Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor

Hairy root cultures of Salvia sclarea were grown in shake flasks and 10 L nutrient sprinkle bioreactor, running for 30 days and the effects of methyl jasmonate (MJ) on their growth and capacity to accumulate diterpenoids were measured. We found that MJ concentration and exposure time to the elicitor were factors that strongly affected the diterpenoid production. The highest diterpenoid accumulation (67.5 ± 7.1 mg g⁻¹ dry weight, calculated as a sum of ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) without reduction of biomass, was achieved when the 23-day-old hairy roots in bioreactor culture were exposed to 125 μM MJ for 7 days. The roots produced 9 and 3.8 times as much aethiopinone (40 ± 5.9 mg g⁻¹ dry weight) and salvipisone (12.6 ± 0.4 mg g⁻¹ dry weight), respectively, as roots cultured in shake flasks. Our results imply that cultivation of S. sclarea hairy roots in sprinkle bioreactor after elicitation with MJ may be valuable to enhance production of the bioactive diterpenoids.     

<Emphasis Type="Italic">recA</Emphasis> mediated spontaneous deletions of the <Emphasis Type="Italic">icaADBC</Emphasis> operon of clinical <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> isolates: a new mechanism of phenotypic variations

Phenotypic variation of Staphylococcus epidermidis involving the slime related ica operon results in heterogeneity in surface characteristics of individual bacteria in axenic cultures. Five clinical S. epidermidis isolates demonstrated phenotypic variation, i.e. both black and red colonies on Congo Red agar. Black colonies displayed bi-modal electrophoretic mobility distributions at pH 2, but such phenotypic variation was absent in red colonies of the same strain as well as in control strains without phenotypic variation. All red colonies had lost ica and the ability to form biofilms, in contrast to black colonies of the same strain. Real time PCR targeting icaA indicated a reduction in gene copy number within cultures exhibiting phenotypic variation, which correlated with phenotypic variations in biofilm formation and electrophoretic mobility distribution of cells within a culture. Loss of ica was irreversible and independent of the mobile element IS256. Instead, in high frequency switching strains, spontaneous mutations in lexA were found which resulted in deregulation of recA expression, as shown by real time PCR. RecA is involved in genetic deletions and rearrangements and we postulate a model representing a new mechanism of phenotypic variation in clinical isolates of S. epidermidis. This is the first report of S. epidermidis strains irreversibly switching from biofilm-positive to biofilm-negative phenotype by spontaneous deletion of icaADBC.     

Novel antibacterial diterpenoids from Larix chinensis Beissn

Two novel labdane-type diterpenoids, butanedioic acid mono[(13S)-13-hydroxylabda-8(17),14-dien-19-yl] ester (1) and butanedioic acid bis[(13S)-13-hydroxylabda-8(17),14-dien-19-yl] ester (2), along with the eleven known diterpenoids 3-13 and three other known compounds, were isolated from the bark of Larix chinensis. Their structures were elucidated both spectroscopically and chemically. The major diterpenoids 1-9, 11, and 13, as well as the very abundant phenolic compound 16, were subjected to antibacterial and cytotoxic bioassays. Compounds 7 and 9 showed remarkable in vitro inhibition of Staphylococcus aureus and S. epidermidis (MIC = 37-73 microM).     

Screening of Echinops ellenbeckii and Echinops longisetus for biological activities and chemical constituents

Members of the genus Echinops in the family of Asteraceae are widely used in Ethiopian herbal medicine for the treatment of various diseases and illness such as migraine, diarrhea, heart pain, different forms of infections, intestinal worm infestation and hemorrhoid. Hydroalcoholic extracts of the root, flower head, leaf and stem of Echinops ellenbeckii O. Hoffm. and Echinops longisetus A. Rich were investigated for their chemical constituents and biological activities. The presence of alkaloids, saponins, phytosterols, polyphenols and carotenoids in the different parts of the plants was observed whilst anthraquinones were not detected. The leaf extracts of both plants and stem extract of E. longisetus showed strong inhibitory activity against cultures of Staphylococcus aureus. None of the extracts were found to be active against Gram-positive organisms. The flower extract of E. ellenbeckii showed strong inhibitory activity against Candida albicans. Root and flower extracts of the plants investigated showed lethal activity against earthworms. Moreover, the extracts of the roots of both plants showed molluscicidal activity against schistosome-transmitting snail hosts. The biological activities observed were dose dependent.     

Antimicrobial activity of some Hypericum species

The crude methanolic extracts of six species of Hypericum [H. caprifoliatum Cham. & Schlecht., H. carinatum Griseb., H. connatum Lam., H. ternum A. St. Hil., H. myrianthum Cham. & Schlecht. and H. polyanthemum Klotzsch ex Reichardt] growing in southern Brazil were analyzed for antimicrobial activity against several microorganisms (bacteria and fungi). The most active plant was H. caprifoliatum, which showed activity against Staphylococcus aureus. Only H. polyanthemum and H. ternum extracts were active against Bacillus subtilis. None of the crude methanolic extracts showed activity against S. epidermidis, Escherichia coli or Saccharomyces cerevisiae. Extracts from these species were evaluated chemically and tannin, flavonoid and phenolic acids were the prominent compounds. The plants contained quercitrin, hyperoside (except H. connatum) and, less frequently, isoquercitrin and chlorogenic acid. In contrast to H. perforatum, which has high concentrations of rutin, these species do not produce this flavonoid or it appears as traces. The tannin concentration varied between 5.1 and 16.7% in H. myrianthum and H. ternum, respectively.     

北细辛不同部位内生真菌的多样性与抑菌活性

为探讨北细辛(Asarum heterotropoides)不同部位可培养内生真菌种群结构的差异,采用植物组织平板分离法分离北细辛根、根状茎、越冬芽三个部位内生真菌,结合形态学和分子生物学方法鉴定菌株,并系统分析和比较其种类和多样性。从北细辛三个部位共分离得到117株内生真菌,其中97株归属于6纲6目8科9属17种。相似性和多样性分析表明,北细辛不同部位内生真菌的组成结构上存在差异,根部内生真菌的多样性指数最高,越冬芽最低。采用菌饼法检测其抑菌活性,G 3-3、G 4-10-1、G 2-2-1、GZJ 2-12-2对金黄色葡萄球菌(Staphylococcus aureus)有抑制作用,G 3-3、G 2-2-1、GZJ 2-12-2、GZJ 1-6、G 3-8对枯草芽胞杆菌(Bacillus subtilis)有抑制作用。GZJ 2-12-2对金黄色葡萄球菌、大肠埃希菌(Escherichia coli)、枯草芽胞杆菌均有抑制作用。     

Plantlet differentiation in leaf and root cultures of birch (Betula Pendula roth.)

The newly-formed leaves on plantlets differentiated from shoot bud cultures of Betula pendula, when excised and grown on a fresh medium produced callus from the margins or regenerated leafy shoots, roots and plantlets. After 4 weeks, upon transfer to murashige and Skoog (MS) medium supplemented with 3-indoleacetic acid (IAA) + 6-(4-hydroxy-3-methyl-trans-2-enyl)aminopurine (zeatin) + 6-aminopurine (adenine), 15–20 plantlets were produced from each explant. Likewise, the roots also showed meristematic activity at several sites, and produced nodulated callus on MS + α-naphthaleneacetic acid (NAA) + 6-(3-methyl-2-butenyl-amino)purine (2-iP) + adenine, and ultimately differentiated plantlets. Anatomical studies showed that initiation of callus takes place by meristematic activity in epidermal cells of leaves, and cortical cells of roots. Cytological investigations revealed no change in chromosomal complement.     

Isolation,structural characterization,and immunological evaluation of a high-molecular-weight exopolysaccharide from Staphylococcus aureus

Colonization of implanted medical devices by coagulase-negative staphylococci such as Staphylococcus epidermidis is mediated by the bacterial polysaccharide intercellular adhesin (PIA), a polymer of beta-(1-->6)-linked glucosamine substituted with N-acetyl and O-succinyl constituents. The icaADBC locus containing the biosynthetic genes for production of PIA has been identified in both S. epidermidis and S. aureus. Whereas it is clear that PIA is a constituent that contributes to the virulence of S. epidermidis, it is less clear what role PIA plays in infection with S. aureus. Recently, identification of a novel polysaccharide antigen from S. aureus termed poly N-succinyl beta-(1-->6)-glucosamine (PNSG) has been reported. This polymer was composed of the same glycan backbone as PIA but was reported to contain a high proportion of N-succinylation rather than acetylation. We have isolated a glucosamine-containing exopolysaccharide from the constitutive over-producing MN8m strain of S. aureus in order to prepare polysaccharide-protein conjugate vaccines. In this report we demonstrate that MN8m produced a high-molecular-weight (>300,000 Da) polymer of beta-(1-->6)-linked glucosamine containing 45-60% N-acetyl, and a small amount of O-succinyl (approx 10% mole ratio to monosaccharide units). By detailed NMR analyses of polysaccharide preparations, we show that the previous identification of N-succinyl was an analytical artifact. The exopolysaccharide we have isolated is active in in vitro hemagglutination assays and is immunogenic in mice when coupled to a protein carrier. We therefore conclude that S. aureus strain MN8m produces a polymer that is chemically and biologically closely related to the PIA produced by S. epidermidis.     

黑毛雪兔子通气组织发育相关基因ShCTR1的克隆及表达

黑毛雪兔子(Saussurea inversa Raab-Straube)是一种典型的高山植物,具有发达的通气组织。该研究以黑毛雪兔子为材料,利用同源克隆和RACE技术克隆了通气组织形成相关基因(ShCTR1),并对其进行序列分析、系统进化分析、表达分析和亚细胞定位分析,以探讨该基因的功能及其与通气组织的关系。结果表明:(1)成功克隆获得ShCTR1基因cDNA全长为2891 bp(NCBI登录号为:ON081649),包含2550 bp的开放阅读框,编码849个氨基酸,理论等电点5.90,分子式C_(4066)H_(6417)N_(1159)O_(1268)S_(43),为疏水蛋白,无跨膜结构。(2)系统进化树分析显示,黑毛雪兔子ShCTR1与菜蓟(Cynara cardunculus L.)CcCTR1的氨基酸序列相似度最高;非编码区序列分析发现ShCTR1基因含有大量的光响应元件,表明ShCTR1基因可能参与对紫外胁迫的响应。(3)实时荧光定量分析显示,ShCTR1基因在根、茎和叶中均有表达,且在根中的表达量最高;在紫外、低温和低氧胁迫下,ShCTR1基因的表达量均上调,证明ShCTR1基因参与了对以上3种环境胁迫的响应。(4)亚细胞定位显示,ShCTR1主要分布于细胞核。研究推测,黑毛雪兔子ShCTR1基因可能在通气组织的形成以及对逆境胁迫的响应中具有重要作用。     

Inoculation of Paenibacillus illinoisensis alleviates root mortality, activates of lignification-related enzymes, and induction of the isozymes in pepper plants infected by Phytophthora capsici

To investigate the variations of the enzymes responsible for lignification, after inoculation with Phytophthora capsici and/or Paenibacillus illinoisensis KJA-424, in relation to biocontrol of Phytophthora blight in pepper, roots of two-month-old plants were inoculated with P. capsici inoculation (P), and co-inoculation of P. capsici and P. illinoisensis cell cultures (P + A). Root mortality of pepper plants induced by inoculation of P. capsici was completely recovered by co-inoculation with antagonistic KJA-424. At day 7, peroxidase (POD) activity increased by 36.7% in P-treated roots but by 7.1% only in P + A-treated, compared with control. Polyphenol oxidase (PPO) activity increased for 3 days and then drastically decreased in P-treated roots but maintained a constant level in control and P + A-treated. At day 7, PPO activity in P-treated leaves decreased but recovered to the level of control in the P + A-treated. Three major POD isozymes (45, 53, and 114 kDa) were shown in P-treated roots, while two major (53 and 114 kDa) in control and P + A-treated, suggesting that the 45 kDa of POD was actively induced in P-treated roots but not induced in P + A-treated roots. A PPO isozyme of 80 kDa was induced in P-treated roots but not induced by co-treated with KJA-424. In leaves, the POD isozyme of 45 kDa appears to be systemically induced in P-treated only. The PPO isozyme of 80 kDa in leaves was not induced by pathogen challenge but recovered by co-inoculated with P. illinoisensis. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, activates of lignification-related enzymes and induction of the isozymes in pepper plants infected by P. capsici.     

Genetic transfer of methycilline resistance in Staphylococus epidermidis and Staphylococcus aureus strains in mixed cultures

In mixed cultures of staphylococci a transfer of the resistance to methicillin and penicillinase plasmids as well as tetracycline and chloramphenicol plasmids was investigated. It was shown that the resistance to methicillin was transferred in mixed cultures from one strain of S. aureus to another and from S. epidermidis to S. aureus. In both cases transfer of methicillin resistance required, the presence of penicillinase plasmid in recipient or donor strain. In the case of other markers transmission was independent. Moreover it was shown that the transfer of resistance genes in mixed cultures was mediated by bacteriophage of the serologic group A.     

Ceriopsins A–D, diterpenoids from Ceriops decandra

Chemical examination of the ethyl acetate solubles of the CH₃OH:CH₂Cl₂ (1:1) extract of the roots of Ceriops decandra collected from Kauvery estuary resulted in the isolation of four new diterpenoids, ceriopsins A–D (1–4). The structures of the new diterpenoids were elucidated by a study of their physical and spectral data as methyl 17-hydroxy-16-oxobeyeran-18-oate (1), methyl 16(R)-16,17-dihydroxybeyeran-18-oate (2), 1β,15(S)-isopimar-7-ene-1,15,16-triol (3), and 8,15(R)-epoxypimarane-1β,16-diol (4).     

Significance of Staphylococcus epidermidis in the Clinical Laboratory

Susceptibility to 11 antibiotics was determined for 63 cultures of Staphylococcus aureus and 63 cultures of Staphylococcus epidermidis obtained at random from the clinical laboratory. The incidence of resistance to nine of these antibiotics was greater for S. epidermidis than for S. aureus. Studies of the minimal inhibitory concentration of these cultures to clindamycin showed that 61 cultures of S. aureus were susceptible whereas only 46 cultures of S. epidermidis were susceptible to this antibiotic. Although cultures of S. aureus were more active in the production of seven virulence factors, some cultures of S. epidermidis produced virulence factors. By successive cultivation in increasing concentrations of clindamycin, resistant variants were obtained for 10 cultures of S. aureus and 3 cultures of S. epidermidis. The presence of subinhibitory concentrations of clindamycin inhibited the production of some virulence factors by the resistant variants. In view of the greater resistance of S. epidermidis to antibiotics and its ability to produce virulence factors, its isolation in the clinical laboratory should not be regarded lightly.     

苦参碱对表皮葡萄球菌生物被膜作用初探

通过中药有效成分苦参碱对表皮葡萄球菌生物被膜抑制作用的研究,为表皮葡萄球菌生物被膜引起的相关感染提供新的治疗途径。利用XTT减低法评价苦参碱对表皮葡萄球菌初始粘附及生物被膜内细菌代谢的影响,镜下观察该药对表皮葡萄球菌生物被膜的形态学影响。结果表明:苦参碱对表皮葡萄球菌生物被膜菌的SMIC50和SMIC80分别为62.5 mg/L和500 mg/L;1 000 mg/L浓度的苦参碱对表皮葡萄球菌早期粘附有抑制作用;250 mg/L浓度的苦参碱对表皮葡萄球菌生物被膜的形态有显著影响。因此可见,苦参碱对表皮葡萄球菌生物被膜的形成与粘附均有抑制作用。     

The superoxide dismutase gene sodM is unique to Staphylococcus aureus: absence of sodM in coagulase-negative staphylococci

Superoxide dismutase (SOD) profiles of clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were determined by using whole-cell lysates and activity gels. All S. aureus clinical isolates exhibited three closely migrating bands of activity as previously determined for laboratory strains of S. aureus: SodM, SodA, and a hybrid composed of SodM and SodA (M. W. Valderas and M. E. Hart, J. Bacteriol. 183:3399-3407, 2001). In contrast, the CoNS produced only one SOD activity, which migrated similarly to SodA of S. aureus. Southern analysis of eight CoNS species identified only a single sod gene in each case. A full-length sod gene was cloned from Staphylococcus epidermidis and determined to be more similar to sodA than to sodM of S. aureus. Therefore, this gene was designated sodA. The deduced amino acid sequence of the S. epidermidis sodA was 92 and 76% identical to that of the SodA and SodM proteins of S. aureus, respectively. The S. epidermidis sodA gene expressed from a plasmid complemented a sodA mutation in S. aureus, and the protein formed a hybrid with SodM of S. aureus. Both hybrid SOD forms as well as the SodM and SodA proteins of S. aureus and the S. epidermidis SodA protein exist as dimers. These data indicate that sodM is found only in S. aureus and not in the CoNS, suggesting an important divergence in the evolution of this genus and a unique role for SodM in S. aureus.     

环链虫草Cordyceps cateniannulata对番茄生长和抗氧化酶活性的影响

【目的】评估环链虫草Cordyceps cateniannulata对植物促生和植物抗氧化酶活性的影响。【方法】本研究利用浸种法将环链虫草接种于番茄植物体,在接种后的第30天和60天,通过番茄株高、根长、地上和地下部分的干鲜重指标评价其对番茄生长的影响;在接种后第10、20、30、60和90天,通过选择性培养基分析其在番茄不同组织中的生存情况,使用形态学及DNA序列比对的方法检验所分离菌株与原有菌株的一致性。在处理后的第30天,检测番茄叶片中的过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)及丙二醛(MDA)含量,观察环链虫草对番茄的抗氧化酶活性影响。【结果】环链虫草可定殖于番茄幼苗且对番茄生长有显著促进作用,菌株对植株的定殖偏好性分别为根部>茎部>叶部。酶活检测结果表明,处理组番茄叶片防御酶活性均呈显著升高的趋势,其中POD、CAT、SOD活性分别比对照增加了52.21%、75.31%和158.59%,MDA含量下降了35.15%。【结论】环链虫草可以通过浸种的方法感染并定殖番茄幼苗的根、茎、叶,促进番茄幼苗的生长并提高番茄抗氧化酶活性,具有较好的田间...     

Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom

The major l-amino acid oxidase (LAAO, EC 1.4.3.2) of king cobra (Ophiophagus hannah) venom is known to be an unusual form of snake venom LAAO as it possesses unique structural features and unusual thermal stability. The antibacterial effects of king cobra venom LAAO were tested against several strains of clinical isolates including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli using broth microdilution assay. For comparison, the antibacterial effects of several antibiotics (cefotaxime, kanamycin, tetracycline, vancomycin and penicillin) were also examined using the same conditions. King cobra venom LAAO was very effective in inhibiting the two Gram-positive bacteria (S. aureus and S. epidermidis) tested, with minimum inhibitory concentration (MIC) of 0.78μg/mL (0.006μM) and 1.56μg/mL (0.012μM) against S. aureus and S. epidermidis, respectively. The MICs are comparable to the MICs of the antibiotics tested, on a weight basis. However, the LAAO was only moderately effective against three Gram-negative bacteria tested (P. aeruginosa, K. pneumoniae and E. coli), with MIC ranges from 25 to 50μg/mL (0.2-0.4μM). Catalase at the concentration of 1mg/mL abolished the antibacterial effect of LAAO, indicating that the antibacterial effect of the enzyme involves generation of hydrogen peroxide. Binding studies indicated that king cobra venom LAAO binds strongly to the Gram-positive S. aureus and S. epidermidis, but less strongly to the Gram-negative E. coli and P. aeruginosa, indicating that specific binding to bacteria is important for the potent antibacterial activity of the enzyme.     

Biocontrol of Sclerotinia lettuce drop by Coniothyrium minitans and Trichoderma hamatum

Fungal isolates, with known activity against Sclerotinia spp. in laboratory assays, were tested for their ability to control Sclerotinia minor in four field experiments (1998–2000). In the first experiment, eight fungal isolates (Trichoderma hamatum LU595, LU593, LU592, Trichoderma virens LU555 and LU556, Coniothyrium minitans LU112, Clonostachys rosea LU115 and Trichoderma rossicum LU596) were evaluated by incorporating spore suspensions into transplant potting mix and planting lettuce seedlings into a S. minor infested field site. At harvest, Trichoderma hamatum LU595, LU593, T. virens LU555 and C. minitans LU112 reduced disease by 30–50% compared with the untreated control under very high disease pressure (100%). In further field experiments C. minitans LU112 and T. hamatum LU593, applied as maizemeal–perlite soil amendments or incorporated into the potting mix, reduced S. minor disease over a range of disease pressures (29–91%). Disease control was equivalent or greater than that achieved with the standard carbendazim fungicide treatment. Both isolates were shown to effectively colonize the lettuce rhizosphere and surrounding soil and this colonization may have protected the roots from infection by S. minor. Multiple applications of C. minitans LU112 or T. hamatum LU593 formulations gave no added disease control compared with a single application at planting. Commercial formulations of both C. minitans LU112 and T. hamatum LU593 applied as transplant treatments, solid substrate soil amendments or as a spore drench gave consistent disease control and are currently being developed further.     

秋华柳和枫杨幼苗对镉的积累和耐受性

以秋华柳和枫杨当年实生幼苗为研究对象,采用向土壤添加外源镉(CdCl₂ · 2.5H₂O)的形式设置了0(对照组)、10 、20 、50、100 mg/kg 5个处理,研究了镉胁迫下秋华柳和枫杨幼苗的生长、生物量变化和根茎叶镉含量,并评价了两树种的耐性指数(Ti)、转移系数(Tf)和生物富集系数(BCF)。结果表明:(1)在镉含量为10 mg/kg时,秋华柳和枫杨幼苗基于生长和生物量参数的耐性指数(Ti)分别为91.72和91.62,与对照组相比无显著变化,其余各组(20、50、100 mg/kg)则显著低于对照植株(P<0.05);(2) 土壤镉浓度小于20mg/kg时,秋华柳植株茎、叶镉积累量分别高达61.73 mg/kg、163.04 mg/kg,根镉积累量为91.05 mg/kg;枫杨植株茎、叶镉积累量最高分别为7.9 mg/kg、5.25 mg/kg,仅为秋华柳茎、叶的12.8%和3.2%,根镉积累量高达190.68 mg/kg;(3) 除对照外,秋华柳幼苗各部分镉含量为叶＞根＞茎,转移系数(Tf)介于0.789-1.513之间,枫杨幼苗各部分镉含量为根＞茎＞叶,转移系数(Tf)介于0.037-0.044之间,远远小于秋华柳Tf;(4)秋华柳和枫杨幼苗在土壤镉浓度为10 mg/kg时具有很高的生长适应性和耐性,秋华柳根吸收的镉向地上部分转移能力、地上部分积累镉的能力都远远大于枫杨,生物富集系数(BCF)进一步证实了这一特性。研究证明,秋华柳植株具有很高的镉耐性、镉转移能力及地上部分积累镉的能力,适合于镉污染严重区域的植物修复。