Isoenzyme Patterns Research Articles

How to easily detect plant NADH-glutamate dehydrogenase (GDH) activity? A simple and reliable in planta procedure suitable for tissues, extracts and heterologous microbial systems

Plant NADH glutamate dehydrogenase (GDH) is an intriguing enzyme, since it is involved in different metabolic processes owing to its reversible (anabolic/catabolic) activity and due to the oligomeric nature of the enzyme, that gives rise to several isoforms. The complexity of GDH isoenzymes pattern and the variability of the spatial and temporal localization of the different isoforms have limited our comprehension of the physiological role of GDH in plants. Genetics, immunological, and biochemical approaches have been used until now in order to shed light on the regulatory mechanism that control GDH expression in different plant systems and environmental conditions. We describe here the validation of a simple in planta GDH activity staining procedure, providing evidence that it might be used, with different purposes, to determine GDH expression in plant organs, tissues, extracts and also heterologous systems.

Isoenzyme profiles and phylogenetic analysis of Giardia duodenalis isolates from Iranian patients.

The main objective of this study was to characterize the Giardia duodenalis isolates from Iranian patients in Fars Province, south of Iran by biochemical and molecular methods. Fifteen mass cultivated of G. duodenalis isolates in modified TYI-S-33 medium were analyzed using isoenzyme electrophoresis and PCR genotyping. Polyacrylamide gel electrophoresis (PAGE) of five different enzyme systems was used to characterize isolates: (i) glucose-6-phosphate dehydrogenase, (ii) glucose phosphate isomerase, (iii) malate dehydrogenase, (iv) malic enzyme, and (v) phosphoglucomutase. As well, a fragment of the SSU-rDNA (292bp) gene was amplified by PCR using the primers RH11 and RH4. The sequencing of the PCR products and phylogenetic tree were performed. The isoenzyme electrophoretic profiles divided fifteen G. duodenalis isolates into four zymodemes. G6PD, GPI, MDH, ME, and PGM enzyme systems showed 1, 2, 2, 3, and 3 enzyme pattern, respectively. G6PD isoenzyme pattern had the most homogeneity, while isoenzyme patterns of ME and PGM had the most heterogeneity in our study. Genotyping results indicated that the zymodemes 1-4 were categorized in assemblage A based on the SSU-rDNA gene. Phylogenetic analysis showed that all four zymodemes were distributed within the cluster of assemblage A. Our results indicated that both isoenzyme and DNA analyses were useful to characterize the isolates of Giardia and distinguishing various zymodemes and assemblages. It could be suggested that the genetic diversity among isoenzymes profiles of G. duodenalis may explain the variable clinical manifestations, pathogenicity, host response, drug susceptibility, and treatment efficacy of human giardiasis.

Agarose gel electrophoresis pattern of serum alkaline phosphatase isoenzymes in Holstein cows during lactation.

A recent study found that an agarose gel electrophoresis (AGE) method yielded two distinct major bands corresponding to the hepatic and bone ALP isoenzymes (ALP2 and ALP3, respec-tively) in bovine serum treated with protease and neuraminidase (PN-treatment), although there were concerns that the intestinal ALP isoenzyme (ALP5) often overlapped with ALP3 in human serum treated with neuraminidase. Because ALP5 was separated from ALP3 in bovine serum treated with protease alone (P-treatment), we used a modified method employing both P- and PN-treated bovine sera to measure the activities of the three ALP isoenzymes in 53 lacta-ting Holstein cows: 24 primiparous and 29 multiparous. Upon electrophoresis, 51 of 53 samples (96.2%) subjected to P-treatment yielded a distinct fraction corresponding to ALP5, as did the control serum. All PN-treated sera yielded a definite ALP2 fraction. The ALP3 fraction was calculated as the remainder after excluding ALP2 and ALP5. The activities of total ALP (t-ALP) and ALP3 in primiparous cows were higher than those in multiparous cows (p ⟨ 0.001) at early-to-peak [10-110 days in milk (DIM)] and mid (111-220 DIM) lactation. In the multi-parous cows, the ALP3 activity at late lactation (221-477 DIM) was significantly higher than that at early-to-peak lactation. Thus, the modified AGE method described here is able to discrimi-nate three fractions of ALP isoenzymes in the sera of lactating cows. The AGE pattern of circu-lating ALP isoenzymes will contribute to the understanding of the physiological bone metabolism status in lactating cows.

Clinical and Diagnostic Significance of Lactate Dehydrogenase and Its Isoenzymes in Animals.

Lactate dehydrogenase (LDH) is widely distributed enzyme in cells of various living systems where it is involved in carbohydrate metabolism catalyzing interconversion of lactate and pyruvate with NAD+/NADH coenzyme system. Cells of tissues are direct source of lactate dehydrogenase isoenzymes that are naturally distributed in blood plasma/serum of animals and humans producing characteristic profile. This profile depends on intracellular isoenzyme concentration in all tissues that contribute to the common pool of lactate dehydrogenases in plasma/serum as a consequence of natural cell degradation. LDH is widely distributed in the body, high activities are found in the heart, liver, skeletal muscle, kidney, and erytrocytes, whereas lesser amounts are found in the lung, smooth muscle, and brain. Because of its widespread activities in numerous body tissues, LDH is elevated in a variety of disorders. There are many conditions that contribute to increased activity of LDH. An elevated total LDH value is a rather nonspecific finding. Therefore, LDH assays assume a more clinical significance when separated into isoenzyme fractions. The activity of LDH and its serum and tissue patterns and composition show great variations between the species. These differences do not allow using catalytic activities of LDH isoenzymes from one species to another. Instead, the pattern of serum LDH isoenzymes should be interpreted in respect to its species origin that is important in particular in veterinary medicine. Determination of total LDH activity and its isoenzyme pattern in serum of mammals had become one of the biochemical indicators in the assessment of organ disorders. When the content of cells is released from tissue to plasma, as on cell injury, the LDH isoenzyme pattern of the serum changes in favour of the profile of the affected organ (tissue) that can be used in the diagnostic practice.

Transglutaminase 2 Has Metabolic and Vascular Regulatory Functions Revealed by In Vivo Activation of Alpha1-Adrenergic Receptor.

The multifunctional tissue transglutaminase has been demonstrated to act as α1-adrenergic receptor-coupled G protein with GTPase activity in several cell types. To explore further the pathophysiological significance of this function we investigated the in vivo effects of the α1-adrenergic receptor agonist phenylephrine comparing responses in wild type and TG2-/- mice. Injection of phenylephrine, but not a beta3-adrenergic agonist (CL-316,243), resulted in the long-term decline of the respiratory exchange ratio and lower lactate concentration in TG2-/- mice indicating they preferred to utilize fatty acids instead of glucose as fuels. Measurement of tail blood pressure revealed that the vasoconstrictive effect of phenylephrine was milder in TG2-/- mice leading to lower levels of lactate dehydrogenase (LDH) isoenzymes in blood. LDH isoenzyme patterns indicated more damage in lung, liver, kidney, skeletal, and cardiac muscle of wild type mice; the latter was confirmed by a higher level of heart-specific CK-MB. Our data suggest that TG2 as an α1-adrenergic receptor-coupled G protein has important regulatory functions in alpha1-adrenergic receptor-mediated metabolic processes and vascular functions.

Suppressed expression of LDHB promotes age-related hearing loss via aerobic glycolysis

Age-dependent decrease of mitochondrial energy production and cellular redox imbalance play significant roles in age-related hearing loss (ARHL). Lactate dehydrogenase B (LDHB) is a key glycolytic enzyme that catalyzes the interconversion of pyruvate and lactate. LDH activity and isoenzyme patterns are known to be changed with aging, but the role of LDHB in ARHL has not been studied yet. Here, we found that LDHB knockout mice showed hearing loss at high frequencies, which is the typical feature of ARHL. LDHB knockdown caused downregulation of mitochondrial functions in auditory cell line, University of Bristol/organ of Corti 1 (UB/OC1) with decreased NAD+ and increased hypoxia inducing factor-1α. LDHB knockdown also enhanced the death of UB/OC1 cells with ototoxic gentamicin treatment. On the contrary, the induction of LDHB expression caused enhanced mitochondrial functions, including changes in mitochondrial respiratory subunits, mitochondrial membrane potentials, ATP, and the NAD+/NADH ratio. Thus, we concluded that suppression of LDHB activity may be closely related with the early onset or progression of ARHL.

Can preoperative serum lactate dehydrogenase levels predict postoperative pulmonary complication following an emergency abdominal surgery? An observational study

Background: Postoperative pulmonary complications (PPC) are one of the commonest complications following gastrointestinal surgery. They lead to increase in morbidity and mortality. Lactate dehydrogenase (LDH) is an enzyme present in essentially all major organ systems. Studies have shown measurement of its activity levels and its isoenzyme pattern may provide additional information about lung and pulmonary endothelial cell injury. The objectives of the present study were to study the levels of serum LDH in patients with and without post-operative pulmonary complications following emergency abdominal surgery.Methods: The study was designed as an observational study. All patients ≥18 years of age undergoing gastrointestinal surgery, excluding those with prior lung pathology were included in the study. The demographic parameters, clinical parameters and laboratory parameters along with details of pulmonary complications were recorded. Serum LDH level were assessed on admission. Levels of serum LDH were compared between patients with and without post-operative pulmonary infections and were assessed for significance.Results: Incidence of PPC was 28% in our study. There was significant difference in the mean age in the group with and without PPC (p=<0.001). Smoking habit, serum albumin total protein and upper abdomen incision surgery were associated with increased incidence of PPC. Pleural effusion was the commonest PPC seen in patients. Serum LDH was not significantly associated with the incidence of PPC.Conclusions: Pre-operative serum LDH level is not a predictive factor for occurrence of postoperative pulmonary complication. Age, smoking, total protein, serum albumin, upper abdomen incision were found to associated with increased risk of PPCs.

Evaluation of total LDH and its isoenzymes as markers in preeclampsia.

Preeclampsia, a rapidly progressing pregnancy-specific multi-systemic syndrome is globally the leading cause of maternal and neonatal morbidity and mortality. This study aims to evaluate the serum total Lactate dehydrogenase levels in women with preeclampsia when compared to normotensive pregnant women and assess the electrophoretic pattern of the LDH isoenzymes in normal pregnancy, preeclampsia and eclampsia. The study, carried out in the Department of Biochemistry of MVJ Medical College, included 30 patients of preeclampsia and 30 normotensive gestational age-matched pregnant women admitted to the Department of OBG. Serum total LDH was analysed by DGKC method. Serum and cord blood samples for isoenzyme distribution analysis were collected from a normal pregnant woman undergoing delivery, a woman with mild eclampsia, two women with eclampsia, and analysed by slab gel electrophoresis followed by activity staining. LDH was significantly elevated in cases as well as between the case (mild and severe) groups, showed a moderate positive statistically significant correlation with systolic, diastolic blood pressure and a sensitivity of 50% and a specificity of 80%. Further, the isoenzyme pattern showed a decreasing distribution of aerobic forms of LDH in preeclampsia-eclampsia. Serum total LDH may serve as a robust and affordable marker of preeclampsia. Serum total LDH, along with its isoenzyme profile, might serve as a predictor and a stronger marker of preeclampsia when compared to serum LDH analysis alone. It may also be used to assess the severity of preeclampsia and hence help in predicting and preventing adverse maternal and foetal outcomes.

Serum lactate dehydrogenase activity and its isoenzyme patterns in patients with pectus excavatum.

In previous study, we found elevated serum total lactate dehydrogenase (LDH) before correction of pectus excavatum and a decrease in total LDH after the deformity correction. In the present study, we analyzed total LDH activity and its isoenzyme patterns to investigate the causes of these laboratory findings in patients with pectus excavatum. Between March 2014 to December 2018, 85 patients with pectus excavatum who had undergone the Nuss procedure (NP) and bar removal (BR) were included into this study. We analyzed (I) total LDH and its isoenzyme patterns before the correction, (II) relationships of total LDH and its isoenzymes with age at time of NP, sex, severity of pectus excavatum, and pectus morphology types, and (III) post-corrective changes. The mean age of the patients was 13.6 (±6.5) years at the age of NP and the mean interval between NP and BR was 2.2 (±0.42) years. Seventy-one males and 14 females were included. The pectus types included 54 symmetric and 31 asymmetric cases. The mean Haller index before NP and BR were 3.8±1.45 and 2.7±0.4, respectively. The mean of total LDH before NP (pre-correction) and BR (post-correction) were 404.2±80.8 and 369.2±79.3 IU/L, respectively. Before correction, total LDH was significantly higher than normal values, irrespective of age [the young group (<10 years old), P=0.006, and the old group (≥10 years old), P<0.001]. The proportion of LDH5 was significantly higher than that of LDH4 (P<0.001). Total serum LDH was significantly associated with age at time of NP and Haller index (P<0.001 and P=0.030). There was no significant correlation between severity and total LDH. However, the value of only LDH5 among all isoenzymes had a significant positive correlation with severity (P=0.006) and the proportion of only LDH5 in the severe group was significantly higher (P=0.003). After correction, proportions of each isoenzyme were all within the reference range, however, there were significant decreases in values of LDH1-LDH4, except LDH5 (P=0.020, P<0.001, P<0.001, and P=0.029). This study shows that pectus excavatum is a muscular disease entity and that laboratory findings are associated with compression of internal organs, which was explained by post-corrective changes in LDH activity and its isoenzyme patterns. This study will provide a deeper and wider comprehension of pectus excavatum.

Surface mycobiota of home-made dry cured sausages from the main producing regions of Argentina and morphological and biochemical characterization of Penicillium nalgiovense populations

The characteristics and quality of home-made dry cured sausages can be recognized and associated with the region of origin. The characteristics of this type of sausages result from the superficial mycobiota that spontaneously colonizes the products. The aim of this study was to identify the house mycobiota associated with home-made dry cured sausages from different localities of Argentina and characterize the populations of Penicillium nalgiovense present by morphological and biochemical markers. To this end, 79 samples were collected from 10 localities of three main producing regions (Buenos Aires, Córdoba and La Pampa provinces). A total of 196 isolates belonging to six genera and 17 species were obtained. The predominant genus was Penicillium (134 of the isolates) and the predominant species was P. nalgiovense (108 isolates). The isoenzyme patterns of α-esterase (α-EST; EC 3.1.1.1) and Malate dehydrogenase NADP+(MDHP; EC 1.1.1.40) were characterized in 48 of these isolates (ten from Colonia Caroya, ten from Oncativo, ten from Tandil, nine from Mercedes and nine from La Pampa). A total of 26 bands were observed: 17 for α-EST and 9 for MDHP. α-EST was the most polymorphic isoenzyme, whereas MDPH presented no polymorphism. The results were subjected to numerical analysis. Cluster analysis revealed the formation of two groups: Group I formed by 24 isolates from Córdoba and Buenos Aires provinces and Group II with 24 isolates from La Pampa and Buenos Aires province. These data suggest the existence of morphological and biochemical variations among P. nalgiovense populations with different geographical origin.

ISOENZIMAS Y ACTIVIDAD DE LACASAS PRODUCIDAS POR Pleurotus ostreatus CRECIDO A DIFERENTES TEMPERATURAS

Pleurotus ostreatus produces laccase isoforms depending on the conditions in which it grows, it has been suggested that the presence of inducers, the pH of the culture medium as well as the type and concentration of carbon and nitrogen sources promote the production of these isoforms, however, the effect of temperature of Pleurotus ostreatus growth on its production of laccase isoforms has not been evaluated. In this study, Pleurotus ostreatus was grown in solid-state fermentation at different temperatures (10, 15, 20, 25, 30 and 35 °C) and the activity and isoforms number of laccases produced in each bioprocess was determined. At 10 and 35 °C there was no growth of the fungus. The activity of laccases was different in each fermentation, being at 25 ºC where the highest value was observed (20000 U L-1). The laccase isoenzymes pattern was also different depending on the incubation temperature of the fungus, it was observed in some times of the culture, 4 isoforms in the fermentations incubated at 15 and 20 ºC, 3 isoforms at 25 ºC and 2 isoforms at 30 ºC. These results show that the growth temperature of Pleurotus ostreatus modifies its patterns of activity and of isoforms of laccases.

The Effects of Mg Deficiency on Activities and Isoenzyme Patterns of Antioxidant Enzymes in Cotton Roots

Magnesium (Mg) is an essential nutrient that takes vital roles in biochemical processes related to energy metabolism, regulation of enzyme activity, nucleic acid biosynthesis and carbohydrate metabolism. Mg deficiency in arable lands is an emerging problem. Loss of balance in different metabolic pathways due to Mg deficiency causes increased production of reactive oxygen species (ROS) such as O2.-, H2O2 and HO. in the roots. The aim of this work was to elucidate the antioxidant response of roots of cotton (Gossypium hirsutum) to Mg deficiency. For this aim, antioxidant enzyme activities were measured and changes in their isoenzymes were determined. In addition, NADPH oxidase (NOX) activity was investigated to determine the changes in ROS signaling. Moreover, H2O2 and lipid peroxidation levels were also measured as an indicator of oxidative damage. Findings of this work indicate that Mg deficiency does not cause oxidative load in roots of Gossypium hirsutum plants that overwhelms cellular antioxidant defence system, as evident by H2O2 and TBARS levels. However, to adjust to the new redox status, root cells drastically downregulate NOX dependent ROS production and induced total superoxide dismutase activity. These findings indicate that site of O2.- production is mitochondria in roots. Although, catalase and ascorbate peroxidase activities were decreased with Mg deficiency induced peroxidase activity seems to limit H2O2 accumulation. These adaptive responses and changes in antioxidant defence were sufficient to prevent excess ROS accumulation and occurrence of oxidative damage in roots of Gossypium hirsutum plants under Mg deficiency

Protective Effects of Ammannia baccifera Against CCl4-Induced Oxidative Stress in Rats.

Ammannia baccifera Linn. is commonly used as a traditional medicine in India and China. The antioxidant potential of an ethanolic extract of A. baccifera (EEAB; 250 mg/kg and 500 mg/kg) was evaluated against CCL4-induced toxicity in rats. Antioxidant activity was assessed by measuring the enzymatic and non-enzymatic antioxidants. Phytochemical constituents of EEAB were also analyzed by using UHPLC-QTOF-MS. EEAB treatment markedly reduced CCl4 effects on lipid peroxidation, cholesterol, triacylglycerides, and protein carbonyls. It increased the levels of phospholipids, total sulfhydryl, and antioxidant enzymes, which were reduced by CCl4 intoxication. Treatment with EEAB significantly alleviated the CCl4 effect on non-enzymatic antioxidants. Isoenzyme pattern analyses revealed that significant alterations in superoxide dismutase (SOD1), glutathione peroxidase (GPx2, GPx3), and catalase (CAT) occurred in rats that were exposed to CCl4 and restored post EEAB treatment. Moreover, CCl4-induced down regulation of SOD, CAT, and GPx gene expression was conversely counteracted by EEAB. Its bioactivity may be due to its incorporation of major compounds, such as chlorogenic acid, quercetin, protocatechuic acid, lamioside, crocetin, and khayasin C. These results suggest that EEAB may be used as a potent antioxidant and hepatoprotective agent since it is a rich source of flavonoids and phenolic compounds.

Mechanical Damages and Chemical Treatments in the Quality of Maize Seeds Stored

The current system of seeds production is highly mechanized and these operations can contribute to losses in quality due the incidence of mechanical damages. The objective in this work was to study the effect of the chemical treatment and the storage on quality of maize seeds with mechanical damages. The seeds used were individually analyzed through X-ray test for the obtainment of three lots; 1-seeds without separation, 2-seeds without mechanical damages and 3-seeds with mechanical damages. The lots were evaluated according the physiological quality, sanity and enzymatic analyzes before and after the chemical treatment with the insecticide Maxim Advanced&amp;reg; and the fungicide Cruiser&amp;reg; and also after 90 days of storage. The chemical treatment of maize seeds is efficient in the control of pathogens, like Fusarium sp. The performance of seeds with internal and external damages can be maintained for 90 days when treated with fungicides and insecticides and stored in environment conditions. The isoenzymatic patterns of catalase (CAT), superoxide dismutase (SOD), malate dehydrogenase (MDH) and alcohol dehydrogenase (ADH), confirmed the higher deterioration of non-treated seeds during the storage. The mechanical damages found in seeds contribute to the reduction of viability and vigor of maize seeds.

Effects of hemazin SC 500 (terbuthylazine) on antioxidative enzymes in human erythrocytes in vitro

The aim of this work was to investigate the effect of the commercial formulation hemazin SC 500, an herbicide containing terbuthylazine as the active compound, on the isoenzyme patterns and activities of Cu-Zn superoxide dismutase (SOD1) and catalase (CAT), as well as on the glutathione S-transferase (GST) activity, in human erythrocytes in vitro. The human erythrocytes were treated with hemazin SC 500 over a broad range of terbuthylazine concentrations (37 nmol L-1? ?37 ?mol L-1) for 1 and 3 h at a temperature of 37?C. Native electrophoresis of the control and treated samples revealed two SOD1 and one CAT isoform. Treatment did not affect the SOD1 and CAT isoenzyme profile, but induced a change in their activities. Terbuthylazine at lower concentration induced a significant increase of the total SOD1 activity and decreased the GST activity in samples incubated for 1 and 3 h. On the other hand, the highest increase in the CAT activity was observed for the sample treated for 1 h with a higher concentration of terbuthylazine. Hemazin SC 500 containing terbuthylazine induces changes in the erythrocyte antioxidative system whereby the response of individual enzymatic antioxidants depends on the concentration of the pesticide and the incubation time.

Efficiency of bacterial biosurfactant for biocontrol of Rhizoctonia solani (AG - 4) causing root rot in faba bean (Vicia faba) plants

Biosurfactants are a structurally diverse group of surface-active compounds produced by microorganisms, with numerous applications in different fields. In the present study, we evaluated the antifungal activity of a biosurfactant produced by Bacillus licheniformis against Rhizoctonia solani AG-4 that causes root rot in two cultivars of Vicia faba (Nubaria 1 & Sakha 1). Molasses and yeast extract as carbon and nitrogen sources exhibited maximum emulsification activity and fungal growth inhibition. Treatment with biosurfactant decreased the disease incidence from 62.11 to 20.00% in cv. Nubaria 1 and from 38.93 to 16.51% in case of cv. Sakha 1. Results revealed that growth parameters, photosynthetic pigments and endogenous phytohormones were markedly inhibited in faba bean, particularly in cv. Nubaria 1 plants, due to the root rot disease. Moreover, infection with R. solani caused an increase in lipid peroxidation content, non-enzymatic (phenolic and flavonoids compounds) and enzymatic antioxidants contents (phenylalanine ammonia lyase) as compared with healthy control plants. Biosurfactant application to the healthy and infected plants enhanced all the mentioned parameters except the lipid peroxidation content which showed significant reduction. Electrophoretic patterns of peroxidase, polyphenol oxidase and superoxide dismutase isoenzymes showed wide variations in their intensities and densities among all treatments. Peroxidase and polyphenol oxidase showed increased activities in cv. Sakha 1 by BS application. It appears that application of bacterial biosurfactant was able to enhance the biological control of root rot disease of faba bean plants.

Endosymbiotic green algae in European Hydra strains show quantitative difference on morphological and isoenzyme level

The process of endosymbiosis is one of the promoters of speciation. The green hydra (Hydra viridissima Pallas, 1766) is a typical example of endosymbiosis. Its gastrodermal myoepithelial cells harbour the individuals of unicellular photoautotrophic algae. In this study we have developed healthy laboratory cultures of endosymbiotic green algae isolated from green hydra strains. The Hydra strains were collected from four different geographical localities, two of which were in Croatia (strains BV, T), one in Israel (strain M9) and one in Germany (strain HV). For the first time, endosymbiotic algae isolated from green hydra strains have been visualized and described by scanning and transmission electron microscopy. Isolated endosymbiotic algae were characterized by cytological morphometric parameters, enzyme activity and isoenzyme pattern analysis (catalase, peroxidase and esterase). In addition, cells of endosymbiotic algae were characterized by morphometric measurements of diameter, perimeter and area, and chloroplast area. Endosymbiotic algae HV (collected in Germany) and M9 (collected in Israel) were significantly different when compared with endosymbiotic algae T and BV collected in Croatia. The endosymbionts were more different with referent alga Parachlorella kessleri than with referent alga Chlorella vulgaris. The results obtained by isoenzyme pattern analysis also suggested that there was a difference between Croatian and European algal endosymbionts, i.e. the results indicate biological diversity among algal symbionts isolated from the green hydras from different geographical localities. Ultimately, five isoenzymes of catalase and five isoenzymes of peroxidase were resolved by PAGE electrophoresis. Catalase isoenzyme K1 appeared only in endosymbiotic alga M9 collected in Israel and catalase isoenzyme K3 only in endosymbiotic alga T collected in Croatia. Peroxidase isoenzyme P1 was observed only in endosymbiont HV while peroxidase isoenzyme P5 was observed in both endosymbionts HV and M9. Peroxidase isoenzymes P3 and P4 were visible in endosymbiotic algae BV and T collected in Croatia.

Salicylic acid modulates isoenzyme pattern of enzymatic antioxidants in wheat

The isoenzyme profile of leaf and root tissues of wheat plants grown with different concentrations of salicylic acid (SA) was studied by native polyacrylamide gel electrophoresis (PAGE). Wheat plants were co-cultivated with different concentration of SA (0, 50, 500 and 1000 µM) in Hogland solution. Primary leaves and roots from one week old plants were used for the extraction of enzymes. The crude enzyme extracts were subjected to native PAGE and were stained for the enzymatic antioxidants catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX) and superoxide dismutase (SOD). Three isoforms of CAT were observed in both leaf and root tissues of which the intensity of major isoform was reduced in leaf tissues of plants grown with 1000 µM SA where as induction in band intensity of two minor isoforms were noticed in root tissues of plants grown with 500 and 1000 µM SA. The native-PAGE for peroxidase activity revealed five isoforms in root and two in leaf tissues. One of these isoforms in both the tissues showed a gradual reduction in activity with each concentration of SA. Band intensity of APX isoforms were found to be decreased in both leaf and root tissues of plants grown with 500 and 1000 µM SA. A stimulation in band intensity of two of the APX isoforms was noticed in plants grown with 50 µM SA. The isoenzyme profile of SOD revealed two iso-isoforms in leaf tissues and three isoforms in root tissues of both control and SA grown plants. Inhibitory test confirmed these isoenzymes as Cu/Zn-SOD. The study suggested that the effect of SA on the isoenzyme profile of wheat plants is tissue specific and also varies for different enzymes. The low concentration of SA stimulates the synthesis some isoforms where as the higher concentration has negative effect on the synthesis of some isoforms of enzymatic antioxidants.

Antioxidative and antifungal response of woody species to environmental conditions in the urban area.

The complexity of ecological conditions in urban areas imposes the plant species need for the development of various biochemical and physiological adaptive strategies. The aim of our research was to examine the antioxidative and antifungal metabolism of species Pinus nigra, Picea omorika, Tilia cordata and Betula pendula from the area of Banja Luka City (urban area) during two vegetation seasons (spring and autumn) and compared with the same species from forest habitats. Changes in the protein concentration, activity and isoenzyme profiles of peroxidases (POD, EC 1.11.1.7), content and antioxidative activity of total phenols and antifungal activity in leaves and needles of the plants from the urban area and forest habitats were monitored. The obtained results indicate that urban areas induce changes in antioxidative metabolism in all examined species, but that the response is species specific. The most sensitive parameter that indicates different adaptation strategy of Pinus nigra, Picea omorika, Tilia cordata and Betula pendula to environment conditions in the urban area were peroxidase isoenzyme patterns. Less specific parameter was phenol content even though there are some indications for role of their antioxidative capacity in the adjustment to specific habitat. In addition, each species had different metabolic strategy to cope with the changes caused by the urban environment.

Macromolecular Toxins Secreted by Botrytis cinerea Induce Programmed Cell Death in Arabidopsis Leaves

Botrytis cinerea causes grey mold disease in crops and horticultural plants. It is suspected to kill plant cells via secreted toxins and to derive nutrients from dead or dying cells. However, whether macromolecular phytotoxins (MPs) secreted by B. cinerea induce necrosis or also trigger a programmed cell death (PCD) remains to be determined. We have previously partially characterized MPs secreted by B. cinerea. Here we isolated MPs from B. cinerea culture and applied them to leaf cells, assessing PCD over the following 120 h. Cell death was assessed by propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) staining. Catalase (CAT), peroxidase (POD) activity and the cytochrome c/a ratio were assessed by spectrophotometer. POD isomers were measured using the benzidine acetate method. In Arabidopsis thaliana (L.) Heynh. exposed to B. cinerea MPs, we observed chromatin condensation and marginalization, nuclear substance leakage and accumulation of autofluorescent materials in the cell wall. Furthermore, B. cinerea MPs induced release of cytochrome c from the mitochondria into the cytosol. Moreover, CAT and POD activity was upregulated and the POD isoenzyme pattern was altered. In conclusion, A. thaliana exposed to B. cinerea MPs exhibits multiple hallmarks of PCD, suggesting that B. cinerea induces PCD in host cells through secreted macromolecules.