Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary—or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.     

UV-B and cadmium induced changes in pigments,photosynthetic electron transport activity,antioxidant levels and antioxidative enzyme activities of <Emphasis Type="Italic">Riccia</Emphasis> sp.

UV-B and cadmium, alone and together, induced changes in photosynthetic pigment levels, photosynthetic electron transport activity, enzymatic and non-enzymatic (low molecular weight) antioxidants, level of hydrogen peroxide and lipid peroxidation in Riccia sp. were evaluated. Chlorophyll content was found to decrease with the rising concentration of cadmium and UV-B exposure alone and its level further declined when both the stresses were applied together. In contrast to this, carotenoids exhibited varied response, as it showed enhancement with UV-B (15, 30 and 45 min exposure) and low concentration of Cd (1 and 10 μM) treatment alone and in combination. Both the stresses caused strong inhibitory effect on PS II activity (H₂O → p-BQ), while PS I activity (DCPIP/ASC → MV) appeared to be less sensitive. Total peroxide content increased with simultaneous increase in lipid peroxidation. The level of non-enzymatic antioxidant ascorbate and enzymatic antioxidants superoxide dismutase and peroxidase activity were found to increase with simultaneous decrease in catalase activity following UV-B and Cd treatments. These results indicate that 45 min of UV-B exposure and 10, 100 and 1000 μM cadmium alone and together, strongly arrested electron flow through PS II which caused accelerated generation of reactive oxygen species (H₂O₂) and excess accumulation of H₂O₂ due to significant inhibition of catalase activity, led to the oxidative damage in Riccia sp.     

Antischistosomal action of thioxo-imidazolidine compounds: an ultrastructural and cytotoxicity study

Schistosomiasis is a disease caused by helminthes of the genus Schistosoma, which threatens approximately 207 million people worldwide. Recently, strains of Schistosoma mansoni appear to be developing tolerance and resistance against Praziquantel, the most commonly available drug on the market used in the treatment of disease. This worrisome development justifies studies that seek alternatives for the prevention, treatment and cure of this disease. This study aimed to evaluate the in vitro activity of new imidazolidine compounds 1-benzyl-4-[(4-chloro-phenyl)-hydrazono]-5-thioxo-imidazolidin-2-one (LPSF/PT-5) and 1-(4-chloro-benzyl)-4-[(4-fluoro-phenyl)-hydrazono]-5-thioxo-imidazolidin-2-one (LPSF/PT-11) against adult worms of S. mansoni. LPSF/PT-5 and LPSF/PT-11 imidazolidine derivatives showed relevant schistosomicidal activity in vitro and induced significant ultrastructural alterations in worms and cell death: results similar to praziquantel. Thus, it is possible that these imidazolidine derivatives can be future candidates as schistosomotic drugs, but further studies are needed to elucidate the induced mechanisms behind this response.     

The infestation and dispersion patterns of Carcinonemertes spp. (Nemertea) on their crab hosts

Seasonal changes in the infestation and dispersion patterns of egg predatory nemerteans on their crab hosts were analyzed. Marked differences in the seasonal patterns of infestation were noted between worm species, yet, common patterns in the aggregation of the worms were found. Worm aggregation increased at the onset of the reproductive seasons of the hosts, and at the nadirs of the reproductive seasons for those hosts with year round breeding. The aggregation patterns of two worm species fluctuated with the physical environment of their estuarine hosts. Salinity changes as a result of seasonal rains may have caused changes in the underlying dispersion patterns of Carcinonemertes epialti on Hemiarapsus oregonensis, and C. mitsukurii on Portunus pelagicus. Lastly, the embryogenic cycle of the host species was significant in shaping the infestation and aggregation patterns of C. epialti on Cancer anthonyi and C. regicides on Paralithodes camtschaticus. Worm immigration and emigration were linked to crab embryogenesis and directly influenced the dispersion patterns of the worms.     

Schistosoma mansoni: In vitro schistosomicidal activity and tegumental alterations induced by piplartine on schistosomula

Schistosomiasis is one of the most important parasitic infections in humans that occur in many tropical and subtropical countries. Currently, the control of schistosomiasis rests with a single drug, praziquantel, which is effective against adult worms but not the larval stages. Recent studies have shown that piplartine, an amide isolated from plants of the genus Piper (Piperaceae), reveals interesting antischistosomal properties against Schistosoma mansoni adult worms. Here, we report the in vitro antischistosomal activity of piplartine on S. mansoni schistosomula of different ages (3h old and 1, 3, 5, and 7days old), and examine alterations on the tegumental surface of worms by means of confocal laser scanning microscopy. Piplartine at a concentration of 7.5μM caused the death of all schistosomula within 120h. The lethal effect occurred in a dose-dependent manner and was also dependent on the age of the parasite. Microscopy observation revealed extensive tegumental destruction, including blebbing, granularity, and a shorter body length. This report provides the first evidence that piplartine is able to kill schistosomula of different ages and reinforce that piplartine is a promising compound that could be used for the development of new schistosomicidal agent.     

辛基酚胁迫对雄性泥鳅抗氧化酶及卵黄蛋白原的影响

为研究辛基酚(OP)对雄性泥鳅抗氧化酶活性及血清卵黄蛋白原(VTG)含量的影响,将雄性泥鳅分别暴露于4种不同质量浓度OP(0.12、0.19、0.32、0.52 mg/L)中持续7、14、21 d和28 d,采用试剂盒检测肝脏超氧化物歧化酶(SOD)与过氧化氢酶(CAT)的含量,采用碱不稳定性蛋白结合磷法检测血清VTG的含量。结果表明,0.12 mg/L OP胁迫14 d,肝脏SOD和CAT含量均无显著变化,但是随着胁迫剂量增大和时间延长,SOD和CAT含量降低极其显著,在0.52 mg/L OP胁迫28 d时降到最低水平;泥鳅在0.12 mg/L OP中暴露7 d时,血清VTG含量就有极其显著升高,且随着胁迫剂量增大和时间的延长,VTG含量呈升高趋势。提示OP胁迫对SOD和CAT活性有显著的抑制作用,并随胁迫剂量增大和时间延长而抑制加剧,造成氧化损伤;OP胁迫可诱导VTG合成,并随暴露剂量增大和时间延长而诱导增强,具有明显的雌激素效应,这可能与其氧化损伤有密切关系。     

In Vitro Anthelminthic Activity and Ultrastructural Analysis of Barbatic Acid against Schistosomulae and Juvenile Worms of Schistosoma mansoni**

Schistosomiasis affects about 260 million people worldwide and the search for new schistosomicidal compounds is urgent. In this study we evaluated the in vitro effect of barbatic acid against schistosomulae and young worms of Schistosoma mansoni. The barbatic acid was evaluated through the bioassay of motility and mortality, cellular viability and ultrastructural analysis of juvenile stages through Scanning Electron Microscopy. Barbatic acid showed a schistosomicidal effect against schistosomulae and young worms of S. mansoni after 3 h of exposure. At the end of 24 h, barbatic acid showed 100 %, 89.5 %, 52 % and 28.5 % of lethality for schistosomulae at the concentrations of 200, 100, 50 and 25 μM, respectively. For young worms, barbatic acid showed 100 % and 31.7 % of lethality at the concentrations of 200 and 100 μM, respectively. Motility changes were observed at all sublethal concentrations. There was a significant reduction in the viability of young worms after exposure to barbatic acid at 50, 100 and 200 μM. Extensive damage to the schistosomulae and young worm's tegument, was observed from 50 μM. This report provides data showing the schistosomicidal effect of barbatic acid on schistosomulae and young worms of S. mansoni, causing death, motility changes and ultrastructural damage to worms.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis and antioxidant enzymes activity in <Emphasis Type="Italic">Acanthophyllum sordidum</Emphasis>

The effect of various hormonal combinations on callus formation and regeneration of shoot and root from leaf derived callus of Acanthophyllum sordidum Bunge ex Boiss. has been studied. Proteins and activity of antioxidant enzymes were also evaluated during shoot and root organogenesis from callus. Calli were induced from leaf explants excised from 30-d-old seedlings grown on Murashige and Skoog medium containing 4.52 μM 2,4-dichlorophenoxyacetic acid + 4.65 μM kinetin. Maximum growth of calli and the most efficient regeneration of shoots and roots occurred with 2.69 μM 1-naphthalene acetic acid (NAA), 2.69 μM NAA + 4.54 μM thidiazuron and 2.46 μM indole-3-butyric acid. Protein content decreased in calli and increased significantly during regeneration of shoots from callus. Superoxide dismutase activity decreased in calli comparing to that of seedlings, then increased in regenerated shoots and roots. High catalase activity was detected in seedlings and regenerated shoots, whereas high peroxidase activity was observed in calli and regenerated roots.     

The effect of bile salts on survival and morphology of a potential probiotic strain Lactobacillus acidophilus M92

Bile tolerance is an important criterion in the selection of microbial strains for probiotic use. The survival and morphological changes of a potential probiotic strain, Lactobacillus acidophilus M92, in the presence of bile salts were examined. Lactobacillus acidophilus M92 has shown a satisfactory degree of tolerance against oxgall and individual bile salts tested, especially to taurocholate. The higher resistance of L. acidophilus M92 against taurine-conjugated bile salts relative to deconjugated and glycine-conjugated bile salts was attributed to its reaction to the stronger acidity of the former. Furthermore, bile salt hydrolase (BSH) was active when L. acidophilus M92 was grown in the presence of sodium taurocholate. The rate of BSH activity was highest at the exponential growth phase. It was hypothesised that BSH activity may be important for the bile salt resistance of this strain. The colonial and cellular morphology may also be a valuable parameter in the selection of bile salt-resistant Lactobacillus strains for probiotic use. Smooth (S) and rough (R) colonies, appeared in the original L. acidophilus M92 bacterial culture and demonstrated a different degree of bile tolerance. Rough colonies were more sensitive to bile salts than smooth ones. The R colony cells assumed a round form, probably induced by gaps in the cell wall caused by the cytotoxicity of glycodeoxycholate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Short-and long-term effects of cadmium on transmembrane electric potential (E m) in maize roots

In this study, the effects of Cd on root growth, respiration, and transmembrane electric potential (E _m) of the outer cortical cells in maize roots treated with various Cd concentrations (from 1 μM to 1 mM) for several hours to one week were studied. The E _m values of root cells ranged between −120 and −140 mV and after addition of Cd they were depolarized immediately. The depolarization was concentration-dependent reaching the value of diffusion potential (E _D) when the Cd concentration exceeded 100 μM. The values of E _D ranged between −65 to −68 mV (−66 ± 1.42 mV). The maximum depolarization of E _m was registered approx. 2.5 h after addition of Cd to the perfusion solution and in some cases, partial (Cd > 100 μM) or complete repolarization (Cd < 100 μM) was observed within 8–10 h of Cd treatment. In the time-dependent experiments (0 to 168 h) shortly after the maximum repolarization of E _m a continuous concentration-dependent decrease of E _m followed at all Cd concentrations. Depolarization of E _m was accompanied by both increased electrolyte leakage and inhibition of respiration, especially in the range of 50 μM to 1 mM Cd, with the exception of root cells treated with 1 and 10 μM Cd for 24 and 48 h. Time course analysis of Cd impact on root respiration revealed that at higher Cd concentrations (> 50 μM) the respiration gradually declined (∼ 6 h) and then remained at this lowest level for up to 24 h. All the Cd concentrations used in this experiment induced significant inhibition of root elongation and concentrations higher than 100 μM stopped the root growth within the first day of Cd treatment. Our results suggest that Cd does not cause irreversible changes in the electrogenic plasma membrane H⁺ ATPase because fusicoccin, an H⁺ ATPase activator diminished the depolarizing effect of Cd on the E _m. The depolarization of E _m in the outer cortical cells of maize roots was the result of a cumulative effect of Cd on ATP supply, plasmalemma permeability, and activity of H⁺ ATPase.     

Interrelations Among Habitat Use, Behavior, and Flight-Related Morphology in Two Cooccurring Satyrine Butterflies, Maniola jurtina and Pyronia tithonus

Mobility, activity patterns, habitat use, and some morphological traits of two often cooccurring satyrine butterflies of grasslands—the meadow brown (Maniola jurtina) and the hedge brown (Pyronia tithonus)—were studied by a mark-release-recapture method at two sites. Additionally, someflight-related morphological traits of a series of collected females of P. tithonus were compared between recently colonized and permanent populations. The more active, but less mobile P. tithonus got faster wing damage than did M. jurtina and had more, and more symmetrically spread eyespots onthe wings. For both species, the microdistribution was affected by shelter, long vegetation, and nectar, but this was more pronounced in P. tithonus. It is hypothesized that P. tithonus may traverse the same landscape at a slower rate than M. jurtina.     

Effect of Aeolosoma sp. (Aphanoneura: Aeolosomatidae) on the Population Dynamics of Selected Cladoceran Species

Although oligochaete worms naturally coexist with cladocerans in many shallow freshwater ponds and lakes, their influence on the latter is not well established. In this work we studied the effect of Aeolosoma sp. on the population growth of Alona rectangula, Ceriodaphnia dubia, Daphnia pulex, Macrothrix triserialis and Moina macrocopa. Population growth studies were conducted at one algal food density (1 × 10⁶cells ml^–1 of Chlorella vulgaris). The experimental design was similar for all five cladoceran species, where we used 100 ml capacity transparent jars containing 50 ml of EPA medium with the desired algal density and three replicates for each treatment. The test medium was changed daily and fresh algal food was added. The initial density of each of the cladoceran species in the population growth studies was 0.4 ind ml^–1 while that of the worms 1.0 ind ml^–1. Following inoculation, we estimated daily the number of cladocerans and the worms for duration of 21 days. Regardless of the presence of worms, Moina macrocopa and Macrothrix triserialis showed rapid population growth while A. rectangula took more than 2 weeks to reach peak abundances. With the exception of M. triserialis, all the other our cladoceran species declined in the presence of Aeolosoma sp. The lowest peak population density (about 1 ind ml^–1) was observed for M. triserialisin controls. The remaining species had peak densities of about 3–5 ind ml^–1. The rates of population increase per day varied from 0.03 to 0.19 depending on the cladoceran taxa and the treatment. In general we found that pelagic taxa were more adversely affected by the presence of the worms than were the littoral cladocerans.     

Sources of variation in rapidly inducible responses to leaf damage in the mountain birch-insect herbivore system

Summary Studies on rapidly inducible resistance in trees against insect herbivores show substantial variation in the strength of responses. Here we report the results of a study which examined causes of this variation. We bioassayed the quality of leaves of two developmental phases (young vs. mature) of the mountain birch Betula pubescens ssp. tortuosa by measuring the growth of two instars of Epirrita autumnata larvae. We used only short shoot leaves from trees of a natural stand, uniform in size and age. Damage was caused by larvae and artificial tearing of leaf lamina, varying the scale and time. We separated seasonal changes in plants from instar-dependent effects of the animals by testing experimental larvae in two subsequent growth trials. We found that only larval-made damage induced responses in leaves that made the leaves significantly poorer quality for the test larvae. Artificial damage induced only weak responses, and artificial canopy-wide damage even caused slight improvement of leaf quality. Cumulative leaf damage did not strengthen birch responses. Leaves that were in the expansion phase responded to damage while fully-expanded, mature leaves showed no response. The pattern of responses indicated that there might be physiological constraints: small-scale damage induced resistance against the larvae but largescale damage did not. Prevalent weather conditions might have modified these responses. Larvae of two instars and sexes, of low- and high-density populations responded to leaf damage similarly. However, prior experience of larvae with the host plant may have affected subsequent larval performance. Variation in rapidly inducible responses in birches was caused by plant characters rather than by test animals.     

Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary -- or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.     

Different antimetabolic effects of related lectins towards nymphal stages ofNilaparvata lugens

Insect feeding trials were carried out to determine the effects of a range of mannose-specific lectins on third instar nymphs of the rice brown planthopper (BPH), Nilaparvata lugens. Stål. Dose response curves show that Galanthus nivalis agglutinin (GNA) has the strongest toxic effect of the lectins tested, and is effective at concentrations considerably lower than those previously reported. Narcissus pseudonarcissus agglutinin (NPA) and Allium sativum agglutinin (ASA) exhibit a significant antimetabolic effect towards the insect but were less effective (on a molar basis) than GNA. LC₅₀ values for GNA, NPA and ASA are approximately 4 μM, 11 μM and >40 μM respectively. These mannose-specific lectins are serologically identical, but differ in the number of subunits per protein molecule; ASA is a dimer, NPA is a trimer and GNA is a tetramer. The results obtained support the hypothesis, that the effectiveness of the mannose-binding lectins as antimetabolites is determined by the number of subunits per molecule. Two N-acetylglucosamine binding lectins, the dimeric Oryza sativa agglutinin (OSA) and the monomeric Urtica dioica agglutinin (UDA), were also tested but at a concentration of 0.1% w/v exhibited no significant antimetabolic effect towards BPH, although the related lectin wheatgerm agglutinin (WGA) has previously been demonstrated to be toxic towards the insect.     

In vitro organogenesis and plant formation in cucumber

In vitro organogenesis was achieved from callus derived from hypocotyl explants of Cucumis sativus L. cv. Poinsett 76. Calli were induced from hypocotyl explants excised from 7-d-old seedlings grown on Murashige and Skoog (MS) medium containing 87.64 μM sucrose, 0.8 % agar, 3.62 μM 2,4-dichlorophenoxy acetic acid and 2.22 μM 6-benzyladenine (BA). Regeneration of adventitious buds from callus (25 shoots explant⁻¹) was achieved on MS medium supplemented with 8.88 μM BA, 2.5 μM zeatin and 10 % coconut water after two subcultures in the same medium at 30-d interval. Gibberellic acid (1.75 μM) favoured shoot elongation and indole 3-butyric acid (7.36 μM) induced rooting. Rooted plants were hardened and successfully established in soil.     

颈盲蝽取食对薇甘菊叶片营养物质和防御酶的影响

【目的】薇甘菊是世界最具危害性的入侵杂草之一,对我国生态环境和农业、林业生产造成了严重的危害。颈盲蝽是控制薇甘菊的一种潜在的重要天敌昆虫。本研究旨在探讨薇甘菊被颈盲蝽取食后,叶片防御相关酶系活性、营养物质和叶绿素含量的变化,阐明颈盲蝽取食对薇甘菊生理功能的影响,为利用颈盲蝽防控薇甘菊提供依据。【方法】从云南瑞丽野外采集薇甘菊的本地天敌昆虫颈盲蝽,测定了颈盲蝽取食前及取食12、24、48、96 h后,薇甘菊叶片中过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性,以及可溶性糖、可溶性蛋白及叶绿素含量,以取食前的植株作为对照。【结果】与对照相比,颈盲蝽取食12 h时,薇甘菊叶片中的POD、CAT活性升高,SOD活性降低;之后POD、SOD活性上升,CAT活性降低,取食48 h时,POD和SOD活性达到最高值,CAT活性达到最低值;取食96 h时,POD与SOD活性降低,但仍高于对照,CAT活性与取食48 h时相近。颈盲蝽取食后,薇甘菊叶片中的可溶性糖含量明显上升,取食96 h达到最高值;可溶性蛋白和叶绿素含量显著降低,在96 h达到最低值,分别低于对照39.30%和69.94%。【结论】颈盲蝽取食严重破坏了薇甘菊叶片的正常生理功能,并最终导致其叶片萎蔫和坏疽。     

Biomass production and fatty acid profile of a Scenedesmus rubescens-like microalga

This investigation examined the effects of nitrogen–phosphate combined deficiency on the biomass yield, fatty acid methyl esters (FAME) production and composition from Scenedesmus rubescens-like microalga. A 15-day indoor culture was performed as a 3 × 3 factorial design (NaNO₃ levels: 3, 10 and 20 mM; KH₂PO₄ levels: 20, 50 and 150 μM). The algae grown under medium nitrogen concentration (10 mM) and high phosphate concentration (150 μM) reached the highest biomass (1223.5 ± 152.5 mg/L). Both nitrogen and phosphate had a significant influence on the FAME yield (P < 0.05 and P < 0.0001, respectively). The FAME yield from algae grown under low nitrogen (3 mM) and phosphate concentration (20 μM) increased throughout the experiment and the highest FAME yield (42.2 ± 2.5% of AFDW) as well as C16 and C18 content (95.8 ± 1.6% of AFDW) was achieved under these conditions. Algae grown under medium nitrogen concentration (10 mM) and low phosphate concentration (20 μM) had the highest FAME productivity (426.0 mg/L ± 135.0 mg/L). Thus, the lower nitrogen concentration (3 mM–10 mM) and low phosphate concentration (20 μM) would be an optimal combination tested to produce the most FAME from S. rubescens-like algae.     

Purification and characterization of Plasmodium yoelii adenosine deaminase

Plasmodium lacks the de novo pathway for purine biosynthesis and relies exclusively on the salvage pathway. Adenosine deaminase (ADA), first enzyme of the pathway, was purified and characterized from Plasmodium yoelii, a rodent malarial species, using ion exchange and gel exclusion chromatography. The purified enzyme is a 41 kDa monomer. The enzyme showed K_m values of 41 μM and 34 μM for adenosine and 2′-deoxyadenosine, respectively. Erythro-9-(2-hydroxy-3-nonyl) adenine competitively inhibited P. yoelii ADA with K_i value of 0.5 μM. The enzyme was inhibited by DEPC and protein denaturing agents, urea and GdmCl. Purine analogues significantly inhibited ADA activity. Inhibition by p-chloromercuribenzoate (pCMB) and N-ethylmaleimide (NEM) indicated the presence of functional –SH groups. Tryptophan fluorescence maxima of ADA shifted from 339 nm to 357 nm in presence of GdmCl. Refolding studies showed that higher GdmCl concentration irreversibly denatured the purified ADA. Fluorescence quenchers (KI and acrylamide) quenched the ADA fluorescence intensity to the varied degree. The observed differences in kinetic properties of P. yoelii ADA as compared to the erythrocyte enzyme may facilitate in designing specific inhibitors against ADA.     

N,N′-bis-(2,3-Methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea enhance adventitious rooting in Pinus radiata and affect expression of genes induced during adventitious rooting in the presence of exogenous auxin

We have analyzed the effect of N,N′-bis-(2,3-methylenedioxyphenyl)urea (2,3-MDPU) and N,N′-bis-(3,4-methylenedioxyphenyl)urea (3,4-MDPU), two symmetrically substituted diphenylurea derivatives with no auxin or cytokinin-like activity, on the rooting capacity of Pinus radiata stem cuttings. Results indicate that both diphenylurea derivatives enhance adventitious rooting in the presence of exogenous auxin (indole-3-butyric acid, IBA), even at low auxin concentration, in rooting-competent cuttings, but have no effect on the adventitious rooting of low or null competent-to-root cuttings. Histological analyses show that, in the simultaneous presence of MDPUs and low concentration of exogenous auxin, adventitious root formation is induced in the cell types that retain intrinsic competence to form adventitious roots in response to auxin. The time course of cellular events leading to root formation and the time of root emergence are closely similar to that observed in cuttings treated only with higher auxin concentration. In addition, the mRNA level of a P. radiata SCARECROW-LIKE gene, which is significantly induced in the presence of the optimal concentration (10 μM) of exogenous auxin needed for cuttings to root, is increased in the presence of MDPUs and low concentration of exogenous auxin (1 μM). The expression of a P. radiata SHORT-ROOT gene in rooting-competent cuttings during adventitious rooting is also affected by the presence of MDPUs when combined with auxin. As MDPUs do not affect the expression of either gene in the absence of exogenous auxin, but only in its presence, we suggest that MDPUs could interact, directly or indirectly, with the auxin-signalling pathways in rooting-competent cuttings during adventitious rooting.