Phenylalanine ammonia-lyase-induced freezing tolerance in jack pine (Pinus banksiana) seedlings treated with low, ambient levels of ultraviolet‐B radiation

Phenylalanine ammonia-lyase (PAL) induction in UVB-exposed plants leads to an increased synthesis of UV-absorbing phenols. As phenols, including anthocycanins, are linked to many protective mechanisms in plants, we tested the hypothesis that UVB-induced phenol accumulation, mediated by PAL, may confer freezing tolerance in jack pine ( Pinus banksiana Lamb) seedlings. The hypothesis was tested by applying UVB in the presence and absence of the PAL-inhibitor, 2-aminoindan-2-phosphonic acid (AIP). Jack pine seedlings were grown for 3 weeks with and without 10 µ M aqueous AIP. Each treatment was then divided into two groups. One group received near-ambient UVB (5.5 kJ m⁻² day⁻¹of biologically effective radiation) for up to 30 h. A second, control group of seedlings received no UVB. Anthocyanin concentration declined by > 99% in PAL-inhibited seedlings and other methanol-extractable UV-absorbing phenols declined by > 48%, relative to the controls. A 20-h exposure to UVB increased seedling freezing (−15°C) tolerance in the absence of the PAL-inhibitor, as shown by a 30% reduction in membrane injury, determined by electrolyte leakage measurements. In PAL-inhibited seedlings, by contrast, the same UVB pre-treatment increased freezing injury by 48%. A longer (30 h) UVB exposure was damaging to both AIP-treated and untreated seedlings. Root feeding with 10 µ M AIP during a 3-week exposure of older (6-month-old) seedlings similarly reduced phenol accumulation in UVB-exposed seedlings. The decline in phenol production in PAL-inhibited seedlings correlated with increased freezing injury. These results suggest a role for ambient UVB in seedling frost hardiness, mediated by a PAL-induced production of phenolic compounds.     

Significance of phenols in cadmium and nickel uptake

The effects of 2-aminoindane-2-phosphonic acid (AIP), a potent phenylalanine ammonia-lyase (PAL) inhibitor, on the accumulation of cadmium and nickel in chamomile (Matricaria chamomilla) were examined in this study. In vitro assay of AIP effect showed a 90% reduction in PAL activity. In plants cultured for 7 days in Cd or Ni solutions with AIP, PAL activity was higher in both shoots and roots (in comparison with metals without AIP), and was correlated with changes in free phenylalanine content. Individual amino acids were both positively and negatively affected by AIP, with the accumulation of tyrosine and proline showing increases in some variants. Contents of soluble phenols and flavonoids were not considerably affected, while amounts of coumarin-related compounds, cell wall-bound phenols and phenolic acids were substantially reduced in AIP-treated variants. Lignin accumulation decreased in controls and increased in Cd variants in response to AIP. Shoot Cd content was depleted, but shoot Ni was elevated by AIP. Total root content of Cd and Ni decreased in +AIP variants. AIP also caused more expressive changes in hydrogen peroxide and superoxide content in Cd than in Ni variants. Our results indicate that phenols have important roles in the uptake of Cd and Ni. The present findings are discussed in the context of available data regarding AIP's effect on phenols.     

Variation and inheritance of peroxidase activity and phenol and saccharide content in cacao in relation to susceptibility to black pod disease

The relationship between susceptibility to black pod disease and activity of peroxidases in crude extracts and soluble phenols and saccharides contents was studied in the pod cortex and in the seeds of three cacao clones: SNK413 (lowly susceptible), SNK10 (highly susceptible) and ICS95 (mildly susceptible) and in the F1 (SNK413×SNK10) and the F′1 (SNK10×SNK413) progeny. Phenol content and peroxidase activity in seeds increased as the pods matured; they were not the same in the proximal, middle and distal parts within the same pod at maturity. This variation could be correlated to the stage of development of the seeds. Total soluble saccharides and ketohexoses in the pod cortex did not vary significantly from one clone to another and could not be related to the susceptibility of the pods. Nevertheless, their contents were 2 to 4 times less in the F1 and F′1 progeny. Total soluble phenols, flavanol and hydroxycinnamic derivatives in the pod cortex were higher in the SNK413 clone. Hydroxycinnamic derivatives were not detected in the SNK10 clone and this character was transmitted to the progeny when SNK10 was male (F1 progeny). Phenolic cornpounds decreased in the F1 and F′1 progeny. These results suggest that phenolic compounds and peroxidase activity could be correlated to the susceptibility of cacao clones to black pod disease and that crossing two clones of different susceptibility produces hybrids with lower phenols and saccharides contents which may be responsible for their poor tolerance to black pod disease.     

The impact of Cu treatment on phenolic and polyamine levels in plant material regenerated from embryos obtained in anther culture of carrot.

The influence of copper sulphate on the regeneration of carrot (Daucus carota L.) androgenic embryos and changes in the levels of phenolic substances and polyamines that might be indicative of the response to oxidative stress were investigated. The cultivation on the regeneration medium supplemented with Cu(2+) at the concentrations 1 and 10 microM for 15 weeks resulted in significant dose-dependent inhibition of the growth and organogenic ability of carrot embryos. The total content of phenolic acids (represented by the sum of all soluble and insoluble fractions) in the Cu(2+)-treated carrot cultures did not change in comparison with the control (0.1 microM Cu(2+)). However, the levels of phenolic acids in the individual fractions showed significant differences. The cultivation in the presence of increased Cu(2+) evoked first of all the rise of free chlorogenic and caffeic acids, and the increase in soluble ester-bound ferulic acid. Marked dose-dependent decline in the amount of ferulic acid incorporated into the cell walls of the Cu(2+)-treated carrot cultures was partly compensated by the increase in the content of p-hydroxybenzoic acid. Decline in the total polyamine contents in the carrot tissues cultivated in the presence of increased Cu(2+) concentrations was observed. The most abundant polyamine, both in a free and PCA-soluble conjugated forms, was putrescine, the least abundant was spermine, which occurred in free form only. While the levels of free polyamines slightly decreased in a dose-dependent manner in the Cu(2+)-treated cultures, those of PCA-soluble conjugates markedly rose (enhancement to 135 and 170% in 1 and 10 microM Cu(2+), respectively, compared with the control). The decline in the total polyamine contents was caused mainly by the decline in the levels of PCA-insoluble conjugates. The decrease observed in this fraction was approximately to 70 and 50% in 1 and 10 microM Cu(2+)-treated cultures, respectively, when compared with the control. The role of phenolic acids and polyamines in preventing Cu(2+)stress in the carrot tissues is discussed.     

Identification of phenolic compounds in isolated vacuoles of the medicinal plant Catharanthus roseus and their interaction with vacuolar class III peroxidase: an H₂O₂ affair?

Class III peroxidases (Prxs) are plant enzymes capable of using H(2)O(2) to oxidize a range of plant secondary metabolites, notably phenolic compounds. These enzymes are localized in the cell wall or in the vacuole, which is a target for secondary metabolite accumulation, but very little is known about the function of vacuolar Prxs. Here, the physiological role of the main leaf vacuolar Prx of the medicinal plant Catharanthus roseus, CrPrx1, was further investigated namely by studying its capacity to oxidize co-localized phenolic substrates at the expense of H(2)O(2). LC-PAD-MS analysis of the phenols from isolated leaf vacuoles detected the presence of three caffeoylquinic acids and four flavonoids in this organelle. These phenols or similar compounds were shown to be good CrPrx1 substrates, and the CrPrx1-mediated oxidation of 5-O-caffeoylquinic acid was shown to form a co-operative regenerating cycle with ascorbic acid. Interestingly, more than 90% of total leaf Prx activity was localized in the vacuoles, associated to discrete spots of the tonoplast. Prx activity inside the vacuoles was estimated to be 1809 nkat ml(-1), which, together with the determined concentrations for the putative vacuolar phenolic substrates, indicate a very high H(2)O(2) scavenging capacity, up to 9 mM s(-1). Accordingly, high light conditions, known to increase H(2)O(2) production, induced both phenols and Prx levels. Therefore, it is proposed that the vacuolar couple Prx/secondary metabolites represent an important sink/buffer of H(2)O(2) in green plant cells.     

UV-B对不同发育时期离体蓝莓主要果实品质及相关酶活性的影响

该研究以幼果期、白果期、转色期的离体‘北陆’蓝莓果实为试材,设置0(CK)、5、10、15min紫外光辐照处理,24h后取样分析蓝莓果实中可溶性糖、总酚、类黄酮和花青苷含量,以及苯丙氨酸裂解酶(PAL)和查尔酮异构酶(CHI)活性的变化,探究UV-B紫外照射处理对不同发育时期蓝莓主要果实品质及相关酶活的影响。结果显示:(1)对于幼果期蓝莓,5min UV-B处理可显著增加果实内可溶性糖含量;10min UV-B处理果实PAL活性增加效果最为显著;15min UV-B处理对果实总酚和花青苷积累的促进作用最大,但显著降低了类黄酮含量和CHI活性。(2)对于白果期蓝莓,5min UV-B处理显著增加了果实类黄酮含量和CHI活性,10min处理使果实可溶性糖和总酚含量较对照分别增加25%和18%;15min处理对果实花青苷含量和PAL活性影响作用最大。(3)对于转色期蓝莓,各处理除果实可溶性糖及类黄酮含量降低外,其余物质含量均显著增加。(4)UV-B处理并未改变果实发育过程中可溶性糖、总酚、类黄酮和花青苷含量及PAL、CHI酶活性的积累规律。(5)蓝莓果内PAL活性与其可溶性糖、总酚和类黄酮的积累呈极显著正相关关系,而CHI活性仅与其可溶性糖呈极显著正相关。研究表明,UV-B辐照处理促进了幼果期和白果期可溶性糖的积累,也能促进不同发育时期蓝莓果实总酚和花青苷及白果期类黄酮的积累,对蓝莓果实主要品质能够产生积极的影响。     

Improving callus regeneration of Miscanthus × giganteus J.M.Greef,Deuter ex Hodk., Renvoize ‘M161’ callus by inhibition of the phenylpropanoid biosynthetic pathway

In previous studies, the regeneration rates of Miscanthus × giganteus J.M.Greef, Deuter ex Hodk., Renvoize from callus tissue cultured on semi-solid media significantly declined after 4 mo of culture, which presents problems with germplasm conservation and use as an alternative propagation system. Due to the species’ lignocellulosic nature, it was hypothesized that the accumulation of phenolic compounds in the callus may be responsible for inhibiting regeneration. The current study aimed to optimize regeneration of M. × giganteus callus by culturing the callus tissue in the presence of 2-aminoindan-2-phosphonic acid (AIP), a competitive inhibitor of phenylalanine ammonia lyase (PAL), to reduce the biosynthesis of phenolics. Embryogenic callus was cultured on media supplemented with 9.0- or 11.3-μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0-, 1-, 10-, 100-, or 1000-μM AIP. Every 28 d for 7 mo, the callus tissue was visually classified based on morphology and regeneration rate. Over the duration of the study, regeneration of shoots was consistently highest in callus cultured on 11.3-μM 2,4-D supplemented with 10- and 100-μM AIP (13–58.3%), and in vitro plantlet development from callus cultured on all concentrations of AIP demonstrated tillering and rooting. Total soluble phenolic content of the callus decreased in a dose-dependent manner from 2242.34-μg g⁻¹ dry weight in the control to 1569.71-μg g⁻¹ dry weight in AIP-treated callus. These data indicate that inhibiting PAL in M. × giganteus cultures increased the percentage of calluses exhibiting regeneration over time.

     

A comparative study of the metal ion uptake and antioxidant enzyme activities of Fusarium equiseti and Fusarium acuminatum as a function of external magnesium concentration

The increase of Mg2+, from 1.3 to 3 microM, in growth medium of F. equiseti and F. acuminatum increased intracellular magnesium levels from 0.83 and 0.81 microM to 1.75 and 1.42 microM on the 12th day, respectively. Intracellular magnesium levels also elevated depending upon the number of incubation days. The maximum manganese levels of F. equiseti and F. acuminatum obtained in 1.6 microM Mg2+ culture medium were 0.67 and 1.23 microM, while maximum iron levels were determined to be 1.3 microM Mg2+ as 0.51 and 0.29 microM, respectively. The maximum intracellular iron and manganese levels were decreased significantly with increasing Mg2+ concentration in the culture medium and were increased depending upon the incubation period. However, intracellular zinc levels of these strains didn't change with Mg2+ concentration and incubation period.The maximum superoxide dismutase (MnSOD) activities of F. equiseti and F. acuminatum, related to increased intracellular manganese levels up to 1.6 microM Mg2+ in growth medium, were determined to be 78 and 110 IU/mg, respectively. CAT activity variations showed agreement with SOD activity and reached a maximum at 320 and 225 IU/mg under the same conditions. The minimum LPO levels of the Fusarium strains with the maximum MnSOD and CAT activities were determined as 1.2 and 0.9 nmol MDA/g., wet weight. The higher LPO level of F. equiseti grown at the same condition, in spite of 1.42-fold higher CAT activity due to the 1.41-fold lower SOD activity, as well as a 2.0-fold higher iron level, indicated increases in the generation of reactive oxygen species via the Fenton reaction.     

Response of phenolic metabolism to the application of carbendazim plus boron in tobacco

In view of the essential role of phenolic compounds in the development of pathogen resistance in plants, and given the influence that fungicides and boron (B) exert over phenolic metabolism, the aim of the present study was to determine the individual effect of the application of a fungicide, as well as to determine the joint effect of the fungicide and B on the metabolism of phenolic compounds in tobacco plants ( Nicotiana tabacum L. cv. Tennessee 86). The fungicide applied was carbendazim (carb), a preventative fungicide, with a purity of 100% at a concentration of 2.6 m M . Boron was applied in the form of H₃BO₃ at: 1.6 m M (B₁), 4 m M (B₂), 8 m M (B₃), 16 m M (B₄), 32 m M (B₅), or 64 m M (B₆). In all, there were eight treatments: one without carb and without B (control), one with only carb, and six combinations of carb with each concentration of B. The results indicated that the foliar application with carb alone led to increases in phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity and a foliar accumulation of phenols. This effect of the carb alone could signify an additional tolerance mechanism to pathogenic infection, given the participation of phenolic compounds in the lignification of plant cell walls. The joint application of carb and B increased both the biosynthesis and the oxidation of the phenolic compounds, especially in carb plus B₃, while the application of carb plus B₅ or carb plus B₆ reduced these processes as well as the foliar biomass.     

主要抗蚜小麦品种(系)的抗性类型及其生化抗性机制

通过对10个抗麦蚜品种(系)室内苗期生命参数、抗性类型和抗蚜次生物质的研究,明确了不同抗性级别的品种对麦蚜种群控制力及部分生化抗蚜机制。实验结果表明,参试的抗蚜品种(系)中30%左右为不选择性：表现为爬行频繁,定殖率低,但是定殖个体生长发育良好;70%为抗生性;表现为使麦长管蚜Sitobion avenae(F.)和禾谷缢管蚜只Rhopalosiphum padi(L.)的发育历期分别延长2.1%～28.2%和3.7%～13.9%,若蚜死亡率增加1.0～3.6倍和1.0～2.25倍,平均寿命缩短10.2%～96.5%和37.5～97,1%,繁殖力下降3.4%～72.8%和25%～97.2%。苗期生化测定结果表明：不同抗源的单宁和总酚含量明显高于感蚜品种,其总酚含量与抗麦长管蚜级别呈显著负相关,以抗生性为主的品种其总酚含量亦与麦长管蚜的内禀增长力(r_m)呈显著负相关(P<0.05),表明总酚是小麦抗长管蚜的关键因子之一,而与禾谷缢管蚜抗性水平无关;单宁含量与麦蚜抗性关系不密切。     

Quantitative structure toxicity relationships for phenols in isolated rat hepatocytes

Quantitative structure toxicity relationship (QSTR) equations were obtained to predict and describe the cytotoxicity of 31 phenols using logLD(50) as a concentration to induce 50% cytotoxicity of isolated rat hepatocytes in 2 h and logP as octanol/water partitioning: logLD(50) (microM)=-0.588(+/-0.059)logP+4.652(+/-0.153) (n=27, r(2)=0.801, s=0.261, P<1 x 10(-9)). Hydroquinone, catechol, 4-nitrophenol, and 2,4-dinitrophenol were outliers for this equation. When the ionization constant pK(a) was considered as a contributing factor a two-parameter QSTR equation was derived: logLD(50) (microM)=-0.595(+/-0.051)logP+0.197(+/-0.029)pK(a)+2.665(+/-0.281) (n=28, r(2)=0.859, s=0.218, P<1 x 10(-6)). Using sigma+, the Brown variation of the Hammet electronic constant, as a contributing parameter, the cytotoxicity of phenols towards hepatocytes were defined by logLD(50) (microM)=-0.594(+/-0.052)logP-0.552(+/-0.085)sigma+ +4.540(+/-0.132) (n=28, r(2)=0.853, s=0.223, P<1 x 10(-6)). Replacing sigma+ with the homolytic bond dissociation energy (BDE) for (X-PhOH+PhO.-->X-PhO.+PhOH) led to logLD(50) (microM)=-0.601(+/-0.066)logP-0.040(+/-0.018)BDE+4.611(+/-0.166) (n=23, r(2)=0.827, s=0.223, P<0.05). Hydroquinone, catechol and 2-nitrophenol were outliers for the above equations. Using redox potential and logP led to a new correlation: logLD(50) (microM)=-0.529(+/-0.135)logP+2.077(+/-0.892)E(p/2)+2.806(+/-0.592) (n=15, r(2)=0.561, s=0.383, P<0.05) with 4-nitrophenol as an outlier. Our findings indicate that phenols with higher lipophilicity, BDE, or sigma+ values or with lower pK(a) and redox potential were more toxic towards hepatocytes. We also showed that a collapse of hepatocyte mitochondrial membrane potential preceded the cytotoxicity of most phenols. Our study indicates that one or a combination of mechanisms; i.e. mitochondrial uncoupling, phenoxy radicals, or phenol metabolism to quinone methides and quinones, contribute to phenol cytotoxicity towards hepatocytes depending on the phenol chemical structure.     

The effects of UV-B radiation on genetic and biochemical changes of Pelargonium graveolens L′Her

Ultraviolet radiation induces biochemical and genetic changes in plants. The aim of this study was to investigate the effects of UV-B radiation on genetic stability, phenolic compounds and antioxidant activity of Pelargonium graveolens L′Her. Plant cuttings were exposed to 0, 0.12. 0.26 and 0.38 W/m2 of UV-B radiation. Results indicated that by increasing the UV-B radiation intensity, total phenols, flavonoids and anthocyanin contents, Phenylalanine ammonia lyase activity and antioxidant capacity were increased. Analysis of four flavonols (quercetin, myricetin, kaempferol and rutin) contents of leaves extract by HPLC indicated that these four flavonols were enhanced in all treated plants and also the ratio of quercetin to kaempferol (Q/K) showed a significant increase (P ≤ 0.05) in UV-B treated plants in compare to control. To evaluate the genetic variation in treated plants, 10 ISSR primers were used. The highest level of percentage of polymorphism (P%), Shannon index (I), number of effective allele (Ne) and Nei’ genetic diversity (He), were observed at the highest UV-B radiation (0.38 W/m2). The AMOVA analysis also showed a significant genetic differentiation (P ≥ 0.001) among the studied groups, and confirmed the differentiation of groups obtained by the cluster analysis of molecular data. Overall, these results showed that biochemical changes in different intensities of UV-B were in line with genetic variations, so that the highest biochemical and genetic variations were observed in 0.38 W/m2 treatment.     

Restoration of secondary metabolism in birch seedlings relieved from PAL-inhibitor

Phenylalanine ammonia lyase (PAL) plays a key role in phenylpropanoid metabolism, catalyzing the deamination of phenylalanine (Phe) to form trans-cinnamic acid. Inhibitors of PAL have been used to study the physiological role of the different compounds derived from trans-cinnamic acid, and to test theories about a trade-off between growth and defence in plants. In a previous study with birch (Betula pubescens Ehrh.) seedlings, the PAL inhibitor 2-aminoindane-2-phosphonic acid monohydrate (AIP) caused an accumulation of Phe and a strong decrease in the quantity of simple phenolics, soluble condensed tannins and growth, whereas flavonol glycosides were generally not affected. The present study demonstrates restoration of secondary metabolism in the previously AIP treated birch seedlings. Our results indicate that Phe accumulated during PAL inhibition could be partly used to increase the content of the phenolic acids, flavan-3-ols and to some extent the soluble condensed tannins. Seedling growth also increased when the supply of PAL inhibitor ceased. We thereby show that the inhibition of PAL by AIP in vivo is reversible, at least for moderate AIP concentrations and the rate of restoration is dependent on the inhibitor concentration.     

A Ca(2+)-dependent cysteine protease is associated with anoxia-induced root tip death in maize

Imposition of anoxia on maize (Zea mays cv. B73) seedlings for 48 h or longer led to the death of the root tip. The necrosis extended into the root axis during postanoxic treatment, leading to the mortality of 30-50% of the seedlings. Using zymography, protease profiles in the root tissues of anoxic seedlings were studied. O2 deprivation for 24 h or longer repressed pre-existing protease activities and induced a novel soluble enzyme in the roots. The anoxia-induced protease (AIP) activity was predominant in the root apex at 24 h of anoxia and, subsequently, became the most abundant soluble activity in the root axis as well. The induction of AIP and its in vitro renaturation were Ca(2+)-dependent. Inhibitor sensitivity studies indicated that AIP is a cysteine protease. In SDS-acrylamide gels, the enzyme activity migrated as a 23.5 kDa polypeptide. The anoxic induction of the activity was repressed by cycloheximide treatment, suggesting that new protein synthesis was required for the AIP appearance. Excision of the root tip (de-tipping) before anoxia led to a superior recovery of seedlings from stress injury. De-tipped seedlings showed lesser root damage and an increased production of lateral roots compared to intact seedlings. Furthermore, the superior anoxia tolerance of de-tipped seedlings was associated with a decreased AIP activity. Thus, the appearance of AIP activity in the root tip at 24 h of anoxia was spatially and temporally associated with the root tissue death. These studies further indicate that the root tip elimination early during anoxia may provide an adaptive advantage.     

A spectrophotometric method for the quantification of an enzyme activity producing 4-substituted phenols: determination of toluene-4-monooxygenase activity

A spectrophotometric method for the quantitative determination of an enzyme activity resulting in the accumulation of 4-substituted phenols is described in this article. Toluene-4-monooxygenase (T4MO) activity in whole cells of Pseudomonas mendocina KR1 is used to demonstrate this method. This spectrophotometric assay is based on the coupling of T4MO activity with tyrosinase activity. The 4-substituted phenol, produced by the action of T4MO on the aromatic ring of a substituted arene, is a substrate for tyrosinase, which converts phenols to o-quinones. The latter react with the nucleophile 3-methyl-2-benzothiazolinone hydrazone (MBTH) to produce intensely colored products that absorb light maximally at different wavelengths, depending on the phenolic substrate used. The incubation of whole cells of P. mendocina KRI with fluorobenzene resulted in the accumulation of 4-fluorophenol. The coupling of T4MO activity with tyrosinase activity in the presence of fluorobenzene resulted in the formation of a colored product absorbing maximally at 480 nm. The molar absorptivity (epsilon) value for the o-quinone-MBTH adduct formed from 4-fluorophenol was determined experimentally to be 12,827 M(-1) cm(-1) with a linear range of quantification between 2.5 and 75 microM. The whole cell assay was run as a continuous indirect assay. The initial rates of T4MO activity toward fluorobenzene, as determined spectrophotometrically, were 61.8+/-4.4 nmol/min/mg P. mendocina KR1 protein (using mushroom tyrosinase), 64.9+/-4.6 nmol/min/mg P. mendocina KR1 protein (using cell extracts Pseudomonas putida F6), and, as determined by HPLC analysis, 62.6+/-1.4 nmol/min/mg P. mendocina KR1 protein.     

Effect of Calcium Ions on Enteropeptidase Catalysis

The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25 degrees C were determined for natural enteropeptidase (K(m) 59.6 microM, k(cat) 6660 min(-1), k(cat)/K(m) 111 microM(-1) x min(-1)), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K(m) 176.9 microM, k(cat) 6694 min(-1), k(cat)/K(m) 37.84 microM(-1) x min(-1)) and trypsin (K(m) 56.0 microM, k(cat) 8280 min(-1), k(cat)/K(m) 147.86 microM(-1) x min(-1)). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD(4)K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca2+. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD(4)K-F(NO(2))G, G(5)DK-F(NO(2))G (where F(NO(2)) is p-nitrophenyl-L-phenylalanine residue), and GD(4)K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.     

Bradykinin and electrical stimulation increase prostaglandin production in the rat vas deferens

The epididymal portion of the rat vas deferens produced prostaglandins (PG) E(2), F(2alpha)and 6-keto F(1alpha). Electrical stimulation (ES, 0.1 Hz, 1 ms) increased such production by 100%, and similar results were obtained in the presence of 1.0 microM bradykinin (Bk). When both stimuli were applied simultaneously, the increases in PG production were 1100% for PGE(2), 800% for PGF(2alpha)and 400% for PG6-keto F(1alpha). Prazosin abolished the effect of ES on PG production. A selective Bk B(2)-receptor antagonist abolished the increase in PG production induced by Bk, both in non-stimulated and in ES tissues. Bk (1.0 microM) elicited contractile responses in non-stimulated as well as in ES tissues, responses that were not modified in the presence of 10 microM indomethacin. In conclusion, the effects of Bk on prostaglandin production appears to depend on the activation of B(2) receptors, while the increase in prostaglandin release induced by ES, and the effects observed with both stimuli simultaneously, should be mediated by the release of noradrenaline and the subsequent activation of alpha(1) adrenoceptors.     

Response of digestive gland cells of freshwater mussel Unio tumidus to phenolic compound exposure in vivo

The exposure of freshwater mussels Unio tumidus to phenolic compounds (tannic, ellagic and gallic acid) in vivo caused changes in proteins and DNA function of digestive gland cells. The mussels were exposed to various concentrations of tested polyphenols (60, 200 and 500 microM) for 24 and 48 h and their antioxidant and pro-oxidant effects were determined. The number of SH-groups was quantified spectrophotometrically using Ellman's reagent. Oxidative modification of proteins increased in the digestive gland cells in a dose- and time-dependent manner. The level of nuclear DNA damage was investigated using the comet assay. The results revealed that polyphenolic acids induce single and double-strand breaks in DNA. The highest changes were observed for tannic and gallic acids and the smallest ones for ellagic acid. 1h of DNA repair process was also studied using the same method. The data obtained in this experiment demonstrate that the most effective DNA repair occurs in the cells exposed to phenolic compounds for 24h. A longer incubation (up to 48 h) does not decrease the capacity of the repair mechanism. The antioxidant activity of the tested phenols was analyzed spectrofluorimetrically using a fluorescence probe DCFH-DA (dichlorofluorescein-diacetate). The experimental data showed that the tested acids can act as antioxidants when used at higher doses (200 and 500 microM) against the reactive oxygen species present in the digestive gland cells. The most effective was ellagic acid, also applied at the smallest dose of 60 microM, in comparison with tannic and gallic acids. In conclusion, our results demonstrate that chosen water-soluble polyphenols, which are located in various plant tissues and are also found in the aquatic environment, can influence organisms living in the water. They can be exposed to these chemicals that cause morphological alterations and changes in certain physiological processes in their organs (i.e. digestive gland cells of bivalve molluscs).     

Effect and analysis of phenolic compounds during somatic embryogenesis induction in Feijoa sellowiana Berg

Summary. The effect of phenolic compounds on somatic embryogenesis in Feijoa sellowiana was analysed. The results showed that caffeic acid (140–560 μM) significantly increased somatic embryogenesis induction compared with the control. The presence of phloridzin, even at lower concentrations (11.5 μM), or caffeic acid or phloroglucinol at concentrations greater than 140.0 and 197.5 μM, respectively, inhibited somatic embryo development beyond the globular stage. When somatic embryos were transferred to the germination medium, the highest rates of germination (81.9%) were obtained with embryos induced in the presence of phloroglucinol (79.0 μM). At all concentrations tested, somatic embryos induced in medium containing phloroglucinol germinated at higher rates than those induced in the presence of caffeic acid. Histological and ultrastructural studies showed that somatic embryos were formed in close association with phenolic-rich cells which, in more advanced stages of development, formed a zone isolating the embryo from the maternal tissue. A comparative analysis of total phenolic content indicated that phenolics reached a peak by the third week of culture, independently of the medium used. However, after that period, the amount of phenolic compounds was significantly higher in explants cultured in the presence of phloroglucinol than in those cultured in the control or in caffeic acid-containing medium. Attempts to identify the type of phenolic compounds showed that flavan-3-ols and gallic acid derivatives were mainly produced in phloroglucinol-containing medium, whereas flavanones and dihydroflavonols were also present in medium containing caffeic acid. Flavones were the main phenols detected in the control. The ways in which phenolic compounds may affect somatic embryogenesis are discussed. Correspondence: J. M. Canhoto, Departamento de Botanica, Faculdade de Ciências e Tecnologia, Universidade de Coimbra, Cal?ada Martim de Freitas, 3001-455 Coimbra, Portugal.