Peroxidase Patterns Research Articles

Abstract 2550: Investigating glutathione peroxidases as potential prognostic factors in uterine corpus endometrial cancer

Abstract Introduction: Uterine corpus endometrial cancer (UCEC) is one of the deadliest women’s reproductive cancers in the United States, and mortality rates have not improved in the last 30 years due to poor prognostic methodologies. Altered expression patterns of Glutathione Peroxidases (GPX1-8) have been connected to the outcome of many cancers. Still, GPX expression patterns and their use as prognostic biomarkers in UCEC have yet to be explored. This project aimed to evaluate the potential utility of GPX1-8 as prognostic biomarkers for UCEC. Methods: To identify GPX1-8 expression alterations in UCEC tumors, the UCEC tissue samples (n=543) from the Cancer Genome Atlas Database (TCGA) dataset were analyzed using the University of Alabama at Birmingham Cancer Data Analysis portal (UALCAN) software to statistically compare mRNA levels between UCEC tumor samples and non-tumorigenic uterine tissue. To determine the impacts of GPX1-8 expression on overall survival (OS) and relapse-free survival (RFS), the UCEC TCGA dataset was analyzed using the pan-cancer RNA seq platform using Kaplan-Meier Plotter (KM plotter). The patient cohort was split into groups according to high GPX expression status and low GPX expression status using the trichotomization function in KM plotter. Hazard ratio (HR), 95% confidence interval (95% CI), and log-rank P value were calculated using the KM plotter platform. Results: GPX1, GPX2, GPX4, GPX7, and GPX8 were differentially expressed in UCEC tumor tissue compared to non-cancerous uterine tissue. However, unfavorable OS in patients associated with low GPX4 expression in UCEC tumor samples (P=0.0043, HR=0.39, 95% CI=0.2-0.76) only. Unfavorable RFS in UCEC patients only correlated with low GPX1 expression (P=0.019, HR=0.43, 95% CI=0.21-0.89) or high GPX6 expression (P=0.0032, HR=2.37, 95% CI=1.31-4.27) in UCEC tumor samples. Conclusion: Low GPX1 and GPX4 expression and high GPX6 expression may be novel unfavorable UCEC prognostic markers. Citation Format: Alexis Dukesherer, Calli A. Davison-Versagli. Investigating glutathione peroxidases as potential prognostic factors in uterine corpus endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2550.

Kinetic Characterization and Partial Purification of Peroxidase in Eucalyptus F1 Hybrids

A differential pattern of peroxidase (E.C. 1.1.11.7) has been observed in reciprocal F1 hybrids of Eucalyptus (FRI-4 and FRI-5) produced using parental combination of E. tereticornis and E. camaldulensis. The hybrids were characterized for the qualitative, kinetic parameters (total and specific activity, units, Km and Vmax) and partial purification of peroxidase. The qualitative study showed the presence of high molecular weight peroxidase isoforms i.e. 260 KD, 250 KD and 110KD bands. A strong maternal inheritance was observed for 260 KD loci in FRI-5. Kinetic characterization of peroxidase revealed that F1 genotypes had efficient peroxidase system over their parental combination resulting in 8 -10 times more heterotic vigour. The FRI-5 genotypes were found to be having efficient peroxidase system with lowest Km values (0.12mM-0.17mM) and higher Vmax values ranging from 0.07-0.14mM min-1mg prot-1. The study revealed the role of maternal inheritance and was found that better genotypes are produced when superior parents are used as seed parent. The results suggest the efficient use of peroxidase marker for the identification of the better genotypes in forest tree species.

CHARACTERIZATION OF Exserohilum turcicum INFECTION SITES IN MAIZE GENOTYPES

ABSTRACT Northern leaf blight caused by Exserohilum turcicum is an important disease of maize (Zea mays L.), and its severity depends more closely on growth lesions than on spot number. Here, we characterized the infection sites of E. turcicum on resistant and susceptible maize genotypes by analyzing the histology of lesions as well as the structural and biochemical mechanisms of infection. Maize leaves were inoculated with the pathogen at specific points and incubated in a microhumidity chamber. Samples were obtained to follow fungal development and host tissue lignification using light and electron microscopy, and the activity and electrophoretic patterns of peroxidases were determined. The time course of spore germination and appressorium formation was essentially the same for both genotypes; however, a delay of 12 h in fungal penetration, accompanied by host tissue lignification, was noted in the resistant genotype, as opposed to that in the susceptible one. Scanning electron microscopy revealed fungal mycelium in the xylem vessels of both genotypes; however, in the resistant genotype, pathogen colonization was restricted to mesophyll cells around the penetration point, where chlorotic flecks were produced. Meanwhile, in the susceptible genotype, following penetration and chlorotic fleck formation, the pathogen continued to grow inside the xylem vessels and profusely colonized mesophyll tissue distant from the penetration point, resulting necrotic lesion development. Electrophoretic patterns of peroxidases were similar between the two genotypes, with three isoenzymes present in all tissues. In addition, two novel isoenzymes were detected in chlorotic flecks, necrotic lesions, and green tissue around the lesions.

An intracerebroventricular injection of amyloid-beta peptide (1–42) aggregates modifies daily temporal organization of clock factors expression, protein carbonyls and antioxidant enzymes in the rat hippocampus

Alzheimer disease (AD) is the most frequent form of dementia in the elderly. It is characterized by the deterioration of memory and learning. The histopathological hallmarks of AD include the presence of extracellular deposits of amyloid beta peptide, intracellular neurofibrillary tangles, neuron and synapse loss, in the brain, including the hippocampus. Accumulation of Aβ peptide causes an increase in intracellular reactive oxygen species (ROS) and free radicals associated to a deficient antioxidant defense system. Besides oxidative stress and cognitive deficit, AD patients show alterations in their circadian rhythms. The objective of this work was to investigate the effects of an intracerebroventricular injection of amyloid beta peptide Aβ(1–42) aggregates on temporal patterns of protein oxidation, antioxidant enzymes and clock factors in the rat hippocampus. Four-month-old male Holtzman rats divided into the groups control (CO) and Aβ-injected (Aβ), were maintained under 12 h-light12h-dark conditions and received water and food ad-libitum. Hippocampus samples were isolated every 6 h during a 24 h period. Our results showed daily patterns of protein carbonyls, catalase (CAT) and glutathione peroxidase (GPx) expression and activity, as well as Rorα and Rev-erbß mRNA, in the rat hippocampus. Interestingly, an intracerebroventricular injection of Aβ aggregates modified daily oscillation of protein carbonyls levels, phase-shifted daily rhythms of clock genes and had a differential effect on the daily expression and activity of CAT and GPx. Thus, Aβ aggregates might affect clock-mediated transcriptional regulation of antioxidant enzymes, by affecting the formation of BMAL1:CLOCK heterodimer, probably, as a consequence of the alteration of the redox state observed in rats injected with Aβ.

Influence of Cyanobacterial Biofertilizer on the Response of Zea mays Plant to Cadmium-stress

CADMIUM (Cd) pollution is a thoughtful problem that alters crop yield and agricultural soils worldwide. Cyanobacteria are emerging candidates as eco-friendly biofertilizers. The present study evaluates the capability of super blue green biofertilizer (cyanobacteria biofertilizer (CB); Nostoc sp. and Anabaena sp.) to alleviate the Cd toxicity on 21 d-old Zea mays plants (applied as 2 and 10mM CdSO4). Application of CB significantly improved maize growth and reduced Cd-accumulation. Cadmium-induced oxidative stress indicated by increment of malondialdehyde (MDA), H2O2 contents and electrolyte leakage (EL) was diminished upon application of CB. Cd-stress significantly induced L-phenylalanine ammonia-lyase (PAL) specific activity and phenolics accumulation in the leaves and roots of maize plants. The results revealed that Cd-stress markedly enhanced GSH/GSSG ratio, gltuthione reductase (GR), superoxide dismutase (SOD) and catalase (CAT) specific activities, while it decreased that of ascorbate peroxidase (APx) and guaiacol peroxidase (GPx) and insignificantly increased polyphenol oxidase (PPO). The isozyme pattern of peroxidase was changed upon Cd-exposure. CB application significantly reduced both of the Cd-induced accumulated phenolics content and reduced glutathione (GSH) while it increased total glutathione (TG). CB application significantly induced all tested antioxidant enzymes. The data revealed that CB application could reduce Cd toxicity in maize plants.

Molecular Cloning and the Expression Pattern of a Phospholipid Hydroperoxide Glutathione Peroxidase in Kalidium foliatum under NaCl Treatment

The halophyte Kalidium foliatum (Pall.) Moq. exhibits strong tolerance to salinity. Salt stress leads to reactive oxygen species (ROS) production, and high contents of ROS are harmful to plants. The phospholipid hydroperoxide glutathione peroxidase (PHGPx) responds positively to damage induced by ROS. To understand the mechanism of tolerance to salt stress in halophytes, a PHGPx gene of K. foliatum was cloned, and its expression pattern was analysed under salt treatment. Treatments of whole plants were carried out with 0, 150, 200, 250, 300, 350 or 400 mM sodium chloride for 72 h. The extraction of total RNA from 4‑month-old seedlings was conducted using the leaves of both treated and untreated plants. The full-length cDNA of KfPHGPx was cloned with reverse transcription PCR (RT-PCR), the KfPHGPx expression patterns were analysed using semi-quantitative RT-PCR and real-time PCR, and the activities of phospholipid hydroperoxide glutathione peroxidase enzyme in K. foliatum were examined. The full-length cDNA clone encoding the full-length sequence consisted of 714 bp of nucleotides and included an open reading frame (ORF) that encoded a polypeptide of 237 amino acids. The deduced amino acid sequence of the KfPHGPx gene has significant similarity with Spinacia oleracea (XP_021837527.1) and Sesuvium portulacastrum (ADE07246.1). The semi-quantitative RT-PCR and real-time PCR analyse showed that KfPHGPx expression can be markedly induced by moderate salt treatment.

Genetic and biochemical variations among sugar beet cultivars resistant to Cercospora leaf spot

The goal of the study was to construct a reliable molecular and biochemical tool for the selection of resistant sugar beet cultivars to minimize the loss in productivity caused by Cercospora leaf spot disease. Three PCR marker systems: inter simple sequence repeats (ISSR), and sequence-related amplified polymorphisms (SRAP), and random amplified polymorphic DNA (RAPD) were used to assess the genetic diversity among eight cultivars of sugar beet and to detect the molecular markers linked to resistance to Cercospora beticola. Molecular tools successfully differentiated resistant from susceptible cultivars and detected 10 specific DNA markers in the resistant cultivars. Euclidean distance analysis based on physiological traits grouped the eight cultivars into two main clusters. Protein and isozyme patterns of peroxidase, polyphenol oxidase, chitinase, super oxide dismutase, glutamate oxalo-acetate transaminase and esterase were validated as quick means to select resistant cultivars of sugar beet. Specific protein and isozyme bands were lacking or were induced in cultivars based on their resistance phenotype. Our Results show that molecular markers and isozyme patterns are powerful tools for distinguishing resistant sugar beet cultivars resistant against Cercospora leaf spot.

Cadmium accumulation and alkaloid production of Narcissus tazetta plants grown under in vitro condition with cadmium stress

The capacity to accumulate cadmium (Cd) in Narcissus tazetta was investigated. Narcissus, an ornamental and medicinal plant can be used for the production of highly valuable alkaloids with anti-tumor, anti-viral and anti-cholinergic activities. For rapid propagation, in vitro micropropagation of this plant was done by three procedures: induction, proliferation and bulb production from twin scales as explants. After 4 months, micropropagated plants were treated by two concentrations of cadmium chloride (0.5, 1.0 mM) in the MS + 9% sucrose solidified media. After 3 weeks, growth, total proteins, peroxidase activity, Cd accumulation, quality and quantity of alkaloids were studied. Results showed that this plant accumulated 2778.13 μg g−1 DW, 801.87 μg g−1 DW and 162.8313 μg g−1 DW Cd in roots, bulbs and leaves respectively without any impact on growth. In order to study the ability of this plant in phytoremediation, tolerance index (TI), bioconcentration factors (BCF) and translocation factor (TF) were measured. Due to the TI > 0.6, BCF > 1 and TF < 1 concluded that Narcissus is suitable as phytostabilizer for remediation of Cd from contaminated media. Peroxidase activity increased under Cd stress. Also, isozyme pattern of peroxidase was changed and new anionic isoenzyme was appeared. Cd enhanced alkaloid content and altered its constituents as homolycorine was found instead of 9-O-demethylhomolycorine under Cd stress. This results presented in this study include the first report on the status of Narcissus alkaloids under Cd treatment.

Changes in activity and isozyme patterns of peroxidase and chitinase in kiwifruit pollen

Changes in activity and isozyme patterns of peroxidase and chitinase in kiwifruit pollen

Expression of dehydrin and antioxidant genes and enzymatic antioxidant defense under drought stress in wild relatives of wheat

The aims of this study were to investigate different species of Triticum and Aegilops in terms of the expression pattern of ascorbate peroxidase, superoxide dismutase, and guaiacol peroxidase encoding genes along with the wheat dehydrin gene, wdhn13, which is involved in the induction of tolerance to drought stress. The study also explored the enzyme activity of these three antioxidant enzymes under drought stress. There was a significant difference in the expression level of the four studied genes, Wdhn13, APX, SOD and GPX, between the well-watered and drought stress (FC = 100% and FC = 25%) treatments and among the examined species. The relative expression of Wdhn13 under conditions of drought was higher in T. boeoticum and Ae. crassa compared to the tolerant reference cultivar (Sirvan) and the rest of the wild species. Ae. tauschii showed the highest expression of the APX gene, and the SOD and GPX genes were up-regulated in Ae. tauschii and T. boeoticum at the drought-stressed conditions. The highest APX, GPX and SOD enzyme activities under the drought-stressed conditions were observed in Ae. crassa, T. boeoticum and Ae. neglecta, respectively. The results indicated that there was relevant correspondence between the genetically determined tolerance of wheat species (i.e. the gene transcript levels) and their antioxidant enzyme activities. The species having Ab (T. boeoticum), D (Ae. tauschii) and D (Ae. tauschii and Ae.crassa) genomes had a higher expression of GPX, SOD, and APX genes, respectively.

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.

Use of Electrophoretic Patterns of Proteins and Isozymes to Characterize Trichoderma Isolates from Cotton Roots

Proteins of 15 isolates of T. longbrachiatum and T. harzianum were compared by polyacrylamid gel electrophoresis (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels were stained with silver nitrate. Protein banding patterns of the isolates were subjected to cluster analysis by the unweighted pair-group method based on arithmetic mean (UPGMA). Apart from T. harzianum isolates T6 in PAGE and T27 in SDS-PAGE, which occupied separate positions in the phenograms, it was possible to delineate the isolates of each species on the basis of the results of PAGE and SDS-PAGE. Peroxidase (EC 1.11.1.7) and esterase (EC 3.1.1.1) isozymes from mycelium and conidia of Trichoderma isolates were separated by PAGE, and the obtained banding patterns were visualized by using specific staining systems. Cluster analysis indicated that delineation of the isolates of each species was not possible on the basis of the electrophoretic patterns of peroxidase. On the contrary, patterns of esterase isozymes provided a reliable method for grouping the isolates of each species with the exception of T9 and T14 of T. longbrachiatum, which occupied separate positions in the phenogram.

Morphological characteristics and isozyme banding patterns of Cucurbita moschata at different altitudes

Hidayati NR, Suranto, Sajidan. 2018. Morphological characteristics and isozyme banding patterns of Cucurbita moschata at different altitudes. Biodiversitas 19: 1683-1689. Aims of this research were to investigate the morphological character and isozyme banding patterns of Cucurbita moschata plants grown at three different altitudes. Samples in this study consisted of leaf, stem, and flowers. The morphological characters were conducted by direct observation in the field and analyzed descriptively as well as statically by one way ANOVA. The isozyme bands appearance of esterase and peroxidase of leaf samples were conducted using polyacrylamide gel electrophoresis (PAGE). Qualitative approach was used to analyze the presence and the absence of isozyme bands, while Retardation factor (Rf) was used to analyze quantitatively. The results showed that most plants grown at middle altitude (351-750 m asl.) were well-developed in terms of length of leaves, stems and flowers. Accordingly, the isozyme banding pattern of peroxidase was also found varied in plants grown at middle altitudes from which the presence of very unique bands was detected. Conversely, the band detected in plants grown at the lower and the highest altitudes was similar in term of band's number but it was different in the quality of the bands. Meanwhile, esterase isozyme banding pattern of plants grown at the lower and higher altitude had more bands than the middle altitude. Based on this result it is obvious that the isozyme data could be used to support in understanding the diversity morphological characters of plants grown in three different altitudes. This early result suggests that altitudes as a crucial factor in contributing the expression of isozyme appearance, which is useful for further pumpkin characterizations.

Influence of PEG induced drought stress on molecular and biochemical constituents and seedling growth of Egyptian barley cultivars

In order to investigate the effects of drought stress on germination components of barley cultivars, a laboratory experiment was conducted in a factorial randomized complete design with four replications. The controlled experiment included ten of Egyptian barley cultivars namely; (Giza 123, 124, 125, 126, 127, 129, 130, 134, 135 and 2000) as first factor. The second factor included 4 levels of drought stress inducer by applying 0, 5, 10 and 20% of polyethylene glycol-6000 (PEG) which is equivalent to four osmotic potential levels including −0.001, −0.27, −0.54 and −1.09 MPa, respectively. The results showed that, the highest reduction was related to the drought level of 20% PEG among the barley cultivars. The best cultivars in terms of germination traits were Giza 134, Giza 127, and Giza 126 this indicate their tolerance to drought stress and Giza 130, 135, 2000 cultivars was moderately tolerance and remaining is less tolerance. The protein band 27 kDa and 78 kDa showed high intensity after stress in almost all cultivars. Those two protein bands their exciting was very clear in treated barley leaf tissue. It could be related to dehydrine and oxygen evolving enhancer protein 2 (OEE2) which involved in drought stress tolerance response. Cultivars Giza 127, 130 and 134 showed highest tolerance response under drought stress. The antioxidant enzymes PAGE pattern of Peroxidase (POX), Sodium dismutase (SOD) and Ascorbate peroxidase (APX) for Barley cultivars under drought stress revealed a high activities for Giza 126, 127, 134, 136 and 2000 under −0.5 MPa osmotic stress by PEG in most of their isoforms. Based on similarity coefficient values the highest values were 1.0 with 100% similarly between tolerant cultivars Giza 130 and Giza 127. Similarly between the susceptible cultivars 125 and Giza 129 was 60%.These data confirmed by the growth parameters which we ranked as tolerant to drought stress.

Influence of anti- and prooxidants on rhizogenesis from hypocotyls of Mesembryanthemum crystallinum L. cultured in vitro

The enrichment with antioxidants (glutathione or ascorbate) or prooxidants (alloxan, methylviologen, hydrogen peroxide) of root inducing medium significantly decreased rhizogenesis frequency (alloxan, hydrogen peroxide) or inhibited roots regeneration (ascorbate, methylviologen) during the in vitro culture of Mesembryanthemum crystallinum L. hypocotyls. The adventitious roots morphology, root hairs length and density, was also influenced. Changes in the rhizogenesis course were related to the differences in hydrogen peroxide concentration during following days of culture between explants exhibiting morphogenic potential and those without the ability to form adventitious roots. In explants with morphogenic potential, rhizogenesis induction was always accompanied by a high level of hydrogen peroxide followed by the decrease in H2O2 content in following days. In contrast, in the explants without regeneration potential, the level of hydrogen peroxide was increasing during the culture period. Activity patterns of superoxide dismutase (SOD) and guaiacol peroxidase (POX) in the following days of culture were similar in the explants exhibiting regeneration potential cultured on different media. Total activity of SOD decreased during initial days of culture and then increased due to the activation of additional SOD isoform described as MnSODII. The activity of POX was low during the rhizogenesis induction, and then increased during following days of culture; the increase was correlated with the decrease in hydrogen peroxide content. In the explants without the ability to regenerate roots, the total activity of SOD was low throughout the whole culture period, whereas the POX activity was significantly higher than in hypocotyls with regeneration potential. It might be concluded that the increase in hydrogen peroxide during initial stages of rhizogenesis and the induction of MnSODII are prerequisites for adventitious roots formation from hypocotyls of M. crystallinum, independently in the presence of anti- or prooxidant in the culture medium.

Modification of antioxidant systems in cell walls of maize roots by different nitrogen sources

Antioxidant systems of maize root cell walls grown on different nitrogen sources were evaluated. Plants were grown on a medium containing only NO3- or the mixture of NO3-+NH4+, in a 2:1 ratio. Eleven-day old plants, two days after the initiation of lateral roots, were used for the experiments. Cell walls were isolated from lateral roots and primary root segments, 2-7 cm from tip to base, representing zones of intense or decreased growth rates, respectively. Protein content and the activity of enzymes peroxidase, malate dehydrogenase and ascorbate oxidase ionically or covalently bound to the walls, as well as cell wall phenolic content and antioxidant capacity, were determined. Cell walls of plants grown on mixed N possess more developed enzymatic antioxidant systems and lower non-enzymatic antioxidant defenses than cell walls grown on NO3-. Irrespective of N treatment, the activities of all studied enzymes and protein content were higher in cell walls of lateral compared to primary roots. Phenolic content of cell walls isolated from lateral roots was higher in NO3--grown than in mixed N grown plants. No significant differences could be observed in the isozyme patterns of cell wall peroxidases isolated from plants grown on different nutrient solution. Our results indicate that different N treatments modify the antioxidant systems of root cell walls. Treatment with NO3- resulted in an increase of constitutive phenolic content, while the combination of NO3-+NH4+ elevated the redox enzyme activities in root cell walls.

Identification and characterization of Memecylon species using isozyme profiling

Background:The protein/isozyme fingerprint is useful in differentiating the species and acts as a biochemical marker for identification and systematic studies of medicinal plant species.Objective:In the present study, protein and isozyme profiles for peroxidase, esterase, acid phosphatase, polyphenol oxidase, alcohol dehydrogenase, and alkaline phosphatase of five species of Memecylon (Melastomataceae), Memecylon umbellatum, Memecylon edule, Memecylon talbotianum, Memecylon malabaricum, and Memecylon wightii were investigated.Materials and Methods:Fresh leaves were used to prepare crude enzyme extract for analyzing the five enzymes isozyme variations. Separation of isozymes was carried out using polyacrylamide gel electrophoresis (PAGE) and the banding patterns of protein were scored. Pair-wise comparisons of genotypes, based on the presence or absence of unique and shared polymorphic products, were used to regenerate similarity coefficients. The similarity coefficients were then used to construct dendrograms, using the unweighted pair group method with arithmetic averages.Results:A total of 50 bands with various Rf values and molecular weight were obtained through PAGE analysis. Among the five Memecylon species, more number of bands was produced in M. wightii and less number of bands was observed in M. edule. The results of similarity indices grouped M. malabaricum and M. wightii in one cluster with 98% similarity and M. umbellatum, M. edule, and M. talbotianum are grouped in another cluster with 79% similarity showing close genetic similarities which is in accordance with the morphological identification of Memecylon species.Conclusion:The protein/isozyme fingerprint is useful in differentiating the species and acts as a biochemical marker for identification of Memecylon species.SUMMARY Biochemical characterization of Memecylon species was evaluated by SDS-PAGE of extracted protein and isozyme profiling on native PAGE.After electrophoresis, each gel was stained with specific stains. Genetic distance relationships were evaluated based on the banding patterns of protein on isozymes.Unique banding pattern of esterase, peroxidase, acid phosphatase, alcohol dehydrogenase and polyphenol oxidase are observed in all the five species of Memecylon, which represent the fingerprint of Memecylon species.SDS-PAGE and isozyme profiling of five Memecylon species revealed that M. malabaricum and M. wightii grouped in one cluster and M. umbellatum, M. edule and M. talbotianum grouped in another cluster showing close genetic similarities which is in accordance with the morphological identification of Memecylon species.This is the first report on the comparison of protein and isozyme profile of five different Memecylon species. Abbreviations Used: SDS-PAGE: Sodium docecyl sulfate polyacrylamide gel electrophoresis; NTSYS PC2: Numerical taxonomy system, version 2.2 for Windows XP, Vista, Win7, Win 8 and Win10 including 64 bit

Assessment of Genetic Diversity in Mustard (Brassica juncea L.) Genotypes using Biochemical and Molecular Markers

Indian mustard (Brassica juncea L.) is an important source of edible oil in India. Quality parameters such as moisture, oil, protein, erucic acid and sinigrin were analysed in sixty genotypes of mustard (Brassica juncea L.) and from which 15 genotypes with desired proximate parameters were selected and analysed further for anti nutritional factors, genetic diversity and isozymes. Erucic acid and Glucosinolate (Sinigrin) was analysed by gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography (UPLC), respectively. SKM 9033 showed the highest (30.88%) oil where as RAYAD-9602 had the lowest (20.80%) oil. The protein content and true protein in mustard cultivars ranged from 27.90–33.69% and 10.66–20.01%, respectively. HNT-33 genotype had the highest concentration of erucic acid where as NRCM120 showed the lowest. Bio-Q-44-279 had the highest sinigrin concentration (46.80 μmole/g) while SKM-9033 had the lowest sinigrin concentration (2.49 μmole/g). Isozyme pattern of peroxidase and esterase showed significant correlation amongst the clusters. Electrophoresis of seed protein by SDS-PAGE showed a total of 28 bands with varied intensity in mustard genotypes. Both Simple Sequence Repeat (SSR) and Inter Simple Sequence Repeat (ISSR) markers were found to be efficient to assess genetic diversity within species of mustard with average polymorphism of 82.91% and 96.02%, respectively. NRCM-120 and SKM-9033 had low content of erucic acid and sinigrin respectively and hence were found to be useful for the development of double zero variety by breeding program. The SW-1-9017 and Bio-341-92 genotypes showed high genetic diversity compared to other genotypes and found to be useful for breeding purpose.

Glutathione peroxidase 1 expression, malondialdehyde levels and histological alterations in the liver of Acrossocheilus fasciatus exposed to cadmium chloride

Cadmium (Cd) is known as a widespread pollutant in aquatic environment. The accumulation of reactive oxygen species (ROS) is attributed to Cd exposure, which may affect the growth, development and physiological metabolism of aquatic organisms. In response to these unfavorable damages, antioxidant systems have been developed to protect against oxidative stress. In this study, we investigated the expression pattern of glutathione peroxidase 1 genes (GPx-1a and GPx-1b) in the liver of Acrossocheilus fasciatus after Cd administration. Total RNA extraction, reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were performed in order to clone the A. fasciatus GPx-1a and GPx-1b full-length cDNA sequences and partial fragment of β-actin cDNA from the liver for the first time. Tissue-specific expression analysis proved that GPx-1 genes were widely expressed in the liver, kidney, gill, testis, muscle, spleen, heart and brain. The changes of GPx-1 mRNA and malondialdehyde (MDA) levels in the liver treated with Cd were measured. In addition, the acute toxic effects of Cd on the microstructure of the liver were studied using light microscopy. These results suggest that GPx-1, MDA and liver histology which represent molecular, biochemical and histological levels, can be used as potential biomarkers to monitor Cd pollution. The overall findings also highlight the potential use of those three bio-indicators combined together as a multi-level tool (molecular, biochemical and histological levels) when monitoring Cd contamination and other possible exogenetic pollutants in aquatic environment.

Patterns of laccase and peroxidases in coarse woody debris of Fagus sylvatica, Picea abies and Pinus sylvestris and their relation to different wood parameters

Lignin and its degradation, particularly in forest ecosystems, play a major role in the global carbon cycle. Filamentous fungi equipped with extracellular oxidoreductases (oxidative enzymes), i.e., laccase, manganese-dependent peroxidases and several other peroxidases, are the key players in the bioconversion of lignin. In particular, for coarse woody debris (CWD), this process is poorly understood and the activities of laccase and peroxidases have never been studied on a large field scale. We investigated the activities of these enzymes in 701 samples of Fagus sylvatica, Picea abies and Pinus sylvestris CWD across three regions in Germany and analyzed their dependence on pH, water content, wood density, total lignin, organic extractives, metals, water-soluble lignin fragments and fungal species richness. Respective enzyme activities were present in 79 % of all samples, and the activities were highly variable and more frequent in F. sylvatica than in coniferous wood. Logistic regressions and correlations between enzyme activities and the variables revealed that the fungal community structure and the amount of water-soluble lignin fragments are most important determinants, and that the prevalent acidic pH in CWD is suitable to facilitate laccase and manganese peroxidase activities. Concentrations of metals (manganese, copper, iron) were sufficient to ensure synthesis and functioning of relevant enzymes. Based on this large field study, we conclude that laccase and peroxidases in CWD are highly relevant for lignin degradation, but the variable pattern of their secretion is the result of a complex array of wood parameters and the fungal community structure, which could only partly be resolved.