Isozyme Spectrum Research Articles

The spectrum of lactate dehydrogenase isozymes in ovarian tissue of cows under different physiological states and pathologies

The article presents the results of studies on the lactate dehydrogenase (LDH) activity and content of its isozymes in ovarian tissue under various physiological and pathological states of cow gonads. Cow ovaries of the Ukrainian dairy black-spotted breed (age 5–8 years, live weight 450–500 kg) of different physiological states were used for research: with “early yellow body”, diameter 10–20 mm, red or brown color (EYB; n = 41); with “late yellow body”, diameter 5–15 mm, color yellow (LYB; n = 32); “follicular growth”, without corpus luteum (FG; n = 84) and with hypofunction (n = 21). The activity and content of lactate dehydrogenase (LDH) isozymes were determined in the supernatant after centrifugation of gonadal tissue homogenates prepared according to standard methods. The content of LDH isozymes (%) was determined after vertical protein electrophoresis of tissue samples in plates of 7.5 % polyacrylamide gel (PAAG) and specific staining according to J. Garbus in our modification and calculated with TotalLab TL120 software. LDH activity was determined by the rate of NADH oxidation and was calculated in µcatal/mg of protein. In all samples, 5 catalytically active enzyme bands were detected: LDH1 – LDH5. It was established that the activity and content of LDH isozymes depends on the physiological state of the gonad. Thus, in the ovarian tissue of cows, LDH activity was the same (2.8–3.0 µcatal/mg protein) under the physiological state of “follicular growth” and pathology – “hypofunction” and is lower by 0.6 and 1.0 µcatal/mg protein under conditions of “early” and “late” corpus luteum (2.0 and 2.2 µcatal/mg protein). The biggest content of LDH1 was (49.3 ± 1.64 %) in the tissue in the physiological state of “late yellow body”. The greatest amount of LDH3, LDH4 and LDH5 (16.4 ± 1.70, 11.2 ± 2.42 and 13.2 ± 3.06 %) – at state “early yellow body”. The content of LDH2 was high (27.5 ± 4.26 %) in the case of ovarian "hypofunction", and in the other physiological states, the value was lower and almost had no difference (19.8 and 22.1 %). The analysis of correlations shows the moderate dependence of LDH activity and the content of isozymes on the physiological state and hypofunction of the gonad (LDH total activity – η = 0.387, LDH 1 – η = 0.465; LDH3 – η = 0.551; LDH4 – η = 0.336; LDH5 – η = 0.347).

Distribution of isozyme spectra of lactate dehydrogenase in tissues of invasive species mammals of Karelia

Distribution of isozyme spectra of lactate dehydrogenase in tissues of invasive species mammals of Karelia

CKD-581 Downregulates Wnt/β-Catenin Pathway by DACT3 Induction in Hematologic Malignancy.

The present study evaluated the anti-cancer activity of histone deacetylase (HDAC)-inhibiting CKD-581 in multiple myeloma (MM) and its pharmacological mechanisms. CKD-581 potently inhibited a broad spectrum of HDAC isozymes. It concentration-dependently inhibited proliferation of hematologic cancer cells including MM (MM.1S and RPMI8226) and T cell lymphoma (HH and MJ). It increased the expression of the dishevelled binding antagonist of β-catenin 3 (DACT3) in T cell lymphoma and MM cells, and decreased the expression of c-Myc and β-catenin in MM cells. Additionally, it enhanced phosphorylated p53, p21, cleaved caspase-3 and the subG1 population, and reversely, downregulated cyclin D1, CDK4 and the anti-apoptotic BCL-2 family. Finally, administration of CKD-581 exerted a significant anti-cancer activity in MM.1S-implanted xenografts. Overall, CKD-581 shows anti-cancer activity via inhibition of the Wnt/β-catenin signaling pathway in hematologic malignancies. This finding is evidence of the therapeutic potential and rationale of CKD-581 for treatment of MM.

The effects of temperature changes on the isozyme and Hsp70 levels of the Amur sturgeon, Acipenser schrenckii, at two acclimation temperatures

The isozyme spectrum expressions and Hsp70 expressions in the muscle and the isozyme activities in the liver were measured to evaluate the stress response of the Amur sturgeon, Acipenser schrenckii, to rapid temperature changes at two acclimation temperatures. The fish (body weight 6.18 ± 0.21 g) were subjected to an increasing temperature change (from 13 °C to 23 °C, Tinc treatment, 60 individuals) or to a decreasing temperature change (from 23 °C to 13 °C, Tdec treatment, 60 individuals) for 72 h. After 0 h, 1 h, 3 h, 12 h, 24 h, 48 h and 72 h of exposure, the fish (n = 7) were removed and prepared for further analysis. The results showed that the levels of Hsp70 in the muscle were significantly upregulated after 48 h of heat exposure (P < 0.05) in the Tinc treatment; however, the level of Hsp70 increased quickly after 1 h of cold exposure (P < 0.05) and returned to initial levels after 3 h of cold exposure in the Tdec treatment (P < 0.05). The expressed levels of alkaline phosphatase (ALP), superoxide dismutase (SOD), lactate dehydrogenase (LDH), acid phosphatase (ACP) and esterase (EST) in the muscle showed significant changes in both the Tinc and Tdec treatments (P < 0.05). The activities of ALP, LDH, ACP and peroxidase (POD) in the liver were significantly lower in the Tinc treatment than that in the Tdec treatment at 0 h after exposure (P < 0.05). The activities of ALP, SOD, LDH, ACP and POD in the liver showed significant changes after different heat or cold exposure times (P < 0.05). The results suggest that qualitative and quantitative changes in isozymes and Hsp70 during temperature changes may significantly contribute to the metabolic reorganization that occurs during temperature acclimation, which imply that the Amur sturgeon has shown good suitability for aquaculture due to its metabolic regulatory ability to respond to acute temperature changes.

Assessment of potential winter hardiness in winter bread wheat cultivars and genotypes by analyzing autofluorescence in seedling tissues

Background.Winter crops are the most productive component of agricultural biocenoses. In Russia, winter wheat suffers the greatest losses in winter, so a search for traits marking high or low winter hardiness in autumn-sown genotypes, including improved cultivars, is needed to assess their potential for overwintering. One of such markers of high winter hardiness is an increased lignin content in plant tissues. The terminal enzyme in the phenylpropanoid pathway of metabolism, wherein lignin components are formed, is cinnamyl-alcohol dehydrogenase (CAD, EC 1.1.1.195). In plants, the CAD enzyme is one of the links in the aromatic metabolism, which generates, in addition to lignin, a number of aromatic compounds, such as lignans, aromatic glycosides, etc. Many of these compounds, like lignin, contain chromophore groups and are capable of autofluorescence.Correlations of the genotypes that incorporate CAD1-F with overwintering are studied in this work.Materials and methods.The winter bread wheat cultivars ‘Zitnica’ (Yugoslavia) and ‘Novosibirskaya 9’ (ICG SB RAS, Russia), contrasting in winter hardiness and CAD isozyme spectra, their hybrids, and 28 improved winter cultivars developed in Krasnodar were selected for the study. Fluorescence analysis of 28 winter wheat cultivars was also performed. Correlation coefficients between fluorescence and frost tolerance were calculated using the results of the analysis of 7 most contrasting cultivars.Conclusions. The tested winter bread wheat genotypes demonstrated the interplay between CAD1-F and successful overwintering: a correlation was found in the genotypes carrying the 00 CAD1-F allele with higher percentage of overwintered plants. This dependence was not observed in every season. The analysis of seedling sections for fluorescence can also be used for preliminary assessment of winter tolerance in winter bread wheat under laboratory conditions.

Research Advances of Leptotrombidium scutellare in China.

Leptotrombidium scutellare is one of the 6 main vectors of scrub typhus in China. It has been found in more than 15 provinces of China. Especially in Yunnan, it was found to be mainly distributed in some mountainous areas with high altitude, low temperature and low precipitation. Rodents and some other small mammals were the most common hosts of L. scutellare. To date, more than 40 host species of L. scutellare have been recorded with very low host specificity, and the main hosts varied in different geographical regions. L. scutellare had a strong resistance against the cold environment, and the temperature and humidity were 2 important factors affecting its growth and development. Among different individuals of their rodent hosts, L. scutellare mites often showed an aggregated distribution pattern, which reflected the interspecific cooperation of the mites. The chromosome karyotype of L. scutellare was 2n=16 and all the 8 pairs of chromosomes were short rod-shaped with metacentric or sub-metacentric types. The isozyme spectrum supported that L. scutellare, L. deliense and L. rubellum were in the same species group. Based on the natural infection, experimental transmission and epidemiological evidence, L. scutellare has been eventually confirmed as the second major vector of scrub typhus in China, which is second only to L. deliense.

COMPARATIVE CHARACTERISTICS OF THE TERMINAL LINK OF GLYCOLYSIS IN THE MUSCLES OF ADULT ANIMALS AND THEIR OFFSPRING

The organism of sexually mature individuals differs significantly from the organism of immature individuals in a number of anatomical, physiological and biochemical parameters. The aim of the work was to investigate the state of the terminal link of glycolysis in the muscles of sexually mature animals and their offspring. The isoenzyme spectrum of LDH in the myocardium of sexually mature animals is characterized by a high content of LDH1 and LDH2 isozymes rapidly migrating to the anode. The isoenzyme spectrum of LDH in skeletal muscles of mature animals is represented mainly by the LDH 5.A feature of the isozyme spectrum of LDH in the tissues of rat pups is that the content of LDH1 and LDH2 is significantly reduced in the myocardium. Their number is 1.2 and 1.13 times, respectively, less than in sexually mature animals. Against this background, the content of LDH3 increases slightly, the content of LDH4 exceeds twice, and that of LDH5 is more than 6 times the indicators of sexually mature animals. In skeletal muscles of rat pups, the dominant content of LDH5 and LDH4 increases and this occurs due to a decrease in the activity of LDH3 (more than 1.5 times), LDH2 (more than 2.3 times) and LDH1 (2.2 times) compared with sexually mature animals. The data obtained indicate that in the myocardium and skeletal muscles of rat pups there is a greater percentage of isoenzymes formed from M-subunits functioning under anaerobic conditions, and with age, as a result of epigenetic transformations, the content of H-subunits increases.

Lactate Dehydrogenase Isozymes in the Tissues of Hibernating Bats (Chiroptera)

A comparative electrophoretic assay of lactate dehydrogenase (EC 1.1.1.27) isozymes has been carried out in the homogenates of the tissues of cardiac and skeletal muscles, liver, kidneys and lungs of five species of hibernating bats of the order Chiroptera: the northern bat Eptesicus nilssonii Keyserling and Blasius, the brown long-eared bat Plecotus auritus L., Brandt’s bat Myotis brandtii Eversmann, Daubenton’s bat Myotis daubentonii Kuhl, and the whiskered bat Myotis mystacinus Kuhl, which live in Karelia near the northern border of their distribution area. High contents of aerobic lactate dehydrogenase 1 and lactate dehydrogenase 2 isozymes have been detected in the skeletal muscle of the studied bats. The lactate dehydrogenase isozyme spectra of the tissues of kidneys and skeletal muscles from the smaller representatives of bats (the whiskered and Brandt’s bats) contained the highest content of H subunits among the studied species. In contrast, the predominance of M subunits has been revealed in the lactate dehydrogenase isozyme spectra of the kidneys of the northern and the brown long-eared bats. The discovered interspecies differences are discussed in the context of the adaptation of bats to hibernation.

The isozyme spectra of lactate dehydrogenase in the tissues of the raccoon dog Nyctereutes procyonoides in the autumn

An electrophoretic assay of lactate dehydrogenase (EC 1.1.1.27) isozymes in the tissue homogenates of cardiac and skeletal muscles, kidney, lungs, spleen, and liver of the raccoon dog Nyctereutes procyonoides from two different geographic zones, viz., northwestern Russia and Poland, as well as the Arctic blue fox Alopex lagopus L. and the red fox Vulpes vulpes L. was performed during the preparatory period to the winter season. Raccoon dogs, which hibernate under natural conditions, differ from other canids (the red fox and Arctic blue fox) to which they are close taxonomically by their body weight and by the higher proportion of aerobic H subunits of lactate dehydrogenase in all organs except for the heart. A higher content of “fast” anode fractions, lactate dehydrogenase-1 and lactate dehydrogenase-2, in the heart, kidney, lungs, liver, and spleen was detected in the raccoon dogs from the northern region compared to those from the southern geographic zone. The shift in the reaction catalyzed by lactate dehydrogenase towards the production of pyruvate indicates that this metabolite is necessary for the synthesis of fatty acids during lipogenesis in the autumn.

AGE-RELATED CHANGES OF PHYSIOLOGICAL AND BIOCHEMICAL PARAMETERS IN RODENTS WITH DIFFERENT ECOLOGICAL SPECIALIZATION

We performed the comparative study of age-related changes in some physiological parameters of the muskrat, which can perform dives and therefore well adapted to the lack of oxygen, and in the non-diving laboratory rat. Analysis revealed the considerable interspecific differences in the antioxidant system and the energy supply level in the heart and kidney tissues. The age-related changes in the studied values were different between species. Results showed the unbalance in the activities of superoxide dismutase (SOD) and catalase, and also compensatory change in the level of such nonenzymatic antioxidant as vitamin E in rats during aging. The muskrat had marked age-related increase of the antioxidant enzymes activities in heart and kidney. Young muskrat of the second generation had a lower specific SOD and catalase activities compared with the first generation animals, possessed the same values as the adults. We identified the interspecific differences in the differential blood leukocyte counts. During ontogenesis the size of erythrocytes and leukocytes increase in rat, but decrease in muskrat, that is necessary for improvement of blood rheological properties and appears as an adaptation to semi-aquatic lifestyle. Age-related changes in the tissue isozyme spectrum of lactate dehydrogenase, in tissue concentration of vitamin E and of leukograms were expressed in muskrats to the lesser extent than in laboratory rats.

Violation of the law of uniformity of the first generation of hybrids

Variability was revealed in the isozyme spectra in the seed progeny from crosses of two beet plants homozygous for contrasting alleles of marker enzyme loci.

INFLUENCE OF DRYING AND GENEBANK STORAGE ON INITIAL GROWTH, PEROXIDASE ACTIVITY AND REGENERATION CAPACITY OF WHEAT SEEDS

Seeds of three winter wheat bread cultivars (Triticum aestivum L.): Sadovo 1, Sadovo 772 and Katya, and three durum wheat cultivars (Triticum durum Desf): Progress, Vazhod and Beloslava were used. The accessions have been submitted to sorbtion drying to low moisture content (water activity aw = 0,116). No essential changes were observed in the initial seedling growth. In evaluation of peroxidase isozyme spectra of seedlings no significant changes were detected as a result of sorption drying to low seed moisture and subsequent storage at -18оС. Field evaluation of the main structural plant yield elements uncovered no statistically significant unfavorable effects on the regeneration capacity of wheat seeds as a result of drying (water activity aw = 0,116) and cool storage (-18oC).

INFLUENCE OF DRYING AND GENEBANK STORAGE ON INITIAL GROWTH, PEROXIDASE ACTIVITY AND REGENERATION CAPACITY OF WHEAT SEEDS

Seeds of three winter wheat bread cultivars (Triticum aestivum L.): Sadovo 1, Sadovo 772 and Katya, and three durum wheat cultivars (Triticum durum Desf): Progress, Vazhod and Beloslava were used. The accessions have been submitted to sorbtion drying to low moisture content (water activity aw = 0,116). No essential changes were observed in the initial seedling growth. In evaluation of peroxidase isozyme spectra of seedlings no significant changes were detected as a result of sorption drying to low seed moisture and subsequent storage at -18оС. Field evaluation of the main structural plant yield elements uncovered no statistically significant unfavorable effects on the regeneration capacity of wheat seeds as a result of drying (water activity aw = 0,116) and cool storage (-18oC).

Effect of environmental and genetic factors on the stability of pea (Pisum sativum L.) isozyme and DNA markers

Environmental (geographic location, year-to-year effect) and genetic (intravarietal variation) effects on the stability of the isozyme patterns of esterase (EST), acid phosphatase (ACP), alcohol dehydrogenase (ADH), leucine aminopeptidase (LAP), shikimate dehydrogenase (SDH) and glucose-6-phosphate isomerase (PGI) used for pea cultivar genotyping were studied. In addition, selected DNA markers (RAPD, SSR, ISSR, IRAP) were used to study intravarietal genetic homogeneity/variation at a DNA level. Five commercial dry-seed pea (Pisum sativum L.) cultivars Canis, Gotik, Komet, Sonet and Zekon were grown during four years (2001 &amp;ndash; 2004) in three locations in the Czech Republic (&amp;Scaron;umperk, Č&amp;aacute;slav, Uhersk&amp;yacute; Ostroh) that differed in soil and climatic characteristics. Mature dry seeds were used as a standard input sample for analyses. No variation in isozyme spectra was found within particular cultivars between years, locations and fruiting nodes in enzymes providing in general a low polymorphism in pea cultivars (ACP, ADH, LAP, SDH, PGI); similarly, these enzymes also exhibited high stability as related to intravarietal variation. In contrast, EST &amp;ndash; highly polymorphic in pea cultivars &amp;ndash; showed certain qualitative variation within particular cultivars as related to both environmental and genetic factors. The intravarietal variation detected by selected DNA markers was negligible and mostly quantitative. Possible reasons for the instability/variation of isozyme markers are discussed from the aspect of cultivar genotyping used in pea breeding and seed production.

Analysis of linkage between genes controlling enzymes in sunflower

The polymorphism of leucine aminopeptidase in 23 inbred lines of sunflower of mutant origin was studied. The isozyme spectrum of leucine aminopeptidase is presented as a single zone of enzyme activity controlled by one gene. This gene was shown to be inherited independently from genes of esterase and 6-phosphogluconate dehydrogenase.

FLAX PLANTS REGENERATED FROM UNPOLLINATED OVULES CULTURED IN OVARY SEGMENTS

We aimed to observe and to compare the induction rate of calli and regeneration rate of shoots originated from unpollinated ovules cultured in flax ovary segments. The percentage of induced calli in ovary culture varied from 15,4% in genotype AC Emerson to 32,4% in genotype Viking. Conversely the regeneration rate was higher in AC Emerson (8,8%) rather than in genotype Viking (1,8%). After four month in culture we screened for somaclonal variation in the calli. Flow cytometry analysis showed that the yellow-green calli in culture were mostly diploid in genotype Viking (83,3%) and in AC Emerson (100%). The dark green calli were of higher ploidy level and contained cells of 4n, 6n, 8n, 16n. Regeneration of shoots was performed exclusively from the diploid cells in the calli as we found out using, karyological analysis on squashes on root tip cells. The origin of regenerated plants was proved using isozyme spectra analysis.

Enzyme Polymorphism in Two Japanese Quail (Coturnix Coturnix Japonica) Lines with Different Concentrations of Plasma Lactate

Japanese Quail (Coturnix coturnix japonica) lines selected for different physiological traits such as metabolic rate are expected to be more suitable for experiments than random populations. The general level of lactate in a randomly bred Japanese Quail population varies within the limits 3.6 mM•L-1. Breeding experiments were performed in order to reduce the variation of plasma lactate. The two quail lines, exhibiting low (L line with 5.281–5.840 mM•L-1) and high (H line with 3.157–4.529 mM•L-1) plasma lactate concentration were divergently selected. During the selection process, genetic variation in loci for liver and heart tissue malate dehydrogenase (Mdh), malic enzyme (Me), esterase (Es) as well as for plasma glucose-6-phosphate dehydrogenase (G-6-Pdh) and common serum proteins was investigated in individual animals. A phenotypic difference in the spectrum of MDH isozymes was detected between the two quail lines L and H. Our data suggest that the genetic constitution of quail lines and the control pop...

Studying allozyme variation in sexual and apomictic Taraxacum and Pilosella (Asteraceae) populations

Allozyme spectra of peroxidase, esterase, superoxid dismutase, tyrosinase, alcohol dehydrogenase, lactate dehydrogenase, and acid phosphatase were examined in populations of sexual (Taraxacum serotinum and Pilosella echioides) and apomictic (T. officinalis and P. officinarum) plant species. The heterozygosity in these populations (0.455-0.620) proved to be considerably higher than the average level characteristic of plant populations (0.058-0.185). The populations examined did not differ in the mean phenotype number mu, i.e., they exhibited the same diversity (3.213-3.380). The proportion of rare phenotypes h also did not differ between the sexual and apomictic species of the same genus, whereas this parameter in the Pilosella populations (0.150-0.174) was significantly higher than in the Taraxacum ones (0.093-0.114). The populations were characterized by numerous isozyme spectra (more than 11 per populations) and displayed multiple allelism (the mean allele frequency was 3.63-4.38 per locus). They exhibited a high percentage of rare (occurring at a frequency lower than 5%) spectra (35-80%). This indicates that agamic complexes, to which these populations belong, may have a more complicated genetic structure of both apomictic and sexual populations than the species that do not belong to agamic complexes.

Genetic Relationship and Clustering of Some Sugarcane Genotypes Based on Esterase, Peroxidase and Amylase Isozyme Polymorphism

Three isozyme systems viz. esterase, peroxidase and amylase were assayed electrophoretically from extracts of leaf tissues of some elite genotypes of India to unravel their genetic relationship. Twenty five isozyme phenotypes were resolved in the 9 genotypes; 8 in esterase (EST), 12 in peroxidase (POX) and 5 in amylase (AMY). The genotypes could be distinguished on the basis of polymorphic isozyme spectra and multimeric isozyme profiles. The most polymorphic isoenzyme system was peroxidase (POX), which provided a discriminatory tool for evaluating and characterizing genotypes. The banding similarly index based on the number of polymorphic bands common between each genotype pair, ranged from 0.38 to 0.89. The genotypes were clustered in 2 distinct groups having 3 and 6 genotypes each by subjecting the genetic dissimilarity between them to UPGMA (Unweighted Pair Group Mean Analysis) statistical treatment and bootstrap analysis. The consistency Index of 8 different nodes present in phenogram representing percentage useable isozyme loci per node showed the node 8 to be the most important and consistent node followed by the node 6. Some esterase and peroxidase isozyme phenotypes were specific to the genotypes and were found to be suitable for cultivar identification.

Peroxidase isozyme polymorphism in Cucurbita pepo cultivars with various morphotypes and different level of field resistance to powdery mildew

A set of 31 Cucurbita pepo L. commercial cultivars with different fruit shapes (morphotypes) was evaluated for resistance to powdery mildew of cucurbits ( Erysiphe cichoracearum and Sphaerotheca fuliginea) under field conditions of a natural infection. During vegetative growth, the infection degree (ID) was assessed four times on leaves (L) and twice on stems and petioles (S). The `Area below the curve' values of the disease infection progress were calculated separately for leaves (ABC-L) and stems and petioles (ABC-S). Data were subjected to one-way analyses of variance and mean separation was performed using LSD multiple range tests. Significant differences in field resistance were found between morphotypes. Cultivars with scallop and acorn fruit shapes were the most resistant. The straightneck, crookneck and ornamental gourd groups showed medium levels of field resistance. The zucchini, cocozelle, pumpkin and vegetable marrow groups of morphotypes were the most susceptible. Isozyme spectra of peroxidases for 11 selected C. pepo cultivars representing different groups of morphotypes with various levels of field resistance were analysed as well. They could be ranged into three basic groups which correspond with the cultivar's level of field resistance. This phenomenon is discussed as a potential biochemical marker in C. pepo selection.