The activity of peroxidase in various cell fractions of wheat plants infected with Septoria nodorum berk.

The activities of peroxidase isoforms and hydrogen peroxide content in leaf cuttings of wheat (Triticum aestivum L., cv. Diamant) resistant to Septoria blotch were studied during aging and following the infection with Septoria nodorum Berk. The differential activation of peroxidase isoforms was regulated by hydrogen peroxide level in the tissue. At early stages of fungus development in plant tissues, the decrease in the activities of soluble, membrane and ion-bound fractions of peroxidase elevated the level of hydrogen peroxide in infected tissues and rapidly activated peroxidase isoforms in infected tissues as compared to the aging ones even before disease symptoms appeared. The anionic peroxidases, which were first to respond to the pathogen, seem to stand for wheat resistance to fungal infections and the protection of leaf tissues from oxidative stress.     

Genotypic variation in cytokinin oxidase from phaseolus callus cultures

Genotypic variation in cytokinin oxidase has been detected in enzyme preparations from Phaseolus vulgaris L. cv Great Northern and Phaseolus lunatus L. cv Kingston callus cultures. Although cytokinin oxidase preparations from Great Northern and Kingston callus tissues appear to have very similar substrate specificities, the cytokinin oxidase activities from the two callus tissues were found to differ in a number of other properties. The cytokinin oxidase from P. vulgaris cv Great Northern callus tissue exhibited a pH optimum of 6.5 (bisTris) and had a strong affinity for the lectin concanavalin A. The cytokinin oxidase from P. lunatus cv Kingston callus tissue exhibited a pH optimum of 8.4 (Taps) and did not bind to concanavalin A. The two enzymes also differed in position of elution when chromatographed on DEAE-cellulose. Both cytokinin oxidase activities exhibited enhanced activity and lower pH optima in the presence of copper-imidazole complexes, but the optimum copper-imidazole ratio and the magnitude of enhancement differed for the two activities. In both callus tissues, transient increases in the supply of exogenous cytokinins induced increases in cytokinin oxidase activity. The differences in pH optima and in glycosylation (as evidenced by the observed difference in lectin affinity) of the cytokinin oxidases from Great Northern and Kingston callus tissues suggest that the compartmentation of cytokinin oxidase may differ in the two callus tissues. The possibility that enzyme compartmentation and isozyme variation in cytokinin oxidase may play a role in the regulation of cytokinin degradation in plant tissues is discussed in relation to known differences in the rates of cytokinin degradation in Great Northern and Kingston callus tissues.     

Participation of the enzymes involved in the biosynthesis of biogenic amines in biochemical interactions between wheat (Triticum aestivum; Poaceae) and bird cherry-oat aphid (Rhopalosiphum padi; Aphididae)

The studies concerned changes in the activities of ornithine decarboxylase (ODC), lysine decarboxylase (LDC) and tyrosine decarboxylase (TyDC) in tissues of wheat (Triticum aestivum L.) infested with bird cherry-oat aphid (Rhopalosiphum padi L.).Obtained results showed that the activities of the enzymes were stimulated in the less susceptible wheat Kontesa cv. infested by the aphids. In the case of the more susceptible Tonacja cv., on most occasions a decrease in the enzyme activities occurred. Such responses were especially clear for TyDC in both analysed cvs., and for LDC and ODC in the case of Kontesa cv. Thus it may be concluded that amino acid decarboxylation plays an important part in the biochemical defence developed in wheat tissues in response to R. padi infestation. The changes in the activities of the decarboxylases were dependent on the wheat genotype as well as the duration of the infestation.     

Glutathione enzymes,glutathione content and t-butyl hydroperoxide induced lipid peroxidation in the gill and digestive gland of the estuarine clam,Rangia cuneata

1. Reduced glutathione (GSH), glutathione reductase (GSSG-reductase) and glutathione peroxidase (GSH-peroxidase) activities were measured in the gill and digestive gland of Rangia cuneata.2. Substantial GSH concentrations were found in both gill (820 ± 80 nmole/g tissue) and digestive gland (930 ± 130 nmole/g tissue). The digestive gland exhibited 2.5-fold greater GSSG-reductase activities and 0.5-fold lower GSH-peroxidase activities relative to the gill.3. In vivo exposure to t-butyl hydroperoxide (BHP) elicited a dose-dependent increase (P < 0.05) in lipid peroxidation in both tissues. Lipid peroxidation occurred earlier and to a greater extent in the digestive gland versus the gill. GSH concentrations in both tissues were unaffected by BHP exposure.4. The study results indicate that gill and digestive gland differ in susceptibility to BHP induced oxidative damage, and the difference is accounted for by differences in tissue GSH metabolism.     

Multifunctional flavonoid dioxygenases: Flavonol and anthocyanin biosynthesis in Arabidopsis thaliana L.

Flavonols and conditionally also anthocyanins, aside from flavonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, flavonol synthase and anthocyanidin synthase, are “multifunctional” enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present final proof of the complementing activities of these synthases for flavonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modification on the biosynthetic pathway and the pattern of flavonoids in different plant tissues are discussed.     

Antioxidative response of Golden Agave leaves with different degrees of variegation under high light exposure

Variegated mutants are spotted rare in wild, and especially in the high light (HL) conditions. In addition, these plants are cultivated by a growing number of horticulturists due to their high economic value. In this experiment, the antioxidative response of both albino and green tissues from a mutant plant Golden Agave, which possesses leaves with different degrees of variegation, was investigated under normal or HL exposure, respectively. Severe oxidative stress was observed in green tissues of high albinism leaves (30?C40?% albino tissues, muy) over low albinism counterparts (less than 10?% albino tissues, mug). Compared with green tissues, low oxidative damage (relative electrolyte leakage and protein damage) and antioxidant enzyme (SOD, CAT, APX, and GR) activities were observed in the albino than in the adjacent green tissues. Photoinactivation of the CAT and the APX enzyme activities were observed in the albino tissues to a significantly level under HL exposure. In addition, lower redox values (ASC/DHA and GSH/GSSG) and higher levels of reactive oxygen species (ROS) were monitored in the green tissues of the muy over mug mutant even under normal light conditions. This work could help us to understand the HL sensitivity of variegated mutant plants better and allow us to improve variegated mutant plant cultivation skills. At last, the ??oxidative stress hypothesis?? was introduced to illustrate the reduced evolutionary advantage of plant variegation phenomenon in the discussion.     

The Biosynthesis of Sucrose and Nucleoside Diphosphate Glucoses in Phaseolus aureus

Sucrose-phosphate synthetase is detectable only in intact chloroplast preparations of Phaseolus aureus. In contrast, sucrose synthetase and uridine diphosphate glucose (UDP-glucose) pyrophosphorylase activities are low in extracts of photosynthetic tissues of P. aureus but are high in extracts of nonphotosynthetic tissues. Activities for ADP-, dTDP-, CDP-, and GDP-glucose pyrophosphorylases are generally higher in extracts of photosynthetic tissues of P. aureus than in extracts of nonphotosynthetic tissues. The high levels of sucrose synthetase and of UDP-glucose pyrophosphorylase found in dark-grown hypocotyls begin to decline about 4 hours after exposure to light at a rate of 50% every 3 hours.     

Changes in ribosephosphate isomerase and ribosephosphate pyrophosphokinase activities in tobacco infected with PVY

Changes in ribosephosphate isomerase and ribosephosphate pyrophosphokinase activities occurring in tobacco leaf tissues infected with the potato virus Y (PVY) were studied at the stage of acute infection. The results obtained have shown that during the entire experimental period the activities of both enzymes were at the end of a dark phase much higher in virus-infected tissues compared with the values found in healthy control plants. The courses of the activity curves of both enzymes were consistent and correlated with the reproduction curve of PVY. The results obtained suggest a direct involvement of both enzymes inde novo biosynthesis of the virus RNAvia the oxidative pentose phosphate pathway.     

Characterization of the enzyme responsible for nopaline and ornaline synthesis in sunflower crown gall tissues

Extracts prepared from sunflower (Helianthus annuus L.) crown gall tissues induced by Agrobacterium tumefaciens strains C58 and T37 (nopaline utilizers) catalyze the synthesis of nopaline and ornaline. These compounds are not synthesized in extracts of crown gall tissues induced by strains B6, 15955 (octopine utilizers), and AT1 (utilizes neither octopine nor nopaline) or in extracts of habituated sunflower callus. Both synthetic activities require NADPH, α-ketoglutarate, and either arginine or ornithine; histidine and lysine will not substitute. Incorporation of arginine or ornithine into product is inhibited by the other substrate but not by histidine or lysine. On the basis of inhibition and K_m data, both activities appear to be catalyzed by one enzyme and the same enzyme is apparently present in crown gall tissues induced by strains C58 and T37.     

The effect of Myrtus communis L. ethanol extract on the small intestine and lungs in experimental thermal burn injury

Thermal trauma can damage organs away from the skin burn site and lead to multiple organ dysfunction. Following thermal injury, all tissues are exposed to ischemia, and as a result, resuscitation and reperfusion occur during the burning shock. Burn damage starts systemic inflammatory reactions that produce toxins and reactive oxygen radicals that lead to peroxidation. This study aimed to investigate, for the first time, the possible antioxidant effects of Myrtus communis ethanol extract on burn-induced oxidative distant organ injury orally. The thermal trauma was generated under ether anesthesia by exposing the dorsum of rats to 90 ^°C water bath for 10 s. 100 mg/kg/day Mrytus communis ethanol extract was applied orally for two days. Malondialdehyde (MDA) and glutathione (GSH) levels, glutatinone-S-transferase (GST), superoxidedismutase (SOD) and catalase (CAT) activities were determined to detect the possible antioxidant effects of myrtle on small intestine and lung tissues. Burn damage significantly increased MDA levels in lung and small intestine tissues, and significantly decreased GSH levels, CAT and GST activities in the small intestine and lung tissues compared to control group. Mrytus communis ethanol extract decreased MDA level and increased GSH level, SOD, CAT and GST activities significantly in either small intestine or lung tissues. Mrytus communis extract may be an ideal candidate to be used as an antioxidant adjunct to improve oxidative distant organ damage to limit the systemic inflammatory response and decreasing the recovery time after thermal injury.     

Effects of temperature and nitrate on phosphomonoesterase activities between carbon source and sink tissues in Zostera marina L.

Inorganic phosphorus (P_i) is important in the regulation of many carbon and nitrogen metabolic processes of plants. In this study, we examined alterations of phosphomonoesterase activity (PA; both alkaline and acid) in a submersed marine angiosperm, Zostera marina, grown in P_i non-limiting conditions under elevated temperature and/or nitrate enrichment. Control plants (ambient water-column NO₃⁻ < 2.5 μM, with weekly mean water temperatures between 26.5-27.0 °C based on a 20-yr data set in a local embayment) were compared to treated plants that were exposed to increased water-column nitrate (8 μM NO₃⁻ above ambient, pulsed daily at 0900 h), and/or increased temperature (ca. 3 °C above weekly means) over eight weeks in late summer-fall. Under both nitrate regimes, increased temperature resulted in periodic increased leaf and root-rhizome tissue carbon content, and increased acid and alkaline PA activities (AcPAs and AlPAs, respectively). There was a positive correlation between AlPA and AcPA activities and sucrose synthase activities in belowground structures, and a negative correlation between AlPA activities and sucrose concentrations. There were also periodic changes in PA partitioning between carbon source and sink tissues. In high-temperature and high-nitrate treatments, AcPAs significantly increased in leaves relative to activities in root-rhizome tissues (up to 12-fold higher in aboveground than belowground tissues in as little as 3 weeks after initiation of treatments). These responses were not observed in control plants, which maintained comparable AcPA activities in above- and belowground tissues. In addition, AlPA activity was significantly higher in leaf than in root-rhizome tissues of plants in high-temperature (weeks 3 and 6) and high temperature combined with high nitrate treatments (week 8), relative to AlPA activities in control plants. The observed changes in PAs were not related to P_i growth limitation, and may allow Z. marina to alter its carbon metabolism during periods of increased carbon demand/mobilization. This response would make it possible for Z. marina to meet short-term P requirements to maximize carbon production/allocation. Such a mechanism could help to explain the variability in PA activities that has been observed for many plant species during periods when environmental P_i exceeds requirements for optimal growth.     

Mixed-function oxygenase enzymes as tools for pollution monitoring: Field studies on the french coast of the Mediterranean sea

• 1.1. MFO enzyme activities were measured in microsomes from whole mussels (Mytilus galloprovincialis) comber livers (Serranus cabrilla), or Posidonia oceanica etiolated tissues, and PAH contents were determined in sediments collected in coastal locations of the French Riviera and Corsica during 3 oceanographic cruises in 1987–1988.
• 2.2. BaP activities in mussel and EROD activities measured in fish were strongly correlated to the log of PAH content in sediments. The first results for CA4H in Posidonia showed significant differences related to PAH pollution levels. The increase in MFO activities measured in Corsica in summer 1988 indicated a recent petroleum contamination.

     

Co-purification of type I alkaline phosphatase and type I phosphoprotein phosphatase from various animal tissues

A purification procedure, which included ethanol treatment as a step for dissociating the large molecular forms of type I phosphoprotein phosphatase, was employed for the studies of the alkaline phosphatase and phosphoprotein phosphatase activities in bovine brain, heart, spleen, kidney, and uterus, rabbit skeletal muscle and liver, and lobster tail muscle. The results indicate that the major phosphoprotein phosphatase (phosphorylase a as a substrate) and alkaline phosphatase (p-nitrophenyl phosphate as a substrate; Mg²⁺ and dithiothreitol as activators) activities in the extracts of all tissues studied were copurified as an entity of M_r = 35,000. The purified enzymes from different tissues exhibit similar physical and catalytic properties with respect to either the phosphoprotein phosphatase or the alkaline phosphatase activity. The present findings indicate that (a) the M_r = 35,000 species, which represents a catalytic entity of the large molecular forms of type I phosphoprotein phosphatase, is widespread in animal tissues, indicating that it is a multifunctional phosphatase; (b) the association of type I alkaline phosphatase activity with type I phosphoprotein phosphatase is a general phenomenon.     

Tropolone—a compound that can aid in differentiating between tyrosinase and peroxidase

In studies dealing with melanogenesis in mammalian tissues, ultrastructural localization of enzymes, identification of subcellular organelles, differentiation and lignification in plant tissues, it is important to have means to differentiate between tyrosinase and peroxidase activities. For a variety of reasons, established criteria used for this purpose are not always reliable. We suggest that tropolone can aid in differentiating between tyrosinase and peroxidase activities since: (a) it is a very effective inhibitor of tyrosinase; (b) in the presence of hydrogen peroxide it can serve as a substrate for peroxidase; (c) at concentrations that inhibit tyrosinase, it does not inhibit peroxidase activity; and (d) it inhibits tyrosinase activity even in the presence of hydrogen peroxide and peroxidase. In a system containing a mixture of tyrosinase and peroxidase, tropolone can differentiate reliably between peroxidase and monohydroxyphenolase or o-dihydroxyphenolase activities of tyrosinase. Moreover, tropolone can differentiate reliably between peroxidase and tyrosinase activities using slices or crude dialysed extracts of various plant tissues.     

Characterization of the root-predominant gene promoter HPX1 in transgenic rice plants

Gene promoter(s) specialized in root tissues is an important component for crop biotechnology. In our current study, we report results of promoter analysis of the HPX1, a gene expressed predominantly in roots. The HPX1 promoter regions were predicted, linked to the gfp reporter gene, and transformed into rice. Promoter activities were analyzed in various organs and tissues of six independent transgenic HPX1:gfp plants using the fluorescent microscopy and q-RT-PCR methods. GFP fluorescence levels were high in root elongation regions but not in root apex and cap of the HPX1:gfp plants. Very low levels of GFP fluorescence were observed in anthers and leaves. Levels of promoter activities were 16- to 190-fold higher in roots than in leaves of the HPX1:gfp plants. The HPX1 promoter directs high levels of gene expression in root tissues producing GFP levels up to 0.39 % of the total soluble protein. Thus, the HPX1 promoter is predominantly active in the root elongation region during the vegetative stage of growth.     

Biosynthesis of the Macrocyclic Diterpene Casbene in Castor Bean (Ricinus communis L.) Seedlings : Changes in Enzyme Levels Induced by Fungal Infection and Intracellular Localization of the Pathway

Casbene is a macrocyclic diterpene hydrocarbon that is produced in young castor bean (Ricinus communis L.) seedlings after they are exposed to Rhizopus stolonifer or other fungi. The activities of enzymes that participate in casbene biosynthesis were measured in cell-free extracts of 67-hour castor bean seedlings (a) that had been exposed to R. stolonifer spores 18 hours prior to the preparation of extracts, and (b) that were maintained under aseptic conditions throughout. Activity for the conversion of mevalonate to isopentenyl pyrophosphate does not change significantly after infection. On the other hand, the activities of farnesyl pyrophosphate synthetase (geranyl transferase), geranylgeranyl pyrophosphate synthetase (farnesyl transferase), and casbene synthetase are all substantially greater in infected tissues in comparison with control seedlings maintained under sterile conditions. The subcellular localization of these enzymes of casbene biosynthesis was investigated in preparations of microsomes, mitochondria, glyoxysomes, and proplastids that were resolved by centrifugation in linear and step sucrose density gradients of homogenates of castor bean endosperm tissue from both infected and sterile castor bean seedlings. Isopentenyl pyrophosphate isomerase and geranyl transferase activities are associated with proplastids from both infected and sterile seedlings. Significant levels of farnesyl transferase and casbene synthetase are found only in association with the proplastids of infected tissues and not in the proplastids of sterile tissues. From these results, it appears that at least the last two steps of casbene biosynthesis, geranylgeranyl pyrophosphate synthetase and casbene synthetase, are induced during the process of infection, and that the enzymes responsible for the conversion of isopentenyl pyrophosphate to casbene are localized in proplastids.     

A new treatment for human malignant melanoma targeting L-type amino acid transporter 1 (LAT1): A pilot study in a canine model

L-type amino acid transporter 1 (LAT1), an isoform of amino acid transport system L, transports branched or aromatic amino acids essential for fundamental cellular activities such as cellular growth, proliferation and maintenance. This amino acid transporter recently has received attention because of its preferential and up-regulated expression in a variety of human tumors in contrast to its limited distribution and low-level expression in normal tissues. In this study, we explored the feasibility of using LAT1 inhibitor as a new therapeutic agent for human malignant melanomas (MM) using canine spontaneous MM as a model for human MM. A comparative study of LAT expression was performed in 48 normal tissues, 25 MM tissues and five cell lines established from MM. The study observed LAT1 mRNA levels from MM tissues and cell lines that were significantly (P < 0.01) higher than in normal tissues. Additionally, MM with distant metastasis showed a higher expression than those without distant metastasis. Functional analysis of LAT1 was performed on one of the five cell lines, CMeC-1. [³H]l-Leucine uptake and cellular growth activities in CMeC-1 were inhibited in a dose-dependent manner by selective LAT1 inhibitors (2-amino-2-norbornane-carboxylic acid, BCH and melphalan, LPM). Inhibitory growth activities of various conventional anti-cancer drugs, including carboplatin, cyclophosphamide, dacarbazine, doxorubicin, mitoxantrone, nimustine, vinblastine and vincristine, were significantly (P < 0.05) enhanced by combination use with BCH or LPM. These findings suggest that LAT1 could be a new therapeutic target for MM.     

Succinic Dehydrogenase and Cytochrome Oxidase Activities in Cell Cultures

Succinic dehydrogenase and cytochrome oxidase have been assayed in permanent cell lines (HEP 1, HEP 2, and HLM), in short-term cultures of chick embryo heart cells, and in various tissues. Their activities in different cells are compared by relating them to deoxyribonucleic acid. They are very low in HEP 1, HEP 2, and HLM cells by comparison with the activities in any normal tissues examined. All the succinic dehydrogenase was shown to be located in the mitochondria of the permanent cell lines by staining with tetrazolium derivatives. Both enzymes were more active in tissues of 19-day chick embryos than in those of 11- or 14-day embryos. The increasing activities found during normal development were quickly curtailed or reversed when heart cells were grown as monolayer cultures. Digitonin-treated mitochondria produced preparations with much higher activities of cytochrome oxidase than untreated samples. Activities measured in this way were again very much lower in HEP 1, HEP 2, and HLM cells than in the normal tissues. From the derived ratio of cytochrome oxidase:succinic dehydrogenase, it was apparent that cytochrome oxidase is diminished to a greater extent than succinic dehydrogenase in both permanent cell lines and short-term cultures, by comparison with the corresponding activities in embryonic and adult tissues. The features common to the metabolism of proliferating cells in vitro and malignant cells are discussed.     

Oral lipase activities and fat-taste receptors for fat-taste sensing in chickens

It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis.