Characterization of azadirachtin binding to Sf9 nuclei in vitro

[22,23-(3)H(2)]dihydroazadirachtin was incorporated by Sf9 cells in culture and was bound specifically to the nuclear fraction. The observed association constant of the binding of the radioligand to a purified nuclear fraction was determined to be 0.037 +/- 0.008 min(-1) using a one-phase exponential association equation, and binding appeared to be to a single population of sites. The binding was essentially irreversible, and the dissociation constant was estimated to be 0.00065 +/- 0.00013 min(-1). An association rate constant of 7.3 x 10(6) M(-1) min(-1) was calculated from these data. Binding was saturable, and the receptor number and affinity were determined as B(max) = 23.87 +/- 1.15 pmol/mg protein, K(d) = 18.1 +/- 2.1 nM. The order of potency of semisynthetic azadirachtin analogues for competition for the binding site was as follows (IC(50) in parentheses): azadirachtin (1.55 x 10(-8) M) > dihydroazadirachtin (3.16 x 10(-8) M) > dansyl dihydroazadirachtin (7.40 x 10(-8) M) > DNP-azadirachtin (7.50 x 10(-8) M) > biotin dihydroazadirachtin (1.27 x 10(-7) M) > 11-methoxy 22,23-dihydroazadirachtin (6.67 x 10(-7) M). [Originally published in Volume 34, Archives of Insect Biochemistry and Physiology, 34:461-473 (1997).] Copyright 1997 Wiley-Liss, Inc.     

Effects of different application methods of azadirachtin against sweetpotato whitefly Bemisia tabaci Gennadius (Hom., Aleyrodidae) on tomato plants

Abstract:  Studies on three different neem treatment methods (seed, soil and foliar) and two different commercial neem products (NeemAzal T/S 1% azadirachtin and NeemAzalU 17% azadirachtin) against sweetpotato whitefly (WF) Bemisia tabaci Gennadius (Hom., Aleyrodidae) on tomato plants were conducted in cages in air-conditioned cultivation rooms. All three methods of neem treatments resulted in reduced colonization and oviposition. Overall oviposition intensity was significantly reduced (44%) by the treatment of tomato seeds but an even higher reduction (74%) was achieved through soil drenching both with 3.0 g/l NeemAzalU and foliar spraying (82%) with 10 ml/l of NeemAzal TS compared with control treatments. In contrast, soil and foliar treatment increased fecundity per female up to 33% and 32%, respectively, at the highest tested concentrations. Reduced egg hatch could be observed only at high neem concentrations; 62% and 51% of deposited eggs hatched at the highest dose rates of NeemAzalU in case of seed and foliar treatments, respectively; whereas only 43% of deposited eggs hatched in case of foliar treatments at highest dose rates of NeemAzal T/S. Seed (35%), foliar (93%) and soil treatments (91%) caused high mortality rates of immatures and reduced number of hatching adults compared with control plants treated with a blank formulation or water. The mortality among immatures increased in relation to azadirachtin concentrations. Concerning susceptibility of different developmental stages, young larvae were the most sensitive. Foliar treatment was the most efficient, with 100% mortality for all three larval stages at high concentrations (10 ml/l of NeemAzal T/S) compared with 78–87% mortality with soil treatment (at 3.0 g/l NeemAzalU). The findings are discussed in the context of integrated control of WF in protected cultivation environments in the humid tropics.     

Neem,Azadirachta indica A. Juss. (meliaceae), and its potential for sustainable woodcarving in Kenya

The density of neem trees in parts of Malindi District in eastern Kenya was assessed to determine the sustainability of their use in the local woodcarving industry. Size and age structure of neem populations under different ownership were analyzed to establish the Annual Allowable Cut. Neem is an excellent carving wood and the available quantities along the Kenyan coast are sufficient to sustainably supply the industry of the whole country. However, because the resource comes from small farm holdings and the supply system to carving groups is not well established, further efforts are required to introduce a management regime that could be certified under the standards of the Forest Stewardship Council.     

Extraction of total phenolic content from Azadirachta indica or (neem) leaves: Kinetics study

The objective of this work is to put forth the optimization and kinetics of total phenolic compounds extraction from Azadirachta indica leaves in a stirred batch extraction. Various experiential extraction parameters have been studied for maximum extraction of the total phenolic compounds. The maximum yield of total phenolic compounds was found to be 10.80?mg?g^?1 of dried neem powder under the optimized conditions. The extraction kinetics behavior followed first-order kinetics with diffusion coefficient ranged from 1.8?×?10^?12 to 3.2?×?10^?12?m² s^?1 for all sets. Activation energy (E_a) value for the extraction of the total phenolic compounds was found to be 22.87?kJ?mol^?1. The kinetic expression model developed by Spiro and Siddique showed a good agreement with the experimental outcomes. The obtained results can be used to scale up the operations for industrial purposes.     

Insect growth regulating effects of neem extract and azadirachtin on aphids

Neem,Azadirachta indica (A. Juss.), seed oil (NSO) applied to leaf discs at a concentration of 1.0% resulted in 94% to 100% mortality of second instar nymphs of currant-lettuce aphid,Nasonovia ribis-nigri (Mosley), and green peach aphid,Myzus persicae (Sulzer), after nine days. The equivalent amount of pure azadirachtin (AZA) (≈40 ppm), the principle active ingredient of neem, was as effective as NSO. The survival of adult aphids was unaffected by NSO or AZA, but the survival of offspring from treated adultM. persicae andN. ribis-nigri was reduced significantly. The lethal concentration of AZA resulting in 50% mortality of second instar nymphs of nine species of aphids ranged from 2.4 ppm forM. persicae on pepper to 635.0 ppm for the strawberry aphid,Chaetosiphon fragaefolii (Cockerell), on strawberry. ForM. persicae, the growth regulating effect of AZA was influenced by the host plant and the nymphal instar treated.     

In vitro production of azadirachtin from cell suspension cultures of Azadirachta indica

The present study aimed to elucidate the effect of nutritional alteration on biomass content and azadirachtin production in cell suspensions of the elite neem variety crida-8.Variations in total nitrogen availability in the medium in terms of different ratios of nitrate: ammonium showed that the ratio 4:1 revealed a profound effect, leading to a 1.5-fold increase in the total extracellular azadirachtin production (5.59 mg/l) over the standard MS medium.Reduction in sucrose (15 mg/l) in the medium exhibited a reduction in biomass and absence of azadirachtin, whereas total phosphate reduction raised intracellular azadirachtin production (6.98 mg/l). An altered medium with a nitrate: ammonium ratio of 4:1 coupled with complete elimination of phosphate enhanced biomass by 36% (59.36 g/l).     

Efficacy of a novel neem oil formulation (RP03™) to control the poultry red mite Dermanyssus gallinae

Dermanyssus gallinae (Mesostigmata: Dermanyssidae) is the most harmful ectoparasite of laying hens, represents an occupational hazard for poultry workers, and a growing threat to medical science per se . There is increasing demand for alternative products, including plant‐derived acaricides, with which to control the mite. The present study investigated the efficacy of neem oil against D. gallinae on a heavily infested commercial laying hen farm. A novel formulation of 20% neem oil, diluted from a 2400‐p.p.m. azadirachtin‐concentrated stock (RP03?), was administered by nebulization three times in 1 week. Using corrugated cardboard traps, mite density was monitored before, during and after treatment and results were statistically analysed. Mite populations in the treated block showed 94.65%, 99.64% and 99.80% reductions after the first, second and third product administrations, respectively. The rate of reduction of the mite population was significantly higher in the treated block (P < 0.001) compared with the control and buffer blocks. The results suggest the strong bioactivity of neem, and specifically of the patented neem‐based formulation RP03?, against D. gallinae . The treatment was most effective in the 10 days following the first application and its effects persisted for over 2 months. Further studies will aim to overcome observed side effects of treatment represented by an oily layer on equipment and eggs.     

Bacillus thuringiensis and neem seed oil (Azadirachta indica) effects on the potato tuber moth Phthorimaea operculella zeller in the field and stores

A comparative evaluation for the efficacy of Bacillus thuringiensis and neem seed oil on Phthorimaea operculella has been carried out in the field and store. These two preparations were almost equally effective on the potato tuber moth infestation. The percentage of infestation was reduced through successive application of either preparations in the field up to harvest. No synergism was observed upon using combination of the two preparations. In the store, neem seed oil (500 ppm) was highly protective and was as effective as sevin. A combination of both neem and B.t. (Delfin) significantly protects the tubers. This suggests the possible use of either neem seed oil or B.t. in combating the insect pest in the field or during storage.     

Challenges to using neem (Azadirachta indica var. sianensis Valenton) in Thailand

The use of botanical insecticides based on the neem seed (Azadirachta indica var.siamensis Valeton) shows strong potential in the fight against important agricultural pests. Unfortunately, its use in rural Thailand is extremely limited. This study examines the efficacy of neem extracts processed under rural conditions; identifies factors that influence local farmers’ adoption of neem products; and proposes recommendations to promote the use of neem insecticides. The principal findings are: in field trials conducted under typical rural conditions, neem did not adequately control major pests of yard-long beans (Vigna sesquipedalis); and intensity of vegetable production, knowledge of neem insecticides and availability and accessibility of neem products strongly influence farmers’ adoption of neem products for pest control.     

Insect growth regulating and antifeedant effects of neem extracts and azadirachtin on two aphid species of ornamental plants

Leaf disc choice test bioassay demonstrated that formulated neem seed extracts were highly deterrent and growth regulatory to rose aphid,Microsiphum rosae (L.) and Chrysanthemum aphid,Macrosiphoniella sanbornii (Gillete). Effective concentrations to produce 50% feeding deterrence was 0.80 and 0.84% respectively for 2nd instar nymphs irrespective of bioassay duration. The disruption of aphid feeding was related to the presence of azadirachtin concentration in the extract. The toxicity on contact from the leaf surface or via topical application due to azadirachtin was significantly different and topical treatment was at least 7 times more effective for both species. Thus growth regulatory effects of azadirachtin were influenced by the host plant and the stage of treatment. Field evaluation with formulated neem extracts revealed the effect to be more of growth regulatory nature thereby showing that azadirachtin is a physiological toxin for aphid species. Neem seed extracts reduced the population of aphid on respective host plants significantly, EC₅₀ values being 0.88 and 0.96% forM. rosae andM. sanbornii respectively.     

Chemical characterization of Azadirachta indica grafted on Melia azedarach and analyses of azadirachtin by HPLC‐MS‐MS (SRM) and meliatoxins by MALDI‐MS

Introduction – Melia azedarach adapted to cool climates was selected as rootstocks for vegetative propagation of Azadirachta indica. Cleft grafting of A. indica on M. azedarach rootstock showed excellent survival. Little is known about the chemistry of grafting. Objective – The roots, stems, leaves and seeds of this graft were examined in order to verify if grafted A. indica would produce limonoids different from those found in non‐grafted plants. Intact matured fruits were also studied to verify if they were free of meliatoxins. Methodology – After successive chromatographic separations the extracts afforded several limonoids. HPLC‐MS/MS and MALDI‐MS were used to develop sensitive methods for detecting azadirachtin on all aerial parts of this graft and meliatoxins in fruits, respectively. Results – The stem afforded the limonoid salannin, which was previously found in the oil seeds of A. indica. Salannin is also found in the root bark of M. azedarach. Thus, the finding of salannin in this study suggests that it could have been translocated from the M. azedarach rootstock to the A. indica graft. HPLC‐MS/MS analyses showed that azadirachtin was present in all parts of the fruits, stem, flowers and root, but absent in the leaves. The results of MALDI‐MS analyses confirmed the absence of meliatoxins in graft fruits. Conclusion – This study showed that A. indica grafted onto M. azedarach rootstock produces azadirachtin, and also that its fruits are free of meliatoxins from rootstocks, confirming that this graft forms an excellent basis for breeding vigorous Neem trees in cooler regions. Copyright © 2010 John Wiley & Sons, Ltd.     

Effects of azadirachtin on oogenesis in Aedes aegypti

ABSTRACT. . Azadirachtin in blood fed to adult female Aedes aegypti through an artificial membrane does not cause feeding inhibition over a wide dose range (0–200 ng/female), and high doses of ingested azadirachtin fail to inhibit or delay oviposition. However, significant, transient retardation of oocyte growth is observed for up to 72 h after feeding. Immature oocytes are observed in 86% of azadirachtin-fed females decapitated 10 h after a blood meal, whereas 96% of decapitated control females contain maturing oocytes. This suggests that azadirachtin delays the release of one or more factors from the head that regulate oogenesis. We propose that adult females overcome the effect of azadirachtin by rapid metabolism rather than by excretion of the compound, since by 2 h after a blood meal, only 0.1% of ingested azadirachtin was recovered from excreta and 5% recovered from the body.     

Expression of Bacteriorhodopsin in Sf9 and COS-1 Cells

We report studies on the expression of the archaebacterial membrane protein bacteriorhodopsin in Sf9 insect cells and in COS-1 mammalian cells. In both cell systems, the apoprotein bacterio-opsin was expressed at levels of 1 g/10⁶ cells. Immunofluorescence studies showed that the expressed protein was accumulated in the endoplasmic reticulum. However, upon addition of all-trans retinal to membranes isolated from either Sf9 or COS-1 cells expressing bacterio-opsin, the characteristic bacteriorhodopsin chromophore (_max at 560 nm) was rapidly generated. This is in contrast to bacterio-opsin expressed in E. coli, which cannot be functionally reconstituted with retinal unless it is first denatured, and then renatured in vitro. These studies demonstrate that the bacterio-opsin expressed is correctly folded and show that localization of a heterologously expressed membrane protein in the endoplasmic reticulum does not necessarily imply that it is misfolded.     

Limonoids and Flavonoids from the Flowers of Azadirachta indica var. siamensis,and Their Melanogenesis‐Inhibitory and Cytotoxic Activities

A new limonoid, 7‐O‐acetyl‐7‐O‐debenzoyl‐22‐hydroxy‐21‐methoxylimocinin ( 2 ), and two new flavonoids, 3′‐(3‐hydroxy‐3‐methylbutyl)naringenin ( 7 ) and 4′‐O‐methyllespedezaflavanone C ( 9 ), along with nine known compounds, including two limonoids, 1 and 3 , and seven flavonoids, 4 – 6, 8 , and 10 – 12 , were isolated from a MeOH extract of the flowers of Azadirachta indica A.Juss. var. siamensis Valeton (Siamese neem tree; Meliaceae). The structures of new compounds were elucidated on the basis of extensive spectroscopic analysis and comparison with literature data. All of these compounds were evaluated for their melanogenesis‐inhibitory activities in B16 melanoma cells induced with α‐melanocyte‐stimulating hormone (α‐MSH). Compound 2 (16.9% melanin content at 30 μM ), 6‐deacetylnimbin ( 3 ; 49.6% melanin content at 100 μM ), and kaempferide ( 10 ; 41.7% melanin content at 10 μM ) exhibited inhibitory effects with no, or almost no, toxicity to the cells (81.0–111.7% cell viability). In addition, evaluation of their cytotoxic activities against HL60, A549, AZ521, and SK‐BR‐3 human cancer cell lines, isoazadironolide ( 1 ), 4′‐O‐methyl‐8‐prenylnaringenin ( 5 ), euchrestaflavanone A ( 8 ), 9 , and 3‐methoxy‐3′‐prenylnaringenin ( 12 ) revealed potent cytotoxicities against one or more cell lines with IC₅₀ values in the range of 4.5–9.9 μM .     

Zinc potentiation of androgen receptor binding to nuclei in vitro

Zn2+ potentiates binding of the 4.5S [3H]dihydrotestosterone-receptor complex to isolated rat prostate Dunning tumor nuclei in vitro when assayed in the presence of 300 microM ZnCl2, 3 mM MgCl2, 0.25 M sucrose, 5 mM mercaptoethanol, 0.15 M KCl, and 50 mM tris(hydroxymethyl)aminomethane, pH 7.5. In the presence of 5 mM mercaptoethanol, the concentration of 50 microM total Zn2+ required to promote half-maximal receptor binding to nuclei corresponds to a free Zn2+ concentration of 50 nM. The receptor-nuclear interaction appears to be selective for Zn2+; other divalent cations when added at a concentration of 1 mM to a buffer containing 5 mM mercaptoethanol are less effective (Ni2+) or have essentially no effect (Ca2+, Mg2+, Mn2+, Co2+, Cu2+, and Cd2+). Zn2+ does not alter the sedimentation rate of the 4.5S [3H]dihydrotestosterone receptor in the presence of mercaptoethanol; however, in the absence of mercaptoethanol, Zn2+ causes the receptor to aggregate. Zn2+-dependent nuclear binding of the 4.5S [3H]dihydrotestosterone receptor is saturable at 1.4 X 10(-13) mol of receptor sites/mg of DNA, corresponding to approximately 1150 sites/nucleus. In the presence of excess nuclei, up to 60% of added receptor is nuclear bound. An apparent binding constant for the receptor-nuclear interaction of 10(13) M-1 was approximated. Pyridoxal 5'-phosphate (less than or equal to 10 mM), but not 0.4 M KCl, inhibits Zn2+-dependent nuclear binding of the [3H]dihydrotestosterone receptor. Up to 66% of nuclear-bound receptor can be extracted in buffer containing 3 mM ethylenediaminetetraacetic acid plus either 0.4 M KCl or 10 mM pyridoxal 5'-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

抗黄曲霉毒素B1单链抗体在Sf9昆虫细胞中的表达与性质分析

目的:在原核表达抗黄曲霉毒素B1(aflatoxin B1,AFB1)单链抗体(single chain Fv fragment,scFv)研究的基础上,为进一步了解和提高抗AFB1 scFv的活性,利用Sf9昆虫细胞表达抗AFB1 scFv,并对其活性进行探索研究。方法:构建pFastBac 1-scFv2E6VHVL重组质粒,将重组质粒转化Escherichia coli (E. coli) DH10Bac细胞,进行蓝白斑筛选,挑取阳性克隆。提取相应的重组杆状病毒穿梭载体Bacmid侵染Sf9昆虫细胞,表达scFv,利用镍亲和层析法纯化scFv,并以ELISA检测scFv活性。结果:蓝白斑筛选后,经菌落PCR和测序验证挑取的白斑阳性单克隆含有正确的单链抗体基因。提取相应的重组杆状病毒穿梭载体Bacmid侵染Sf9昆虫细胞,通过Western blot检测得知抗AFB1 scFv在Sf9昆虫细胞中成功表达。AFB1对scFv的抑制中浓度(IC₅₀)为30μg/ml。结论:与E. coli BL21(DE3)表达系统相比,scFv灵敏度转好,但仍有较大提升空间。     

Desensitization of fifth instar Spodoptera litura to azadirachtin and neem

The deterrence of azadirachtin, in its pure form and as a constituent of neem seed extract, to fifth instar Spodoptera litura (Fab.) larvae, was measured using cabbage, Brassica oleraceae (L.) var. capitata, leaf disc assays. Paired-choice assays, in which larvae could choose between feeding on a treated (1.3 ng azadirachtin per square cm leaf area) or an untreated leaf disc for 2 h, were conducted at 24 h intervals throughout the fifth instar. In addition, no-choice assays, in which larvae could feed on only one leaf disc (10 ng azadirachtin per square cm leaf area) for 1.5 h, were conducted consecutively over a six hour period at the beginning of the fifth instar. The effects of hunger and habituation on desensitization in our no-choice tests were partitioned. After repeated exposures, larvae became desensitized to pure azadirachtinal in both choice and no-choice tests, but did not desensitize to neem containing the same absolute amount of azadirachtin in choice tests. Hunger was responsible for approximately one third of the desensitization response in the no-choice tests. Sensitivity to azadirachtin was independent of age within the fifth instar.     

元谋栽培印楝枝叶化学成分研究

从元谋栽培印楝Azadirachta indica的枝叶中分离得到15个化合物,结构类型有三萜、倍半萜、甾体等。经波谱解析鉴定为nimbin（1）,6-deacetylnimbin（2）,6-deacetylnimbinene（3）,nimbinene（4）,azadiradi-one（5）,7-acetoxy-elema-1,3-dien-8-ol（6）,1-naphthalenone（7）,acarusnol（8）,colvane-2β,9α-diol（9）,乌苏酸（10）,马斯里酸（11）,2α-羟基乌苏酸（12）,猕猴桃酸B（13）,2α,3α,4β-trihydroxypregnan-16-one（14）,2β,3β,4β-trihydroxypregnan-16-one（15）。化合物6～15为首次从该植物中分离得到。     

Calcium-binding protein regucalcin inhibits deoxyribonucleic acid synthesis in the nuclei of regenerating rat liver

The effect of regucalcin, a Ca²⁺-binding protein isolated from rat liver cytosol, on deoxyribonucleic acid (DNA) synthesis in the nuclei of regenerating rat liver was investigated. At 1 day after partial hepatectomy, the liver weight was increased about 50% of that of sham-operated rats, and it reached to the same levels as sham operation at 3 days after hepatectomy. Nuclear DNA synthesis was markedly increased at 1 day after hepatectomy, and this increase was also seen at 3 days. Nuclear DNA synthesis was clearly enhanced in the presence of EGTA (0.4 mM) in the incubation mixture. The presence of Ca²⁺ ( 1.0–25 M) caused a significant decrease in the nuclear DNA synthesis of normal rat liver. Regucalcin (0.25 and 0.5 M) clearly inhibited the nuclear DNA synthesis of normal rat liver. This inhibition was also seen in the presence of Ca²⁺ (1.0 M). Moreover, in the liver nuclei obtained at 1 day after partial hepatectomy, the presence of regucalcin (0.05–0.5 M) caused a remarkable inhibition of nuclear DNA synthesis. This effect was also revealed in the presence of EGTA (0.4 mM). Thus, the inhibitory effect of regucalcin was remarkable in regenerating rat liver nuclei in comparison with that of normal rat liver. The present results demonstrate that regucalcin can suppress nuclear DNA synthesis in regenerating rat liver. We suppose that regucalcin may have a role in the regulation of nuclear DNA synthesis in liver cell proliferation.     

Purification and Characterization of Aspartic Protease Derived from Sf9 Insect Cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS–PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K _m of 0.85 μM. The k _cat and k _cat?K _m values were 13 s^?1 and 15 s^?1 μM^?1 respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K _i, of 25 pM.