Salacia campestris root bark extract: peroxidase inhibition, antioxidant and antiradical profile

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

ISOENZYMATIC POLYMORPHISM and ACTIVITY of PEROXIDASES of COMMON BEAN(Phaseolus vulgaris L.) UNDER SALINE STRESS

Uma das utilizações da técnica de cultura de tecidos para o melhoramento vegetal é a identificação de linhas de células que apresentem tolerância ao estresse salino. Para se estudar os mecanismos bioquímicos envolvidos na expressão genética da tolerância a salinidade, calos oriundos de eixos embrionários de quatro cultivares de feijão (Phaseolus vulgaris L.; cultivares IAC - carioca, IAPAR 14, JALO-EEP 558, BAT - 93), foram cultivados em meio sólido Murashige & Skoog (1962), suplementado com NaCl nas concentrações de 0, 20, 40, 60 e 80 mM. Após 14 dias de incubação, os calos foram coletados e analisados quanto aos padrões isoenzimáticos e de atividade das peroxidases. Os cultivares BAT e IAPAR apresentaram duas zonas de atividade em comum na região anódica e apenas uma zona enzimática específica a cada um deles (migração mais rápida).Possivelmente as duas zonas anódicas intermediárias sejam produtos do mesmo loco enzimático, porém com alelos diferentes, consequentemente diferentes mobilidades eletroforéticas. O cv. JALO apresentou duas zonas anódicas de atividade em comum com os cultivares IAC e IAPAR com uma zona anódica exclusiva de migração mais lenta, a qual apresentou atividade mais intensa de todos os cultivares analisados. Este cultivar revelou ainda uma zona catódica provavelmente dimérica e heterozigota nos indivíduos de todos os tratamentos aplicados. Provavelmente, esta é a mesma zona que ocorre em homozigose com fixação do alelo lento para os indivíduos de todos os tratamentos efetuados nos cultivares BAT e IAPAR. O cv. IAC apresentou duas bandas anódicas em comum com os cv. IAPAR e JALO. Apresentou também a banda anódica mais rápida em comum com o cv. IAPAR e uma banda anódica exclusiva de migração mais lenta. Curiosamente, os indivíduos deste cv. mantidos em meio suplementado com 20 mM de NaCl não apresentaram atividade nas três zonas anódicas mais lentas. Ocorreu no cv. IAC uma única zona de atividade catódica, dimérica e heterozigota para os indivíduos provenientes de todos os tratamentos, composta provavelmente de dois alelos diferentes da zona correspondente ao cv. JALO. Amostras provenientes dos tratamentos 40 e 60 mM de NaCl, desta zona catódica, apresentaram maior atividade enzimática. A análise da atividade da peroxidase no extrato bruto, revelou que os cultivares responderam diferentemente ao aumento da concentração salina no meio de cultura, com aumento pronunciado dessa atividade nos cultivares IAC e JALO.

Alterações na atividade da peroxidase e do conteúdo de carboidratos em mandioca cultivada in vitro sob estresse salino

A influência de estresse induzido por cloreto de sódio (75 e 150 mM) sobre o conteúdo de carboidratos solúveis e atividade da peroxidase, foi estudada em plântulas de mandioca cultivadas in vitro. Os resultados mostraram que a atividade da peroxidase diminuiu gradualmente durante o crescimento de plântulas em todos os tratamentos. O conteúdo de açúcares redutores foi menor em plântulas submetidas a 75 mM de NaCl, nas fases mais adiantadas do desenvolvimento, em comparação com a dose mais elevada do sal (150 mM de NaCl) ou sua omissão. Os resultados obtidos indicaram que o NaCl alterou o metabolismo de carboidratos, atividade da peroxidase e o crescimento de plântulas cultivadas in vitro.

POLIAMINAS E ATIVIDADE da PEROXIDASE em FEIJÃO (Phaseolus vulgaris L.) CULTIVADO SOB ESTRESSE SALINO

O teor de poliaminas (putrescina, espermidina e espermina) e a atividade enzimática da peroxidase (EC 1.11.1.7) foram determinados em plantas de Phaseolus vulgaris L. cv Carioquinha, após terem sido submetidas a estresse salino (50 e 100 mM de NaCl). Foram observadas alterações nos teores das poliaminas, principalmente putrescina, que aumentou com o tempo e a concentração de NaCl. Também ocorreu aumento na atividade da peroxidase em ambas concentrações de NaCl utilizadas. Os resultados mostraram alterações no metabolismo de poliaminas e peroxidases nas plantas de feijão cultivadas em meio salino.

Cádmio e a atividade de peroxidase durante a germinação de sementes de feijoeiro

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detecção do gene de peroxidase em sementes de soja pela reação da polimerase em cadeia (PCR)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bromophenol blue discoloration using peroxidase immobilized on highly activated corncob powder

The aim of the present study was to evaluate the efficacy of peroxidase immobilized on corncob powder for the discoloration of dye. Peroxidase was extracted from soybean seed coat, followed by amination of the surface of the tertiary structure. The aminated peroxidase was immobilized on highly activated corncob powder and employed for the discoloration of bromophenol blue. Amination was performed with 10 or 50 mmol.L-1carbodiimide and 1 mol.L-1ethylenediamine. The amount of protein in the extract was 0.235 ± 0.011 mg.mL-1and specific peroxidase activity was 86.06 ± 1.52 µmol min-1.mg-1, using 1 mmol.L-1ABTS as substrate. Ten mmol.L-1and 50 mmol.L-1 aminated peroxidase retained 88 and 100% of the initial activity. Following covalent immobilization on a corncob powder-glyoxyl support, 10 and 50 mmol.L-1aminated peroxidase retained 74 and 86% of activity, respectively. Derivatives were used for the discoloration of 0.02 mmol.L-1bromophenol blue solution. After 30 min, 93 and 89% discoloration was achieved with the 10 mmol.L-1and 50 mmol.L-1derivatives, respectively. Moreover, these derivatives retained 60% of the catalytic properties when used three times. Peroxidase extracted from soybean seed coat immobilized on a low-cost corncob powder support exhibited improved thermal stability. Keywords: Peroxidases. Multipoint immobilization of enzymes. Aminated enzymes. Corncob powder. RESUMO Descoloração de azul de bromofenol utilizando peroxidase imobilizada em pó de sabugo de milho altamente ativado Nesta pesquisa a enzima peroxidase foi extraída do tegumento de sementes de soja, e a superfície da estrutura terciária foi aminada. A peroxidase aminada foi imobilizada em suporte pó de sabugo de milho altamente ativado e utilizado na descoloração de azul de bromofenol. A aminação da peroxidase foi realizada com carbodiimida em concentrações de 10 e 50 mmol.L-1, e 1 mol.L-1de etilenodiamina. A quantidade de proteínas no extrato foi de 0,235 ± 0,011 mg.mL-1, e a atividade específica da peroxidase foi 86,06 ± 1,52 µmol min-1.mg-1, usando 1 mmol.L-1de ABTS como substrato. A peroxidase aminada a 10 mmol.L-1reteve 88% e a aminada a 50 mmol.L-1reteve 100% da atividade inicial. As peroxidases aminadas a 10 ou 50 mmol.L-1foram covalentemente imobilizadas em suporte glioxil-pó de sabugo de milho com atividade recuperada de 74% e 86%, respectivamente. Os derivados obtidos foram utilizados na descoloração de solução de azul de bromofenol 0,02 mmol.L-1. Após 30 min 93% de descoloração foram alcançados com o derivado glioxil-pó de sabugo de milho com a peroxidase aminada 10 mmol.L-1e 89% com a aminada 50 mmol.L-1. Estes derivados mantiveram 60% das propriedades catalíticas, quando utilizado por três vezes. A peroxidase extraída do tegumento da semente de soja imobilizada em suporte de baixo custo pó de sabugo de milho apresentou melhoria na estabilidade térmica da enzima. Palavras-chave: Peroxidases. Imobilização multipontual de enzimas. Aminação de enzimas. Pó de sabugo de milho.

Characterization of peroxidase changes in tolerant and susceptible soybeans challenged by soybean aphid (Hemiptera: Aphididae)

Changes in protein content, peroxidase activity, and isozyme profiles in response to soybean aphid feeding were documented at V1 (fully developed leaves at unifoliate node, first trifoliate leaf unrolled) and V3 (fully developed leaf at second trifoliate node, third trifoliate leaf unrolled) stages of soybean aphid-tolerant (KS4202) and -susceptible (SD76R) soybeans. Protein content was similar between infested and control V1 and V3 stage plants for both KS4202 and SD76R at 6, 16, and 22 d after aphid introduction. Enzyme kinetics studies documented that control and aphid-infested KS4202 V1 stage and SD76R V1 and V3 stages had similar levels of peroxidase activity at the three time points evaluated. In contrast, KS4202 aphid-infested plants at the V3 stage had significantly higher peroxidase activity levels than control plants at 6 and 22 d after aphid introduction. The differences in peroxidase activity observed between infested and control V3 stage KS4202 plants at these two time points suggest that peroxidases may be playing multiple roles in the tolerant plant. Native gels stained for peroxidase were able to detect differences in the isozyme profiles of aphid-infested and control plants for both KS4202 and SD76R.

Bromofhenol blue discoloration using peroxidase immobilized on highly activated corncob powder

The aim of the present study was to evaluate the efficacy of peroxidase immobilized on corncob powder for the discoloration of dye. Peroxidase was extracted from soybean seed coat, followed by amination of the surface of the tertiary structure. The aminated peroxidase was immobilized on highly activated corncob powder and employed for the discoloration of bromophenol blue. Amination was performed with 10 or 50 mmol.L-1 carbodiimide and 1 mol.L-1 ethylenediamine. The amount of protein in the extract was 0.235 ± 0.011 mg.mL-1 and specific peroxidase activity was 86.06 ± 1.52 µmol min-1 . mg-1, using 1 mmol.L-1 ABTS as substrate. Ten mmol.L-1 and 50 mmol.L-1 aminated peroxidase retained 88 and 100% of the initial activity. Following covalent immobilization on a corncob powder-glyoxyl support, 10 and 50 mmol.L-1 aminated peroxidase retained 74 and 86% of activity, respectively. Derivatives were used for the discoloration of 0.02 mmol.L-1 bromophenol blue solution. After 30 min, 93 and 89% discoloration was achieved with the 10 mmol.L-1 and 50 mmol.L-1 derivatives, respectively. Moreover, these derivatives retained 60% of the catalytic properties when used three times. Peroxidase extracted from soybean seed coat immobilized on a low-cost corncob powder support exhibited improved thermal stability.

Transcription and activity of antioxidant enzymes after ionizing irradiation in radiation-resistant and radiation-sensitive mice

The involvement of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase in radiobiological processes has been described at the enzyme activity level. We irradiated radiation-resistant (RR) and radiation-sensitive (RS) mice and studied antioxidant enzymes at the transcriptional and activity level. In addition, aromatic hydroxylation and lipid peroxidation parameters were determined to study radiation resistance at the oxidation level. RS BALB/c/J Him mice and RR C3H He/Him mice were whole-body-irradiated with x-rays at 2, 4, and 6 Gy and killed 5, 15, and 30 min after irradiation. mRNA was isolated from liver and hybridized with probes for antioxidant enzymes and β-actin as a housekeeping gene control. Antioxidant enzyme activities were determined by standard assays. Parameters for aromatic hydroxylation (o-tyrosine) and lipid peroxidation (malondialdehyde) were determined by HPLC methods. Antioxidant transcription was unchanged in contrast to antioxidant activities; SOD and CAT activities were elevated within 15 min in RR animals but not in RS mice, at all doses studied. Glutathione peroxidase activity was not different between RR and RS mice and was only moderately elevated after irradiation. No significant differences were found between RR and RS animals at the oxidation level, although a radiation dose-dependent increase of oxidation products was detected in both groups. We found that ionizing irradiation led to increased antioxidant activity only minutes after irradiation in the absence of increased transcription of these antioxidant enzymes. RR animals show higher antioxidant enzyme activities than do RS mice, but oxidation products are comparable in RS and RR mice. As unchanged transcription of antioxidant enzymes could not have been responsible for the increased antioxidant enzyme activities, preformed antioxidant enzymes should have been released by the irradiation process. This would be in agreement with previous studies of preformed, stored SOD. The finding of higher SOD and CAT activities in RR than in RS animals could point to a role for these antioxidant enzymes for the process of radiation sensitivity.

Identification of haptoglobin as a natural inhibitor of trypanocidal activity in human serum.

Trypanosomes are protozoan parasites of medical and veterinary importance. Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense infect humans, causing African sleeping sickness. However, Trypanosoma brucei brucei can only infect animals, causing the disease Nagana in cattle. Man is protected from this subspecies of trypanosomes by a toxic subtype of high density lipoproteins (HDLs) called the trypanosome lytic factor (TLF). The toxic molecule in TLF is believed to be the haptoglobin-related protein that when bound to hemoglobin kills the trypanosome via oxidative damage initiated by its peroxidase activity. The amount of lytic activity in serum varies widely between different individuals with up to a 60-fold difference in activity. In addition, an increase in the total amount of lytic activity occurs during the purification of TLF, suggesting that an inhibitor of TLF (ITLF) exists in human serum. We now show that the individual variation in trypanosome lytic activity in serum correlates to variations in the amount of ITLF. Immunoblots of ITLF probed with antiserum against haptoglobin recognize a 120-kDa protein, indicating that haptoglobin is present in partially purified ITLF. Haptoglobin involvement is further shown in that it inhibits TLF in a manner similar to ITLF. Using an anti-haptoglobin column to remove haptoglobin from ITLF, we show that the loss of haptoglobin coincides with the loss of inhibitor activity. Addition of purified haptoglobin restores inhibitor activity. This indicates that haptoglobin is the molecule responsible for inhibition and therefore causing the individual variation in serum lytic activity.

Functional expression and characterization of Eehinococcus granulosus thioredoxin peroxidase suggests a role in protection against oxidative damage

A full-length cDNA sequence coding for Echinococcus granulosus thioredoxin peroxidase (EgTPx) was isolated from a sheep strain protoscolex cDNA library by immunoscreening using a pool of sera from mice infected with oncospheres. EgTPx expressed as a fusion protein with glutathione S-transferase (GST) exhibited significant thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Furthermore, the suggested antioxidant role for EgTPx was reinforced in an in vivo assay, whereby its expression in BL21 bacterial cells markedly increased the tolerance and survival of the cells to high concentrations of H2O2 compared with controls. Immunolocalization studies revealed that EgTPx was specifically expressed in all tissues of the protoscolex and brood capsules. Higher intensity of labelling was detected in many, but not all, calcareous corpuscle cells in protoscoleces. The purified recombinant EgTPx protein was used to screen sera from heavily infected mice and patients with confirmed hydatid infection. Only a portion of the sera reacted positively with the EgTPx-GST fusion protein in Western blots, suggesting that EgTPx may form antibody-antigen complexes or that responses to the EgTPx antigen may be immunologically regulated. Recombinant EgTPx may prove useful for the screening of specific inhibitors that could serve as new drugs for treatment of hydatid disease. Moreover, given that TPx from different parasitic phyla were phylogenetically distant from host TPx molecules, the development of antiparasite TPx inhibitors that do not react with host TPx might be feasible. (C) 2003 Elsevier B.V. All rights reserved.

Breastmilk-saliva interactions boost innate immunity by regulating the oral microbiome in early infancy

Introduction Xanthine oxidase (XO) is distributed in mammals largely in the liver and small intestine, but also is highly active in milk where it generates hydrogen peroxide (H2O2). Adult human saliva is low in hypoxanthine and xanthine, the substrates of XO, and high in the lactoperoxidase substrate thiocyanate, but saliva of neonates has not been examined. Results Median concentrations of hypoxanthine and xanthine in neonatal saliva (27 and 19 μM respectively) were ten-fold higher than in adult saliva (2.1 and 1.7 μM). Fresh breastmilk contained 27.3±12.2 μM H2O2 but mixing baby saliva with breastmilk additionally generated >40 μM H2O2, sufficient to inhibit growth of the opportunistic pathogens Staphylococcus aureus and Salmonella spp. Oral peroxidase activity in neonatal saliva was variable but low (median 7 U/L, range 2–449) compared to adults (620 U/L, 48–1348), while peroxidase substrate thiocyanate in neonatal saliva was surprisingly high. Baby but not adult saliva also contained nucleosides and nucleobases that encouraged growth of the commensal bacteria Lactobacillus, but inhibited opportunistic pathogens; these nucleosides/bases may also promote growth of immature gut cells. Transition from neonatal to adult saliva pattern occurred during the weaning period. A survey of saliva from domesticated mammals revealed wide variation in nucleoside/base patterns. Discussion and Conclusion During breast-feeding, baby saliva reacts with breastmilk to produce reactive oxygen species, while simultaneously providing growth-promoting nucleotide precursors. Milk thus plays more than a simply nutritional role in mammals, interacting with infant saliva to produce a potent combination of stimulatory and inhibitory metabolites that regulate early oral–and hence gut–microbiota. Consequently, milk-saliva mixing appears to represent unique biochemical synergism which boosts early innate immunity.

Responses of Scots pine to nickel and copper exposure and herbivory

The goal of this thesis was to examine the ecophysiological responses of Scots pine (Pinus sylvestris L.), with an emphasis on the oxidative enzyme peroxidase and plant phenolics to environmental stresses like elevated levels of nickel (Ni) and copper (Cu), and herbivory. The effects of Ni and Cu were studied in a gradient survey at a sulphur dioxide contaminated site in the Kola Peninsula, and with experiments in which seedlings were exposed to Ni mist or to Ni and Cu amended into the soil. In addition, experimental Ni exposure was combined with disturbance of the natural lichen cover of the forest ground layer. Pine sawfly attack was simulated in the early season defoliation experiment, in which mature Scots pine were defoliated (100 %) during two successive years in a dry, nutrient-poor Scots pine stand. In addition, the effect of previous defoliation on the growth of sawfly (Diprion pini L.) larvae was studied. Apoplastic peroxidase activity was elevated in the needles of pine in a Ni- , Cu- and SO2- polluted environment, which indicated an increased oxidative stress. Increased foliar peroxidase activity due to Ni contamination was shown in the experiment, in which Ni was added as mist. No such response was found in peroxidase acitivity of the roots exposed to elevated Ni and/or Cu in the soil. Elevated Ni in the soil increased the concentration of foliar condensed tannins, which are able to bind heavy metals in the cells. Addition of low levels of Ni in the soil appeared to benefit pine seedlings, which was seen as promoted shoot growth and better condition of the roots. Wet Ni deposition of 2000 mg m-2 reduced growth and survival of pine seedlings, whereas deposition levels 200 mg m-2 or 20 mg m-2 caused no effects in a 2-y lasting experiment. The lichen mat on the forest floor did not act as an effective buffer against the adverse impacts of heavy metals on pine seedlings. However, some evidence was found indicating that soil microbes profited from the lichen mat. Artificial defoliation increased peroxidase activity in the Scots pine needles. In addition, defoliation decreased nitrogen, diamine putrescine and glucose concentrations in the needles and increased the concentrations of several phenolic compounds, starch and sucrose. Previous artificial defoliation led to poor growth of sawfly larvae reared on the pines, suggesting delayed induced resistance in Scots pine. However, there was no consistent relationship between inducibility (proportional increase in a compound following defoliation) and adverse effects on the growth of pine sawfly larvae. The observed inducible responses in needle phenolics due to previous defoliation thus appear to represent non-specific responses against sawflies.

Lignin biosynthesis in Norway spruce: from a model system to the tree

Lignin is a hydrophobic polymer that is synthesised in the secondary cell walls of all vascular plants. It enables water conduction through the stem, supports the upright growth habit and protects against invading pathogens. In addition, lignin hinders the utilisation of the cellulosic cell walls of plants in pulp and paper industry and as forage. Lignin precursors are synthesised in the cytoplasm through the phenylpropanoid pathway, transported into the cell wall and oxidised by peroxidases or laccases to phenoxy radicals that couple to form the lignin polymer. This study was conducted to characterise the lignin biosynthetic pathway in Norway spruce (Picea abies (L.) Karst.). We focused on the less well-known polymerisation stage, to identify the enzymes and the regulatory mechanisms that are involved. Available data for lignin biosynthesis in gymnosperms is scarce and, for example, the latest improvements in precursor biosynthesis have only been verified in herbaceous plants. Therefore, we also wanted to study in detail the roles of individual gene family members during developmental and stress-induced lignification, using EST sequencing and real-time RT-PCR. We used, as a model, a Norway spruce tissue culture line that produces extracellular lignin into the culture medium, and showed that lignin polymerisation in the tissue culture depends on peroxidase activity. We identified in the culture medium a significant NADH oxidase activity that could generate H2O2 for peroxidases. Two basic culture medium peroxidases were shown to have high affinity to coniferyl alcohol. Conservation of the putative substrate-binding amino acids was observed when the spruce peroxidase sequences were compared with other peroxidases with high affinity to coniferyl alcohol. We also used different peroxidase fractions to produce synthetic in vitro lignins from coniferyl alcohol; however, the linkage pattern of the suspension culture lignin could not be reproduced in vitro with the purified peroxidases, nor with the full complement of culture medium proteins. This emphasised the importance of the precursor radical concentration in the reaction zone, which is controlled by the cells through the secretion of both the lignin precursors and the oxidative enzymes to the apoplast. In addition, we identified basic peroxidases that were reversibly bound to the lignin precipitate. They could be involved, for example, in the oxidation of polymeric lignin, which is required for polymer growth. The dibenzodioxocin substructure was used as a marker for polymer oxidation in the in vitro polymerisation studies, as it is a typical substructure in wood lignin and in the suspension culture lignin. Using immunolocalisation, we found the structure mainly in the S2+S3 layers of the secondary cell walls of Norway spruce tracheids. The structure was primarily formed during the late phases of lignification. Contrary to the earlier assumptions, it appears to be a terminal structure in the lignin macromolecule. Most lignin biosynthetic enzymes are encoded for by several genes, all of which may not participate in lignin biosynthesis. In order to identify the gene family members that are responsible for developmental lignification, ESTs were sequenced from the lignin-forming tissue culture and developing xylem of spruce. Expression of the identified lignin biosynthetic genes was studied using real-time RT-PCR. Candidate genes for developmental lignification were identified by a coordinated, high expression of certain genes within the gene families in all lignin-forming tissues. However, such coordinated expression was not found for peroxidase genes. We also studied stress-induced lignification either during compression wood formation by bending the stems or after Heterobasidion annosum infection. Based on gene expression profiles, stress-induced monolignol biosynthesis appeared similar to the developmental process, and only single PAL and C3H genes were specifically up-regulated by stress. On the contrary, the up-regulated peroxidase genes differed between developmental and stress-induced lignification, indicating specific responses.