only floating inflorescences and their flowers produce the largest pollen grains (17,567 μm³) and have the lowest P/O ratio (6752), which is typical of the syndromes of hydroautogamy and epihydrophily. The relatively prolific seed production (about 70%) and wide dispersal in the floating and submerged broad-leaved species and the submerged filliform-leaved species indicate that these two groups are well adapted to the aquatic environment. This is in contrast with the less well-adapted complex and unstable pollination system found in linear-leaved species. We propose that the pollination systems and life forms in the Potamogeton, have undergone modification in response to the demands imposed by the aquatic environment during the evolutionary process in the genus.     

Prenylated flavanones and flavanonols as chemical markers in Glycosmis species (Rutaceae)

Fifteen prenylated or geranylated flavanones and flavanonols were isolated from the leaf extracts of different Glycosmis species collected in Thailand and Malaysia. All structures were elucidated by spectroscopic methods, especially 1D and 2D NMR. Six compounds were described for the first time and two were only known so far as synthetic products. The chemotaxonomic significance of flavanoid accumulation within the genus Glycosmis is highlighted.     

Flavonoids in the species of Cyrtomium (Dryopteridaceae) and related genera

Flavonoids in 19 Cyrtomium, three Cyrtogonellum and two Phanerophlebia taxa were surveyed. Major flavonoids were flavonol O-glycosides based on kaempferol, quercetin, and sometimes myricetin, and C-glycosylflavones, such as isovitexin, vitexin, isoorientin, orientin and their O-glycosides. The C-methylflavanones, farrerol and cyrtominetin, and their 7-O-glucosides were isolated from Cyrtomium devexiscapulae and Cyrtomium laetevirens. Flavanones have been reported from Cyrtomium falcatum sensu lato. Though C. falcatum sensu lato is divided into four taxa, i.e. C. falcatum subsp. falcatum, C. falcatum subsp. australe, C. falcatum subsp. littorale, and C. devexiscapulae, the occurrence of the flavanones was restricted to C. devexiscapulae, and they did not occur in C. falcatum sensu stricto.     

Phenological and chemical differences among hybrid poplar clones (Salicaceae) varying in resistance to Cryptorhynchus lapathi (L.) (Coleoptera: Curculionidae)

Hybrid poplars with Populus maximowiczii parentage have demonstrated antixenotic resistance against adult poplar-and-willow borers, Cryptorhynchus lapathi (L.) (Coleoptera: Curculionidae), in the summer, and antibiosis against C. lapathi larvae in the spring. We examined phenological and biochemical differences among hybrid poplars with and without P. maximowiczii parentage that are resistant and susceptible, respectively, to C. lapathi, to test the hypotheses that resistance was due to physical hydraulic forces, inadequate nutrition, or abundant secondary metabolites. The hybrids with P. maximowiczii parentage flushed earlier than susceptible hybrids, but no differences were found between hybrids with respect to sap flow or bark moisture content that could explain larval mortality in the spring. Similarly, constitutive levels of nutritive compounds (sitosterol, nitrogen, linoleic and linolenic acids, and carbohydrates) were lower or not different in the most susceptible hybrid TN 302-9. Thus, all hybrids appear to be nutritionally sound. TN 302-9 contained the highest constitutive levels of condensed tannins and catechin, but overall phenolic glycoside levels (salicin and salicortin) were similar among all hybrids, with the exception of one sampling date; however, this difference would not likely explain resistance. Neither induced or constitutive polyphenol oxidase (PPO) activity nor induced levels of secondary metabolites could explain resistance. Thus, we conclude hybrids that contain P. maximowiczii parentage have a novel mechanism for resistance.     

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.     

Changes in RACK1 expression induce defects in nodulation and development in Phaseolus vulgaris

RACK1 is a scaffold protein with the ability to interact in a regulated manner with a diverse number of ligands from distinct signal-transduction pathways. This assessment allowed us to infer that it may be involved in different processes such as nodulation. In a recent study we showed by silencing, that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development in Phaseolus vulgaris. On the other hand, we have also observed that its overexpression provokes a dramatic phenotype in: (a) seedlings that have been exposed to heat, in which systemic necrosis is induced; and (b) in Agrobacterium rhizogenes-transformed roots, where nodulation is strongly inhibited and nodules show early senescent symptoms. These findings indicate that PvRACK1 may be an integrator of diverse signal-transduction pathways in processes as varied as nodulation, cell expansion, heat stress responses, and systemic activation of necrosis.     

Unusual iridoid glycosides in Veronica sects. Hebe and Labiatoides

In a chemosystematic investigation of three Southern hemisphere species of Veronica, namely the Australian Veronica derwentiana Andrews and Veronica perfoliata R.Br. (formerly Derwentia species), and the New Zealand Veronica catarractae G. Forster (formerly a species of Parahebe), the water-soluble constituents were isolated and identified by spectroscopic methods. Apart from other iridoid glucosides common to the genus, three unusual substituted benzoyl esters of aucubin (derwentiosides A–C) were obtained from V. derwentiana and a chlorinated iridoid glycoside (catarractoside) from V. catarractae in addition to other iridoids common to the genus. The chemical profile of V. perfoliata is similar to that of Northern hemisphere species of Veronica because of the presence of characteristic 6-O-catalpol esters. The profile of V. derwentiana is unique, since 6-O-esters of aucubin rather than of catalpol dominate, however, the acyl groups are the same as those present in catalpol esters found in some other Veronica sections. V. catarractae also contains one of the catalpol esters characteristic of Veronica, but in addition three 6-O-rhamnopyranosyl substituted iridoid glycosides, one of which is 6-O-rhamnopyranosylcatalpol. Esters of the latter compound are previously only known from the more derived species in recent phylogenetic trees of sect. Hebe to which V. catarractae now also belongs, but as a more basal member.     

Antihepatoma activity of Physalis angulata and P. peruviana extracts and their effects on apoptosis in human Hep G2 cells

Physalis angulata and P. peruviana are herbs widely used in folk medicine. In this study, the aqueous and ethanol extracts prepared from the whole plant of these species were evaluated for their antihepatoma activity. Using XTT assay, three human hepatoma cells, namely Hep G2, Hep 3B and PLC/PRF/5 were tested. The results showed that ethanol extract of P. peruviana (EEPP) possessed the lowest IC50 value against the Hep G2 cells. Interestingly, all extracts showed no cytotoxic effect on normal mouse liver cells. Treatment with carbonyl cyanide m-chlorophenyl hydrazone, a protonophore, caused a reduction of membrane potential (Deltapsim) by mitochondrial membrane depolarization. At high concentrations, EEPP was shown to induce cell cycle arrest and apoptosis through mitochondrial dysfunction as demonstrated by the following observations: (i) EEPP induced the collapse of Deltapsim and the depletion of glutathione content in a dose dependent manner; (ii) pretreatment with the antioxidant (1.0 microg/ml vitamin E) protected cells from EEPP-induced release of ROS; and (iii) at concentrations 10 to 50 microg/ml, EEPP displayed a dose-dependent accumulation of the Sub-G1 peak (hypoploid) and caused G0/G1-phase arrest. Apoptosis was elicited when the cells were treated with 50 microg/ml EEPP as characterized by the appearance of phosphatidylserine on the outer surface of the plasma membrane. The results conclude that EEPP possesses potent antihepatoma activity and its effect on apoptosis is associated with mitochondrial dysfunction.     

Role of the aromatic ring of Tyr43 in tetraheme cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F

Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme cytochrome c(3). The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of cytochrome c(3). Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L cytochrome c(3) by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure.     

锌胁迫对小球藻抗氧化酶和类金属硫蛋白的影响

通过对抗氧化酶活性和类金属硫蛋白的测定,考察在0、5、10、20、50和100 μmol/L Zn²⁺(氯化锌)胁迫下锌对普通海洋小球藻的生物学影响。结果表明:不同浓度Zn²⁺均能抑制小球藻的生长,当Zn²⁺浓度大于10 μmol/L时,小球藻生物量随培养时间延长而迅速下降;过氧化物歧化酶 (SOD)活性随Zn²⁺胁迫浓度的增加而增加,当Zn²⁺浓度为50 μmol/L时SOD活性达到最大,但继续增加Zn²⁺胁迫浓度反而导致SOD活性下降;而过氧化物酶 (POD)活性则随着Zn²⁺胁迫浓度的增加而降低。同时,实验发现藻细胞内有两种主要的锌结合形态,其中Zn结合类金属硫蛋白(Zn-MT-like)与兔肝金属硫蛋白(MT)的分子量相近,且随着Zn²⁺胁迫浓度的增加而出现规律性地增多。因此,藻细胞内Zn-MT-like蛋白的诱导量可作为小球藻受Zn²⁺胁迫的响应指标。     

Kaurenolides and fujenoic acids are side products of the gibberellin P450-1 monooxygenase in Gibberella fujikuroi

The steps involved in kaurenolide and fujenoic acids biosynthesis, from ent-kauradienoic acid and ent-6alpha,7alpha-dihydroxykaurenoic acid, respectively, are demonstrated in the gibberellin (GA)-deficient Gibberella fujikuroi mutant SG139, which lacks the entire GA-biosynthesis gene cluster, complemented with the P450-1 gene of GA biosynthesis (SG139-P450-1). ent-[2H]Kauradienoic acid was efficiently converted into 7beta-hydroxy[2H]kaurenolide and 7beta,18-dihydroxy[2H]kaurenolide by the cultures while 7beta-hydroxy[2H]kaurenolide was transformed into 7beta,18-dihydroxy[2H]kaurenolide. The limiting step was found to be hydroxylation at C-18. In addition, SG139-P450-1 transformed ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid into [14C4]fujenoic acid and [14C4]fujenoic triacid. Fujenal was also converted into the same products but was demonstrated not to be an intermediate in this sequence. All the above reactions were absent in the mutant SG139 and were suppressed in the wild-type strain ACC917 by disruption of the P450-1 gene. Kaurenolide and fujenoic acids synthesis were associated with the microsomal fraction and showed an absolute requirement for NADPH or NADH, all properties of cytochrome P450 monooxygenases. Only 7beta-hydroxy[14C4]kaurenolide synthesis and not further 18-hydroxylation was detected in the microsomal fraction. The substrates for the P450-1 monooxygenase, ent-kaurenoic acid and [2H]GA12, efficiently inhibited kaurenolide synthesis with I50 values of 3 and 6 microM, respectively. Both substrates also inhibited ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid metabolism by SG139-P450-1. Conversely, [14C4]GA14 synthesis from [14C4]GA12-aldehyde was inhibited by ent-[2H]kauradienoic acid and fujenal with I50 values of 10 and 30 microM, respectively. These results demonstrate that kaurenolides and seco-ring B kaurenoids are formed by the P450-1 monooxygenase (GA14 synthase) of G. fujikuroi and are thus side products that probably result from stabilization of radical intermediates involved in GA14 synthesis.     

Pollen dispersal and breeding structure in a hawkmoth-pollinated Pampa grasslands species Petunia axillaris (Solanaceae)

Background and Aims The evolution of selfing is one of the most common transitions in flowering plants, and this change in mating pattern has important systematic and ecological consequences because it often initiates reproductive isolation and speciation. Petunia axillaris (Solanaceae) includes three allopatric subspecies widely distributed in temperate South America that present different degrees of self-compatibity and incompatibility. One of these subspecies is co-distributed with P. exserta in a restricted area and presents a complex, not well-understood mating system. Artificial crossing experiments suggest a complex system of mating in this sympatric area. The main aims of this study were to estimate the pollen dispersal distance and to evaluate the breeding structure of P. axillaris subsp. axillaris, a hawkmoth-pollinated taxon from this sympatric zone.Methods Pollen dispersal distance was compared with nearest-neighbours distance, and the differentiation in the pollen pool among mother plants was estimated. In addition, the correlation between genetic differentiation and spatial distance among plants was tested. All adult individuals (252) within a space of 2800 m² and 15 open-pollinated progeny (285 seedlings) were analysed. Genetic analyses were based on 12 polymorphic microsatellite loci.Key Results A high proportion of self-pollination was found, indicating a mixed-mating system. The maximum pollen dispersal distance was 1013 m, but most pollination events (96 %) occurred at a distance of 0 m, predominantly in an inbreeding system. Both parents among sampled individuals could be identifed in 60–85 % of the progeny.Conclusions The results show that most pollen dispersal in the hawkmoth-pollinated P. axillaris subsp. axillaris occurs within populations and there is a high proportion of inbreeding. This mating system appears to favour species integrity in a secondary contact zone with the congener species P. exserta.     

Pseudocitrobacter gen. nov., a novel genus of the Enterobacteriaceae with two new species Pseudocitrobacter faecalis sp. nov., and Pseudocitrobacter anthropi sp. nov,isolated from fecal samples from hospitalized patients in Pakistan

Four isolates of Gram-negative facultatively anaerobic bacteria, three of them producing NDM-1 carbapenemase, were isolated from hospitalized patients and outpatients attending two military hospitals in Rawalpindi, Pakistan, and studied for their taxonomic position. Initially the strains were phenotypically identified as Citrobacter species. Comparative analysis of 16S rRNA gene sequences then showed that the four strains shared >97%, but in no case >98.3%, 16S rRNA gene sequence similarities to members of the genera Citrobacter, Kluyvera, Pantoea, Enterobacter and Raoultella, but always formed a separate cluster in respective phylogenetic trees. Based on multilocus sequence analysis (MLSA) including partial recN, rpoA, thdF and rpoB gene sequence and respective amino acid sequence analysis it turned out that the strains also here always formed separate clusters. Based on further comparative analyses including DNA–DNA hybridizations, genomic fingerprint analysis using rep- and RAPD-PCRs and physiological tests, it is proposed to classify these four strains into the novel genus Pseudocitrobacter gen. nov. with a new species Pseudocitrobacter faecalis sp. nov. with strain 25 CIT^T (= CCM 8479^T = LMG 27751^T) and Pseudocitrobacter anthropi sp. nov. with strain C138^T (= CCM 8478^T = LMG 27750^T), as the type strains, respectively.     

Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc₁ complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c_tot (cyt c₁ + cyt c₂), cyt b_L, cyt b_H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c₁ and c₂ is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c₁, c₂, b_L, and b_H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc₁ complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc₁ complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.