合萌水提物抗炎镇痛作用的实验研究

目的:观察合萌水提物的抗炎、镇痛作用。方法:采用二甲苯致小鼠耳肿胀、角叉菜胶致小鼠足跖肿胀、醋酸致小鼠腹腔毛细血管通透性增加及醋酸致小鼠扭体实验、热板实验模型,观察合萌水提物的抗炎镇痛作用,并测定丙二醛(MDA)、一氧化氮(NO)的含量及超氧化物歧化酶(SOD)活性。结果:合萌水提物可抑制二甲苯致小鼠耳肿胀,减轻小鼠足跖肿胀,降低小鼠毛细血管通透性,减少醋酸致小鼠扭体次数,增加热板小鼠痛阈值,降低血清MDA、NO含量,提高血清SOD活性。结论:合萌水提物具有抗炎镇痛作用,作用机制可能与降低血管通透性、抑制NO等炎症介质产生及增强清除自由基、抗脂质过氧化能力有关。     

Stem Nodules in Aeschynomene indica and Their Capacity of Nitrogen Fixation

There has been no report on stem nodules with nitrogen fixing activity. Aeschynomene indica produce nodules on the aerial parts, and the stem nodules proved to be capable of considerable nitrogen fixation [C₂H₄ produced c. 4 μmol (g fresh weight)^-1 h^-1]. The stem nodules embed reddish tissues, and a rod-shaped bacterium was isolated from the tissues. The bacterium was ascertained to form root and stem nodules on seedlings of A. indica.     

Photosynthetic Bradyrhizobia from Aeschynomene spp. Are Specific to Stem-Nodulated Species and Form a Separate 16S Ribosomal DNA Restriction Fragment Length Polymorphism Group

We obtained nine bacterial isolates from root or collar nodules of the non-stem-nodulated Aeschynomene species A. elaphroxylon, A. uniflora, or A. schimperi and 69 root or stem nodule isolates from the stem-nodulated Aeschynomene species A. afraspera, A. ciliata, A. indica, A. nilotica, A. sensitiva, and A. tambacoundensis from various places in Senegal. These isolates, together with 45 previous isolates from various Aeschynomene species, were studied for host-specific nodulation within the genus Aeschynomene, also revisiting cross-inoculation groups described previously by D. Alazard (Appl. Environ. Microbiol. 50:732–734, 1985). The whole collection of Aeschynomene nodule isolates was screened for synthesis of photosynthetic pigments by spectrometry, high-pressure liquid chromatography, and thin-layer chromatography analyses. The presence of puf genes in photosynthetic Aeschynomene isolates was evidenced both by Southern hybridization with a Rhodobacter capsulatus photosynthetic gene probe and by DNA amplification with primers defined from photosynthetic genes. In addition, amplified 16S ribosomal DNA restriction analysis was performed on 45 Aeschynomene isolates, including strain BTAi1, and 19 reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii, and other Bradyrhizobium sp. strains of uncertain taxonomic positions. The 16S rRNA gene sequence of the photosynthetic strain ORS278 (LMG 12187) was determined and compared to sequences from databases. Our main conclusion is that photosynthetic Aeschynomene nodule isolates share the ability to nodulate particular stem-nodulated species and form a separate subbranch on the Bradyrhizobium rRNA lineage, distinct from B. japonicum and B. elkanii.     

The aquatic budding bacterium Blastobacter denitrificans is a nitrogen-fixing symbiont of Aeschynomene indica

Blastobacter spp. are freshwater bacteria that form rosette structures by cellular attachment to a common base. Comparative analyses of ribosomal 16S rRNA gene and internally transcribed spacer region sequences indicated that B. denitrificans is a member of the alpha-subdivision of Proteobacteria. Among the alpha-Proteobacteria, B. denitrificans was related to a cluster of genera, including Rhodopseudomonas palustris, Afipia felis, Nitrobacter hamburgensis, and Bradyrhizobium spp. Although the precise phylogenetic relationships among these genera could not be established with a high degree of confidence, the sequences of B. denitrificans and several bradyrhizobial isolates from nodules of Aeschynomene indica were almost identical. Bradyrhizobia are bacteria that form nitrogen-fixing symbioses with legumes, including soybeans (Glycine max) and members of the genus Aeschynomene. From symbiotic infectiveness tests we demonstrated that the type strain for B. denitrificans, IFAM 1005, was capable of forming an effective nitrogen-fixing symbiosis with A. indica. Not only do these results reveal a previously unknown ecological adaptation of a relatively obscure aquatic bacterium, but they also demonstrate how evidence gathered from molecular systematic analyses can sometimes provide clues for predicting ecological behavior.     

Bradyrhizobium aeschynomenes sp. nov., a root and stem nodule microsymbiont of Aeschynomene indica

Aeschynomene indica has a distinctive symbiosis with Bradyrhizobium in which nodulation is Nod factor-independent. In this study, we characterised three Gram-negative and rod-shaped strains (83002^T, 81013 and 83012) isolated from root nodules of Aeschynomene indica in Shandong Peninsula. The major cellular fatty acids of isolates were C_16:0, C_18:0, C_18:1 ω7c 11-methyl, summed feature 3 and summed feature 8. The major polar lipids were phosphatidylethanolamine (PE), aminolipids (AL) and phosphatidylcholine (PC). Phylogenetic analysis based on the 16S rRNA locus showed that they belonged to the Bradyrhizobium genus, and shared the highest similarity to the type strains Bradyrhizobium oligotrophicum S58^T and Bradyrhizobium denitrificans LMG 8443^T. As expected, analysis of concatenated sequences of six housekeeping genes (atpD, recA, glnII, dnaK, gyrB, and rpoB) and nifH gene proposed that these three strains formed a distinct clade within the genus Bradyrhizobium. The highest average nucleotide identity and DNA-DNA hybridization values of the three strains in comparison to the closest Bradyrhizobium species were 87.5% and 65.3%, respectively, which are far below the threshold of species delineation, and thus confirmed the three strains as a new species. The genome size of strain 83002^T is 7.52 Mbp, and the DNA G+C content is 65.42 mol%. Strain 83002^T (=KCTC 82266^T=MCCC 1K04775^T) was chosen as the type strain of the new species, for which the name Bradyrhizobium aeschynomenes sp. nov. was proposed.     

The Aquatic Budding Bacterium Blastobacter denitrificans Is a Nitrogen-Fixing Symbiont of Aeschynomene indica

Blastobacter spp. are freshwater bacteria that form rosette structures by cellular attachment to a common base. Comparative analyses of ribosomal 16S rRNA gene and internally transcribed spacer region sequences indicated that B. denitrificans is a member of the α-subdivision of Proteobacteria. Among the α-Proteobacteria, B. denitrificans was related to a cluster of genera, including Rhodopseudomonas palustris, Afipia felis, Nitrobacter hamburgensis, and Bradyrhizobium spp. Although the precise phylogenetic relationships among these genera could not be established with a high degree of confidence, the sequences of B. denitrificans and several bradyrhizobial isolates from nodules of Aeschynomene indica were almost identical. Bradyrhizobia are bacteria that form nitrogen-fixing symbioses with legumes, including soybeans (Glycine max) and members of the genus Aeschynomene. From symbiotic infectiveness tests we demonstrated that the type strain for B. denitrificans, IFAM 1005, was capable of forming an effective nitrogen-fixing symbiosis with A. indica. Not only do these results reveal a previously unknown ecological adaptation of a relatively obscure aquatic bacterium, but they also demonstrate how evidence gathered from molecular systematic analyses can sometimes provide clues for predicting ecological behavior.     

元江印楝植物内生真菌及其抗菌活性筛选的研究

通过抗菌活性初步筛选,从采自云南元江县的印楝(Azadirachta indica A.Juss)植物茎和果实中已分离到的372株内生真菌中筛选出80株作为复筛菌株,经显微形态特征观察鉴定为5目、6科、29个属。选择16种病原微生物作为指示菌检测复筛菌株发酵产物的抗菌活性,结果表明,其中29株内生真菌对细菌、植物病原真菌和皮肤致病真菌中的一种或多种病原微生物有抑制生长作用,活性菌株比例占复筛菌株的36.25%,并显示种群多样性,其中7株内生真菌显示较强的广谱抗菌作用,活性较好的菌株主要分布在曲霉属和交链孢属。     

The Nod factor-independent symbiotic signaling pathway: development of Agrobacterium rhizogenes-mediated transformation for the legume Aeschynomene indica

The nitrogen-fixing symbiosis between Aeschynomene indica and photosynthetic bradyrhizobia is the only legume-rhizobium association described to date that does not require lipochito-oligosaccharide Nod factors (NF). To assist in deciphering the molecular basis of this NF-independent interaction, we have developed a protocol for Agrobacterium rhizogenes-mediated transformation of A. indica. The cotransformation frequency (79%), the nodulation efficiency of transgenic roots (90%), and the expression pattern of the 35S Cauliflower mosaic virus promoter in transgenic nodules were all comparable to those obtained for model legumes. We have made use of this tool to monitor the heterologous spatio-temporal expression of the pMtENOD11-β-glucuronidase fusion, a widely used molecular reporter for rhizobial infection and nodulation in both legumes and actinorhizal plants. While MtENOD11 promoter activation was not observed in A. indica roots prior to nodulation, strong reporter-gene expression was observed in the invaded cells of young nodules and in the cell layers bordering the central zone of older nodules. We conclude that pMtENOD11 expression can be used as an infection-related marker in A. indica and that Agrobacterium rhizogenes-mediated root transformation of Aeschynomene spp. will be an invaluable tool for determining the molecular basis of the NF-independent symbiosis.     

Relationships of Bradyrhizobia from the Legumes Apios americana and Desmodium glutinosum

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana, Desmodium glutinosum, and Amphicarpaea bracteata, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii.     

一株印楝植物内生真菌Epicoccumsp．次生代谢产物的研究

从印楝植物内生真菌Epicoccum sp.的发酵液中分离得到6个化合物,经波谱数据分析分别鉴定为苔黑酚(1)、4-甲基苔黑酚(2)、苔色酸(3)、对羟基苯乙酸(4)、邻苯二甲酸正丁异丁酯(5)、乙基-β-D-葡萄糖苷(6).以上化合物均为首次从该属真菌中分离得到.     

Bradyrhizobia from wild Phaseolus, Desmodium, and Macroptilium species in northern Mexico

rRNA genetic markers were analyzed in 97 isolates of nodule bacteria from six legume species in Chihuahua, Mexico. The most common genotypes were widely shared across host species and had 16S rRNA sequences identical to those of strains from an eastern North American legume (Amphicarpaea) that are closely related to Bradyrhizobium elkanii.     

Genetic Diversity, Symbiotic Evolution, and Proposed Infection Process of Bradyrhizobium Strains Isolated from Root Nodules of Aeschynomene americana L. in Thailand

The diversity of bacteria nodulating Aeschynomene americana L. in Thailand was determined from phenotypic characteristics and multilocus sequence analysis of the 16S rRNA gene and 3 housekeeping genes (dnaK, recA, and glnB). The isolated strains were nonphotosynthetic bacteria and were assigned to the genus Bradyrhizobium, in which B. yuanmingense was the dominant species. Some of the other species, including B. japonicum, B. liaoningense, and B. canariense, were minor species. These isolated strains were divided into 2 groups-nod-containing and divergent nod-containing strains-based on Southern blot hybridization and PCR amplification of nodABC genes. The divergent nod genes could not be PCR amplified and failed to hybridize nod gene probes designed from B. japonicum USDA110, but hybridized to probes from other bradyrhizobial strains under low-stringency conditions. The grouping based on sequence similarity of nod genes was well correlated with the grouping based on that of nifH gene, in which the nod-containing and divergent nod-containing strains were obviously distinguished. The divergent nod-containing strains and photosynthetic bradyrhizobia shared close nifH sequence similarity and an ability to fix nitrogen in the free-living state. Surprisingly, the strains isolated from A. americana could nodulate Aeschynomene plants that belong to different cross-inoculation (CI) groups, including A. afraspera and A. indica. This is the first discovery of bradyrhizobia (nonphotosynthetic and nod-containing strain) originating from CI group 1 nodulating roots of A. indica (CI group 3). An infection process used to establish symbiosis on Aeschynomene different from the classical one is proposed.     

Bradyrhizobia from Wild Phaseolus, Desmodium, and Macroptilium Species in Northern Mexico

rRNA genetic markers were analyzed in 97 isolates of nodule bacteria from six legume species in Chihuahua, Mexico. The most common genotypes were widely shared across host species and had 16S rRNA sequences identical to those of strains from an eastern North American legume (Amphicarpaea) that are closely related to Bradyrhizobium elkanii.     

AFLP fingerprint analysis of Bradyrhizobium strains isolated from Faidherbia albida and Aeschynomene species

The diversity of Bradyrhizobium isolates from Faidherbia albida and Aeschynomenee species was assessed using AFLP analysis, a high-resolution genomic fingerprinting technique. Reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii and Bradyrhizobium liaoningense were included for comparison. At a similarity level of 50%, a total of 34 different groups were obtained by cluster analysis of the genomic fingerprints. Four of these clusters correspond to the three reference species, demonstrating the large diversity of the isolates studied. Comparison with other data demonstrates that AFLP has a higher resolution than restriction analysis of 16S rRNA genes, SDS-PAGE analysis of proteins and phenotypic analysis. Results of the latter two methods showed little correspondence with the genotypic data.     

Stem and Root Nodulation in Aeschynomene spp

Nodulation ability of 15 Rhizobium strains isolated from root and stem nodules of tropical Aeschynomene species was studied on 20 different Aeschynomene species and four other legumes—Arachis hypogaea, Stylosanthes guianensis, Macroptilium atropurpureum, and Sesbania rostrata. The results of this investigation showed that Aeschynomene species could be divided into three groups according to the cross-inoculation group concept.     

Diversity and relationships of Bradyrhizobia from Amphicarpaea bracteata based on partial nod and ribosomal sequences.

Partial sequences of three nod genes (nodC, nodD1, and nodA 5' flanking region) and of 16S and 23S rDNA were obtained from isolates of Bradyrhizobium sp. associated with the native North American legume Amphicarpaea bracteata. Isolates from Amphicarpaea had identical sequences in the three nod gene regions, but differed from all other Bradyrhizobium taxa at > 10% of nucleotide sites. Parsimony analysis of all nod gene segments indicated a phylogenetic relationship of these bacteria to B. elkanii, with B. japonicum diverging prior to the diversification of these taxa. All Bradyrhizobium isolates from Amphicarpaea were also identical to B. elkanii in the size of the intervening sequence (IVS) in the 5' region of the 23S rRNA gene, while B. japonicum had an IVS length variant with 29 additional nucleotides. Parsimony analysis of both 16S and 23S partial rDNA sequences grouped Bradyrhizobium sp. isolates from Amphicarpaea into a clade together with B. elkanii, consistent with the relationships inferred from nod sequences.     

Flavonoids from Limnophila indica

Two flavonoids, (2S)-5,7,3',4'-tetramethoxyflavanone (1) and 5,7,2',5'-tetramethoxyflavone (2) together with three known flavonoids, 7-O-methylwogonin (3), skullcapflavone I (4) and 5-hydroxy-7,2'-dimethoxyflavone (5) were isolated from the whole plant of Limnophila indica. The structures of compounds 1-5 were elucidated on the basis of spectral and chemical studies.     

Nodulation of Aeschynomene afraspera and A. indica by photosynthetic Bradyrhizobium Sp. strain ORS285: the nod-dependent versus the nod-independent symbiotic interaction

Here, we present a comparative analysis of the nodulation processes of Aeschynomene afraspera and A. indica that differ in their requirement for Nod factors (NF) to initiate symbiosis with photosynthetic bradyrhizobia. The infection process and nodule organogenesis was examined using the green fluorescent protein-labeled Bradyrhizobium sp. strain ORS285 able to nodulate both species. In A. indica, when the NF-independent strategy is used, bacteria penetrated the root intercellularly between axillary root hairs and invaded the subepidermal cortical cells by invagination of the host cell wall. Whereas the first infected cortical cells collapsed, the infected ones immediately beneath kept their integrity and divided repeatedly to form the nodule. In A. afraspera, when the NF-dependent strategy is used, bacteria entered the plant through epidermal fissures generated by the emergence of lateral roots and spread deeper intercellularly in the root cortex, infecting some cortical cells during their progression. Whereas the infected cells of the lower cortical layers divided rapidly to form the nodule, the infected cells of the upper layers gave rise to an outgrowth in which the bacteria remained enclosed in large tubular structures. Together, two distinct modes of infection and nodule organogenesis coexist in Aeschynomene legumes, each displaying original features.