Changes in the location of polyphenol oxidase in potato (Solanum tuberosum L.) tuber during cell death in response to impact injury: comparison with wound tissue

In order to elucidate the nature of the response of potato to impact injury at the biochemical level, changes in the location of the enzyme responsible for the discoloration, polyphenol oxidase, were determined using immunogold location with an antibody specific for potato tuber polyphenol oxidase. Tissue printing revealed that the enzyme was distributed throughout the tuber. Following impact injury, both tissue printing and quantitative electron microscopy indicated that there was no increase in the level of the enzyme although there was subcellular redistribution of polyphenol oxidase. This redistribution was first apparent at 12 h after impact, as determined by the use of confocal immunolocation, and coincided with loss of membrane integrity. These changes were examined in parallel with a number of stress-related parameters in both impact and wound responses. Wounding was accompanied by active gene expression and protein synthesis, leading to metabolic activity and tissue repair. In contrast, the bruising response was characterised by a limited active response and vital-staining methods indicated that after 16 h the tissue undergoes cell death. Received: 4 June 1998 / Accepted: 18 September 1998     

高粱雄性不育与可育花药同工酶及游离组蛋白的比较研究

本文研究了高粱细胞质雄性不育花药、可育花药不同发育时期的COD、PPO、MDH及游离组蛋白变化特征,结果表明,在花粉母细胞减数分裂期,COD、PPO未呈现差异,但到了小孢子单核期,不育花药与可育花药间COD、PPO出现明显差异,并且这种差异一直保持至花粉粒双核—三核期。COD、PPO的变化时期与花粉败育的关键时期(小孢子单核期)相一致。不育花药与可育花药的MDH两者相同,但游离组蛋白在花药发育的不同时期均呈现明显差异。本文作者将不育花药、可育花药的COD、PPO及游离组蛋白中出现的差异归因于不育花药中的细胞质不育基因对核基因表达的调控作用。     

Tissue localization of polyphenol oxidase inSorghum

Summary Plastidic polyphenol oxidase (PPO) was localized in various plastid types ofSorghum bicolor (L.) Moench using cytochemical and biochemical franctionation techniques. PPO was found to be present in the mesophyll plastids yet absent from the bundle sheath and guard cell plastids. Mechanical fractionation of mesophyll and bundle sheath plastids, with subsequent electrophoretic or spectrophotometric assay of the preparations, also indicated that PPO was absent from the bundle sheath but present in the mesophyll fraction. A developmental study revealed that, although all leaf plastids near the basal meristem were ultrastructurally similar, the mesophyll and bundle sheath plastids were already differentiated with respect to PPO activity.     

Oleuropein, a non-toxic olive iridoid, is an anti-tumor agent and cytoskeleton disruptor

Oleuropein, a non-toxic secoiridoid derived from the olive tree, is a powerful antioxidant and anti-angiogenic agent. Here, we show it to be a potent anti-cancer compound, directly disrupting actin filaments in cells and in a cell-free assay. Oleuropein inhibited the proliferation and migration of advanced-grade tumor cell lines in a dose-responsive manner. In a novel tube-disruption assay, Oleuropein irreversibly rounded cancer cells, preventing their replication, motility, and invasiveness; these effects were reversible in normal cells. When administered orally to mice that developed spontaneous tumors, Oleuropein completely regressed tumors in 9-12 days. When tumors were resected prior to complete regression, they lacked cohesiveness and had a crumbly consistency. No viable cells could be recovered from these tumors. These observations elevate Oleuropein from a non-toxic antioxidant into a potent anti-tumor agent with direct effects against tumor cells. Our data may also explain the cancer-protective effects of the olive-rich Mediterranean diet.     

间苯二酚、水杨酸对绿豆下胚轴不定根形成的作用

２０—１００ｍｇＬ（－１）间苯二酚能明显地促进绿豆下胚轴不定根的形成,与２０ｍｇＬ（－１）ＩＢＡ混合处理具加成效应,其作用在于降低生根初期ＩＡＡ氧化酶和多酚氧化酶活性．１０—１００ｍｇＬ（－１）水杨酸抑制下胚轴不定根的形成,随处理浓度的加大,对生根数目、生根范围和根重的抑制作用增加．水杨酸处理后１－３ｄ,能提高ＩＡＡ氧化酶和多酚氧化酶的活性．     

Substrate specificity,de novo synthesis and partial purification of polyphenol oxidase derived from the wood-decay fungus,Coriolus versicolor

Summary Coriolus versicolor, a white-rot Basidiomycete, secretes cellulolytic and ligninolytic enzymes as well as polyphenol oxidase (PPO). Whereas the former degrade wood polymers, the latter can convert diphenols to diquinones and oligomerize syringic acid, a lignin derivative. Certain phenolic compounds can serve as disease-resistance factors controlling the proliferation of wood-decay fungi within host tissues. BecauseC. vesicolor can be batch-cultured, overproduction and enhanced secretion of enzymes of biological and commercial interests are feasible. Reported here are the results of attempts to define the timed appearances of intracellular and extracellular PPO, to assess substrate specificity as well as distinguish synthesis versus activation of intracellular PPO and to partially purify extracellular PPO. These efforts were to provide data enabling cell-free synthesis of PPO, cloning of the gene(s) for the oxidase and the establishment of its subcellular route of secretion. Whereas two protein peaks (6 and 12 days in a 16 day time-course) were observed for dialyzed mycelial homogenates, the homogenates' PPO specific activity rose between 4 and 12 days and then declined. Total extracellular protein content climbed from 6 to 15 days for dialyzed growth medium and the medium's PPO specific activity rose at 4 days post-inoculation and except at 9 days increased linearly to 15 days. When aliquots of dialyzed 12 and 15 day media were added to PPO assay mixtures containing catechol and either syringic or gallic acids, statistically significant differences in PPO specific activity between phenolic substrates were noted. Supplementation of cultures with 1.91 g cycloheximide ml growth medium^–1 (control, growth medium only) together with 0.5 Ci [¹⁴C]-leucine revealed that cycloheximide inhibited PPO activity and suppressed [¹⁴C]-leucine incorporation into TCA-insoluble cytoplasmic protein. As for PPO partial purification, growth medium dialysis followed by 0–30% (NH₄)₂SO₄ fractionation and subsequent 12 000×g dialyzate centrifugation yielded a 3.27-fold enhancement in PPO specific activity within the 12 000×g supernatant. Chromatography of the latter upon DEAE-Sephadex indicated that PPO exchanged with the DEAE counterion as it could be eluted with high ionic strength salt. These results suggest that: the occurrences of intracellular and extracellular PPO are time-dependent, intracellular PPO is de novo synthesized, the preferred substrate for extracellular PPO appears to be catechol and extracellular PPO can be partially purified by a combination of dialysis and ammonium sulfate fractionation as well as possibly DEAE chromatography and/or Sephadex G-150 gel filtration.     

67-kDa Laminin Receptor-dependent Protein Phosphatase 2A (PP2A) Activation Elicits Melanoma-specific Antitumor Activity Overcoming Drug Resistance

The Ras/Raf/MEK/ERK pathway has been identified as a major, druggable regulator of melanoma. Mutational activation of BRAF is the most prevalent genetic alteration in human melanoma, resulting in constitutive melanoma hyperproliferation. A selective BRAF inhibitor showed remarkable clinical activity in patients with mutated BRAF. Unfortunately, most patients acquire resistance to the BRAF inhibitor, highlighting the urgent need for new melanoma treatment strategies. Green tea polyphenol (−)-epigallocatechin-3-O-gallate (EGCG) inhibits cell proliferation independently of BRAF inhibitor sensitivity, suggesting that increased understanding of the anti-melanoma activity of EGCG may provide a novel therapeutic target. Here, by performing functional genetic screening, we identified protein phosphatase 2A (PP2A) as a critical factor in the suppression of melanoma cell proliferation. We demonstrated that tumor-overexpressed 67-kDa laminin receptor (67LR) activates PP2A through adenylate cyclase/cAMP pathway eliciting inhibitions of oncoproteins and activation of tumor suppressor Merlin. Activating 67LR/PP2A pathway leading to melanoma-specific mTOR inhibition shows strong synergy with the BRAF inhibitor PLX4720 in the drug-resistant melanoma. Moreover, SET, a potent inhibitor of PP2A, is overexpressed on malignant melanoma. Silencing of SET enhances 67LR/PP2A signaling. Collectively, activation of 67LR/PP2A signaling may thus be a novel rational strategy for melanoma-specific treatment.     

Characterization of endogenous and recombinant forms of laccase-2, a multicopper oxidase from the tobacco hornworm, Manduca sexta

Laccases belong to the group of multicopper oxidases that exhibit wide substrate specificity for polyphenols and aromatic amines. They are found in plants, fungi, bacteria, and insects. In insects the only known role for laccase is in cuticle sclerotization. However, extracting laccase from the insect's cuticle requires proteolysis, resulting in an enzyme that is missing its amino-terminus. To circumvent this problem, we expressed and purified full-length and amino-terminally truncated recombinant forms of laccase-2 from the tobacco hornworm, Manduca sexta. We also purified the endogenous enzyme from the pharate pupal cuticle and used peptide mass fingerprinting analysis to confirm that it is laccase-2. All three enzymes had pH optima between 5 and 5.5 when using N-acetyldopamine (NADA) or N-β-alanyldopamine-alanyldopamine (NBAD) as substrates. The laccases exhibited typical Michaelis–Menten kinetics when NADA was used as a substrate, with K_m values of 0.46 mM, 0.43 mM, and 0.63 mM, respectively, for the full-length recombinant, truncated recombinant, and cuticular laccases; the apparent k_cat values were 100 min⁻¹, 80 min⁻¹, and 290 min⁻¹. The similarity in activity of the two recombinant laccases suggests that laccase-2 is expressed in an active form rather than as a zymogen, as had been previously proposed. This conclusion is consistent with the detection of activity in untanned pupal wing cuticle using the laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Immunoblot analysis of proteins extracted from both tanned and untanned cuticle detected only a single protein of 84 kDa, consistent with the full-length enzyme. With NBAD as substrate, the full-length recombinant and cuticular laccases showed kinetics indicative of substrate inhibition, with K_m values of 1.9 mM and 0.47 mM, respectively, and apparent k_cat values of 200 min⁻¹ and 180 min⁻¹. These results enhance our understanding of cuticle sclerotization, and may aid in the design of insecticides targeting insect laccases.     

植物多酚的定量分析方法和生态作用研究进展

植物多酚是植物体内最重要的次生代谢物质.由于其特殊的结构特征和生物学活性,植物体中多酚潜在的生态学意义受到广泛关注.本文综述了植物多酚在生态领域的研究进展,并对其未来的研究方向进行了展望和预测;通过分析各种植物多酚的定性定量方法的优缺点,试图为研究植物多酚的非化学专业研究人员提供一些简单通用的分析方法.