Phosphorylated PKM2 regulates endothelium-dependent vasodilation in diabetes / 中南大学学报(医学版)

OBJECTIVES@#Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy.@*METHODS@#The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 μmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels.@*RESULTS@#Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05).@*CONCLUSIONS@#p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.

Population structure and phylogenetic relationships in Brassica rapa L. subspecies by using isozyme markers / Estrutura populacional e relações filogenéticas em subespécies de Brassica rapa L. utilizando marcadores isoenzimáticos

Abstract The present study aimed to assess population structure and phylogenetic relationships of nine subspecies of Brassica rapa L. represented with thirty-five accessions cover a wide range of species distribution area using isozyme analysis in order to select more diverse accessions as supplementary resources that can be utilized for improvement of B. napus. Enzyme analysis resulted in detecting 14 putative polymorphic loci with 27 alleles. Mean allele frequency 0.04 (rare alleles) was observed in Cat4A and Cat4B in sub species Oleifera accession CR 2204/79 and in subspecies trilocularis accessions CR 2215/88 and CR 2244/88. The highest genetic diversity measures were observed in subspecies dichotoma, accession CR 1585/96 (the highest average of observed (H0) and expected heterozygosity (He), and number of alleles per locus (Ae)). These observations make this accession valuable genetic resource to be included in breeding programs for the improvement of oilseed B. napus. The average fixation index (F) is significantly higher than zero for the analysis accessions indicating a significant deficiency of heteozygosity. The divergence among subspecies indicated very great genetic differentiation (FST = 0.8972) which means that about 90% of genetic diversity is distributed among subspecies, while 10% of the diversity is distributed within subspecies. This coincides with low value of gene flow (Nm = 0.0287). B. rapa ssp. oleifera (turnip rape) and B. rapa ssp. trilocularis (sarson) were grouped under one cluster which coincides with the morphological classification.

Resumo O presente estudo teve como objetivo avaliar a estrutura populacional e as relações filogenéticas de nove subespécies de Brassica rapa L. representadas com 35 acessos, cobrindo uma ampla gama de áreas de distribuição de espécies usando análise isoenzimática, a fim de selecionar acessos mais diversos como recursos suplementares que podem ser utilizados para melhoria de B. napus. A análise enzimática resultou na detecção de 14 loci polimórficos putativos com 27 alelos. A frequência média de 0,04 alelo (alelos raros) foi observada em Cat4A e Cat4B, nas subespécies Oleifera CR 2204/79 e nas subespécies trilocularis CR 2215/88 e CR 2244/88. As maiores medidas de diversidade genética foram observadas na subespécie dicotômica CR 1585/96 (a média mais alta observada (H0) e heterozigosidade esperada (He) e número de alelos por locus (Ae). Essas observações tornam esse acesso um valioso recurso genético a ser incluído em programas de melhoramento de oleaginosas B. napus. O índice médio de fixação (F) é significativamente maior que 0 para os acessos à análise, indicando uma deficiência significativa de heterozigose. A divergência entre as subespécies indicou uma grande diferenciação genética (FST = 0,8972), o que significa que cerca de 90% da diversidade genética é distribuída entre as subespécies, enquanto 10% da diversidade é distribuída nas subespécies. Isso coincide com o baixo valor do fluxo gênico (Nm = 0,0287). B. rapa ssp. oleifera (nabo) e B. rapa ssp. trilocularis (sarson) foram agrupados conforme a classificação morfológica.

Distribution of Meloidogyne species in carrot in Brazil / Distribuição de espécies de Meloidogyne em cenoura no Brasil

ABSTRACT: Root-knot nematodes (RKN - Meloidogyne spp.) are one of the most serious threats to carrot production worldwide. In Brazil, carrots are grown throughout the year, and economic losses due to RKN are reported. Since little is known on the distribution of RKN species in carrot fields in Brazil, we collected plant and soil samples from 35 fields across six states. Based on the morphology of perineal patterns, esterase phenotypes and species-specific PCR, three Meloidogyne species were identified: 60% of the fields were infested with Meloidogyne incognita, M. javanica was reported in 42.9% of the areas, whereas M. hapla was detected in 17.1% of carrot fields. Mixed populations were reported in 20% of the areas with a predominance of M. incognita + M. javanica. The combination of morphological, biochemical, and molecular techniques is a useful approach to identify RKN species.

RESUMO: Os nematoides-das-galhas (RKN - Meloidogyne spp.) são uma das mais sérias ameaças à produção de cenoura no mundo. No Brasil, as cenouras são cultivadas ao longo do ano, e as perdas econômicas devido à RKN são frequentemente relatadas. Como pouco se sabe sobre a distribuição de espécies RKN em campos de cenoura no Brasil, coletamos amostras de plantas e solo de 35 campos em seis estados. Baseado na morfologia do padrão perineal, fenótipos de esterase e/ou PCR espécie-específica, três espécies de Meloidogyne foram identificadas: 60% dos campos estavam infestados por Meloidogyne incognita, M. javanica foi encontrada em 42,9% das áreas, enquanto M. hapla foi detectada em 17,1% dos campos de cenoura. Populações mistas foram encontradas em 20% das áreas, com predominância de M. incognita + M. javanica. A combinação de técnicas morfológicas, bioquímicas e moleculares é uma abordagem útil para identificar espécies de RKN.

Sceletium Tortuosum: Effects on Central Nervous System and Related Disease

Sceletium tortuosum is a well-known medicinal plant in South Africa with potential applications. Its rawmaterial, extracts and isolated alkaloids are used as dietary supplements, natural medicines and health food.In this paper, methods of planting, extraction, isolation and identification of Sceletium tortuosum, as well as itschemical structure of main extracted alkaloids, their related pharmacological effects and mechanisms fortreating the disease are reviewed. In general, Sceletium tortuosum is active to central nervous system (CNS) byinhibiting phosphodiesterase isozyme 4 (PDE4), serotonin (5-HT) uptake and acetylcholinesterase (AChE). Italso acts as a monoamine releasing agent for antidepressant effects. Therefore, it is a useful therapeutic agentin clinical use.

Expression characteristics of glutamine synthetase of wheat in Escherichia coli / 生物工程学报

Glutamine synthetase is a key enzyme in plant nitrogen assimilation. To study the structure of wheat glutamine synthetase isoenzymes, GS1, GSr, GSe, GS2 and GS2p of wheat were cloned into pET-21a, and the expression condition was optimized. Although wheat glutamine synthetase isoenzymes had 70%-80% amino acid sequence homology, the isoforms expressed with different characteristics. Induced at 30 °C, the most expression level of GSr, GSe and GS2 was after 3 h, and of GS1 was at the 7 h whereas no GS2p was expressed, and the GS isoenzymes showed different expression level, with the order of GS1 (22%)>GSr (15%)>GS2 (12%)>GSe (5%). GSe expressed as soluble protein, and GS1 expressed mainly as soluble protein whereas GSr and GS2 expressed as insoluble proteins. Induced at 30 °C for 3 h, mRNA transcript levels of GS isoforms were different, with the order of GSr (7.59)>GS2 (1.84)>GS2p (1.66)>GSe (1.46)>GS1 (1.00). The levels of mRNA transcription were not consistent with the level of the protein translation. The analysis of mRNA secondary structure showed the free energy of translation initiation region of glutamine synthetase isoforms was different, with the order of GS1 (14.4)<GSr (17.2)<GS2 (22.6) <GSe (25.4) <GS2p (31.6), the smaller freed energy, the more unstable mRNA secondary structure of translation initiation region and the higher level of protein expression. Soluble expression condition of glutamine synthetase isozymes was also different, with GS1, GSr, GSe and GS2 induced at 30 °C for 5 h, 16 °C for 15 h, 37 °C for 5 h, and 25 °C for 7 h respectively. The soluble protein showed different expression level with GS1 (20%)>GSr (13%)>GS2 (10%)>GSe (7%), and different activities with GS1>GSe>GS2, and the activity of GSr was not detected. The gene sequence of glutamine synthetase isoenzymes determines the amount, status and activity of proteins expressed in prokaryotic cells.

Population structure and phylogenetic relationships in Brassica rapa L. subspecies by using isozyme markers

Abstract The present study aimed to assess population structure and phylogenetic relationships of nine subspecies of Brassica rapa L. represented with thirty-five accessions cover a wide range of species distribution area using isozyme analysis in order to select more diverse accessions as supplementary resources that can be utilized for improvement of B. napus. Enzyme analysis resulted in detecting 14 putative polymorphic loci with 27 alleles. Mean allele frequency 0.04 (rare alleles) was observed in Cat4A and Cat4B in sub species Oleifera accession CR 2204/79 and in subspecies trilocularis accessions CR 2215/88 and CR 2244/88. The highest genetic diversity measures were observed in subspecies dichotoma, accession CR 1585/96 (the highest average of observed (H0) and expected heterozygosity (He), and number of alleles per locus (Ae)). These observations make this accession valuable genetic resource to be included in breeding programs for the improvement of oilseed B. napus. The average fixation index (F) is significantly higher than zero for the analysis accessions indicating a significant deficiency of heteozygosity. The divergence among subspecies indicated very great genetic differentiation (FST = 0.8972) which means that about 90% of genetic diversity is distributed among subspecies, while 10% of the diversity is distributed within subspecies. This coincides with low value of gene flow (Nm = 0.0287). B. rapa ssp. oleifera (turnip rape) and B. rapa ssp. trilocularis (sarson) were grouped under one cluster which coincides with the morphological classification.

Resumo O presente estudo teve como objetivo avaliar a estrutura populacional e as relações filogenéticas de nove subespécies de Brassica rapa L. representadas com 35 acessos, cobrindo uma ampla gama de áreas de distribuição de espécies usando análise isoenzimática, a fim de selecionar acessos mais diversos como recursos suplementares que podem ser utilizados para melhoria de B. napus. A análise enzimática resultou na detecção de 14 loci polimórficos putativos com 27 alelos. A frequência média de 0,04 alelo (alelos raros) foi observada em Cat4A e Cat4B, nas subespécies Oleifera CR 2204/79 e nas subespécies trilocularis CR 2215/88 e CR 2244/88. As maiores medidas de diversidade genética foram observadas na subespécie dicotômica CR 1585/96 (a média mais alta observada (H0) e heterozigosidade esperada (He) e número de alelos por locus (Ae). Essas observações tornam esse acesso um valioso recurso genético a ser incluído em programas de melhoramento de oleaginosas B. napus. O índice médio de fixação (F) é significativamente maior que 0 para os acessos à análise, indicando uma deficiência significativa de heterozigose. A divergência entre as subespécies indicou uma grande diferenciação genética (FST = 0,8972), o que significa que cerca de 90% da diversidade genética é distribuída entre as subespécies, enquanto 10% da diversidade é distribuída nas subespécies. Isso coincide com o baixo valor do fluxo gênico (Nm = 0,0287). B. rapa ssp. oleifera (nabo) e B. rapa ssp. trilocularis (sarson) foram agrupados conforme a classificação morfológica.

Effect of mangiferin on superoxide dismutase isozyme expression in rats with chronic bronchitis / 中国药学杂志

OBJECTIVE: This research was designed to investigate the regulation of mangiferin on superoxide dismutase (SOD) isozyme expressionin rats with cigarettesmoke-induced chronic bronchitis, plus withthe protection on bronchial epithelial cellsultrastructure and anti-inflammatory action. METHODS: The rat model with chronic bronchitis was established by cigarette smoke. Real-time fluorescence RT-PCR was executed for evaluating the SOD1, SOD2 and SOD3 gene expression in lung tissue, and enzyme-linked im-muno sorbent assay (ELISA) for SOD1, SOD2 and SOD3 protein level. As well, interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a) and malondialdehyde (MDA) level in lung tissue were detected by ELISA. Furthermore, the bronchial epithelial cellsultrastructure was observed under transmission electron microscopy. The histopathological images was obtained by lung tissue HE staining slides. RESULTS: The cigarette smokeresulted in a markedly down-regulation expressions of SOD1, SOD2 and SOD3 in lung tissue. The down-regulation expressions of SOD1 and SOD2 in lung tissue cannot be antagonized by mangiferin. However, mangiferin significantly inhibited the down-regulation of SOD3, and markedly decreased IL-1, TNF-a and MDA. Furthermore, the structural completeness of the bronchial epithelial cellsultrastructure was maintainedin a good attitude by mangiferin, while the greatly reduced chronic bronchitis was found. CONCLUSION: Mangiferin can obviously reduce the chronic bronchitis induced by cigarette smoke and keep the bronchial epithelial cells from damage. The protective action might not rely only on anantagonistic action on down-regulated SOD3 expression in lung tissue by mangiferin.

Ultrastructural changes and oxidative stress markers in wild and cultivar Sesamum orientale L. following Alternaria sesami (Kawamura) Mohanty and Behera inoculation.

Alternaria sesami causes leaf spot disease in Sesamum orientale. Conidium germination, inoculation, penetration and colonization of the pathogen on the plant surfaces were studied using scanning electron microscopy. Electron microscopy analysis revealed multiple germ tubes from conidium that spread in all direction across the leaf surfaces. Penetration in the plant surface occured, directly through the epidermis or via stomata with or without the appressoria formation. Hyphal penetration continued through the substomata cavity and some of hyphal branches grew in the intercellular space of mesophyll tissue. Hyphal toxin, caused cell and cell wall damages. Changes in different biochemical parameters in the diseased sesame plants (both in wild and cultivar) were compared to control. Transmission electron microscopy showed structural changes in the chloroplast of diseased plants. Isozyme pattern and assays of different enzymes, namely catalase, acid phosphatase and peroxidase expressed varied level of activities. Meanwhile, esterase, polyphenol oxidase and superoxide dismutase in diseased plants showed remarkable levels compared to control. Due to the infection, chlorophyll content, carbohydrates and total soluble protein decreased whereas free amino acid, proline, phenols and disease-related proteins increased in the host plants. Differential SDS-PAGE band profiling of total soluble proteins were also observed in plants due to the infection.

Enzyme reaction kinetics, metabolic enzyme phenotype, and metabolites of berberine / 中草药

Objective: To study the metabolic characteristics of berberine using the pooled human liver microsomes and recombinant human cytochrome enzymes P450 (CYP) isozymes, to identify CYP isozymes responsible for berberine metabolism and its contribution, and to determine the structures of metabolism. Methods: Pooled human liver microsomes were incubated with berberine (20, 100, 200, 400, 600, 800, and 1 200 ng/mL). The Michaelis-Menten parameters (Km), maximum velocity (Vmax), and clearance (CLint) of pooled liver microsomes were initially estimated by analyzing Lineweave-Brurk plot. Various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of berberine and the certain concentration of berberine was incubated with recombinant human CYP isozymes (CYP3A4, CYP1A2, CYP2D6, and CYP2C9). The concentration of berberine and metabolites in the incubation pool was determined by UPLC method. The P450 isozymes were ranked with the method of total normalized rate (TNR) and the related metabolites were identified by LC-MS/MS. Results: The Vmax, Km, and CLint of berberine in pooled human liver microsomes were 1.51 nmol·mg-1·h-1, 2.69 nmol/mL, and 0.56 mL·mg-1·h-1, respectively. Quinidine (the specific inhibitor of CYP2D6) and Furafylline (the specific inhibitor of CYP1A2) could significantly inhibit the berberine metabolism, and the other CYP inhibitors had no significant effect on the metabolism of berberine. CYP2D6 and CYP1A2 were responsible for 75.253 9% and 23.323 6% of the berberine metabolite M1 (demethyleneberberine), and responsible for 46.893 8% and 8.679 5% of M2 (thalifendine or berberrubine). The major metabolic pathway of berberine in pooled human liver microsomes incubation system is O-demethylated, demethyleneberberine, thalifendine, or berberrubine could be generated in vitro. Conclusion: Bererine is metabolized by CYP2D6 and CYP1A2 in human liver, the metabolites of berberine are demethyleneberberine and thalifendine or berberrubine.

Isozymic variations in specific and nonspecific esterase and its thermostability in silkworm, Bombyx mori L.

Esterase isozymic variations were documented in the haemolymph of developed multivoltine and bivoltine silkworm breeds during unfavorable seed crop seasons of May – September using α- and β- napthylacetate separately to identify specific and nonspecific esterase having thermotolerant potentiality. Variations existed in the isozyme pattern with three bands (Est-2, 3 and 4) in pure Nistari race and other developed multivoltine and bivoltine breeds. Est-2 and Est-3 were non-specific esterases as they were observed when both α- and β-napthylacetate was used as substrates separately. Est- 4 band was observed only with α-napthylacetate as substrate and was therefore confirmed to be specific α-esterase band in the haemolymph of silkworm, Bombyx mori L. Zymograms showed that the non-specific esterase band (Est-3) with Rf of 0.43 and specific α-esterase band (Est-4) with Rf of 0.32 predominately withstood a temperature of 70 ± 2oC for a duration of 10 min and were confirmed as thermostable esterases in haemolymph of silkworm, Bombyx mori L. This also categorized the presence of thermostable esterases in developed multivoltine and bivoltine breeds of silkworm, even though the qualitative activity was more in the former than the latter. The qualitative presence of thermostable esterases and their activity could be adopted as an indicative biochemical marker in relation to thermotolerance in silkworm.

Genetic variability of wild and captivity populations of Colossoma macropomum (Cuvier, 1818) - doi: 10.4025/actascibiolsci.v34i2.7149

Tambaqui (Colossoma macropomum) is among the most important fish species of the Amazon and one of the most cultivated in Brazil. In the present work we have evaluated the genetic variability of wild and captivity populations of C. macropomum. Enzymatic markers were used to estimate the genetic variability of 41 specimens from a wild group; and 30, 33 and 45 from three captivity groups, which came from Pentecostes (Ceará State), Jaboticabal (São Paulo State) and Itacoatiara (Amazonas State), respectively. Nine isoenzymic systems were used to evaluate the genetic variability of these populations. Using zimogram data we obtained the polymorphism level, allele number, allelic frequency, observed and expected heterozigosity, Wright F statistics (FIS, FST), genetic distance, level of similarity and group analysis. The isoenzymic data showed that, from the nine systems, six presented polymorphic loci (Fbp-2, G6pdh-2, G6pdh-3, Pgi-1, Pgi-2 and Pgm-1). The populations from Pentecostes and Jaboticabal presented loss of genetic variability and low heterozigosity, compared to the wild population and to the artificial population acquired at Itacoatiara fish farm. Based on these results and on fish farmer information we could consider the population from Itacoatiara as recently derived from a wild population. Concluding, we suggest that the artificial populations of tambaqui, which contain a

Genetic variability of wild and captivity populations of Colossoma macropomum (Cuvier, 1818) / Variabilidade genética de populações selvagens e de cativeiro de Colossoma macropomum (Cuvier, 1818)

Tambaqui (Colossoma macropomum) is among the most important fish species of the Amazon and one of the most cultivated in Brazil. In the present work we have evaluated the genetic variability of wild and captivity populations of C. macropomum. Enzymatic markers were used to estimate the genetic variability of 41 specimens from a wild group; and 30, 33 and 45 from three captivity groups, which came from Pentecostes (Ceará State), Jaboticabal (São Paulo State) and Itacoatiara (Amazonas State), respectively. Nine isoenzymic systems were used to evaluate the genetic variability of these populations. Using zimogram data we obtained the polymorphism level, allele number, allelic frequency, observed and expected heterozigosity, Wright F statistics (FIS, FST), genetic distance, level of similarity and group analysis. The isoenzymic data showed that, from the nine systems, six presented polymorphic loci (Fbp-2, G6pdh-2, G6pdh-3, Pgi-1, Pgi-2 and Pgm-1). The populations from Pentecostes and Jaboticabal presented loss of genetic variability and low heterozigosity, compared to the wild population and to the artificial population acquired at Itacoatiara fish farm. Based on these results and on fish farmer information we could consider the population from Itacoatiara as recently derived from a wild population. Concluding, we suggest that the artificial populations of tambaqui, which contain animals originated from this funding population at Pentecostes, should be renewed with the introduction of a new group of individuals with genetic variability equivalent to the wild population.

O tambaqui é uma espécie de peixe bastante importante na região amazônica e uma das espécies mais cultivadas no Brasil. O objetivo deste trabalho foi avaliar a variabilidade genética do Colossoma macropomum de cativeiro e selvagem, utilizando marcadores isoenzimáticos. Utilizamos 41 indivíduos de uma população da natureza e 30, 33 e 45 de populações de cativeiro de Pentecoste, Jaboticabal e Itacoatiara, respectivamente. Nove sistemas isoenzimáticos foram utilizados para verificar a variabilidade genética do tambaqui, bem como os níveis de polimorfismos, números de alelos, freqüências alélicas, heterozigosidade observada e esperada, estatística F de Wright (FIS e FST), distância e similaridade genética, e análise de agrupamento. Das nove isoenzimas analisadas apenas seis sistemas apresentaram polimorfismo (Fbp-2, G6pdh-2, G6pdh-3, Pgi-1, Pgi-2 and Pgm-1). Verificamos que as populações de Pentecostes e Jaboticabal estão com falta de heterozigotos e apresentam-se estruturadas geneticamente em relação às populações de Itacoatiara e da Natureza que apresentam excesso de heterozigotos e não são estruturadas. Concluímos que os indivíduos de Itacoatiara são provenientes de populações selvagens e sugerimos que se realize uma renovação dos plantéis de tambaqui de Pentecostes e Jaboticabal, com o intuito de recuperar a variabilidade genética perdida.

Early diagnosis of radiodermatitis using lactate dehydrogenase isozymes in hairless mice (SKH1-hr) / 한국실험동물학회지

In this study, we evaluate a method for the early diagnosis of radiodermatitis for use in the prevention and therapy of this condition. Hairless mice (SKH1-hr) were used to study the early diagnosis of radiodermatitis. Lactate dehydrogenase (LDH, EC 1.1.1.27) isozymes were analyzed using native-polyacrylamide gel electrophoresis and western blotting of blood serum and tissues collected from SKH1-hr mice. Radiodermatitis developed 24 days after the first X-irradiation. Reduced spleen weight was observed after the last X-irradiation (P<0.05). Thereafter the weight increased until 24 days after the first irradiation, finally reaching levels comparable to those in the sham-irradiated control group. LDH activity was the highest in skeletal muscle and lowest in blood serum. LDH C4, A4, A3B, A2B2, AB3, and B4 isozymes were detected, in the mentioned order, from the cathode. This result was similar in other mouse strains. In the irradiated group, LDH A4 isozyme levels were reduced in the serum until inflammation occurred, whereas those of B4 isozyme were elevated. The subunits A and B followed a similar trend to that of LDH A4 and B4 isozyme, respectively. Importantly, antibodies against LDH B4 isozyme could prove useful in the early diagnosis of radiodermatitis.

Studies on morphological and genetical similarities of Medicago murex and M. doliata to M. scutellata.

Lucerne (M. sativa L., 2n = 4x = 32) is susceptible to weevil (Hypera postica Gyll) insect, hence incorporation of desirable gene (s) from M. scutellata (2n = 30) is an important researchable issue. Incompatibility due to incongruous chromosomal arrangements in these two species necessitated the identification of closer species to M. scutellata (possibly progenitors). After screening 197 accessions comprising 50 Medicago species, M. murex (2n = 2x = 14) and M. doliata (2n = 2x = 16) have been identified as morphologically similar having compatible ploidy and genetically closer to M. scutellata as observed with 17 simple sequence repeats (SSR) and 8 enzymes based isozyme markers. The identified accessions namely IL-04-223 and IL-04-151 of M. doliata and M. murex respectively showing low levels (< 5%) of weevil infestation can be contemplated with diploid M. sativa (2n = 2x = 16) to generate weevil resistant lines.

Screening of new human carbonic anhydrase Ⅱ(hCA Ⅱ) inhibitors / 中国药理学通报

Aim To provide practical method for screening human carbonic anhydrase Ⅱ(hCA Ⅱ) inhibitors in drug discovery.Methods hCA Ⅱ protein was obtained from induced BL21(DE3) E.coli containing plasmid pET-28b-hCA Ⅱ.The hCA Ⅱ activity was detected under pH 7.6 and 25℃ by its esterase activity which could decompose PNPA to increase the photoabsorption at 348 nm. After the assay conditions were finally selected, 35 new compounds were tested.Results A practical method for screening hCA Ⅱ inhibitors was successfully constituted by using recombinant hCA Ⅱ protein expressed in E.coli as the source of hCA Ⅱ enzyme.10 new compounds had better inhibitory effect and 9 new compounds had the same inhibitory effect on hCA Ⅱ compared with acetazolamide.Conclusions The hCA II inhibitor screening technique constituted in this work possesses advantages of being reliable, rapid, and practical. 19 new compounds are worth further research for developing high efficiency and low side effect drugs used for high-altitude illness.

Dos nuevas poblaciones de Lutzomyia pseudolongipalpis Arrivillaga y Feliciangeli (Diptera: Phlebotominae) vector de leishmaniasis visceral en Venezuela / Two new populations of Lutzomyia pseudolongipalpis Arrivillaga & Feliciangeli (Diptera: Phlebotominae) vector of visceral leishmaniasis in Venezuela

Dos nuevas poblaciones de Lutzomyia pseudolongipalpis Arrivillaga & Feliciangeli, especie del complejo Lutzomyia longipalpis (Lutz & Neiva), son registradas para Venezuela con base en dos enzimas diagnostico, Adenilato quinasa y Hexoquinasa.

Two new populations of Lutzomyia pseudolongipalpis Arrivillaga & Feliciangeli, species belonging to the Lutzomyia longipalpis (Lutz & Neiva) complex, are reported in Venezuela on diagnostic isoenzymes.

Immunohistochemical Localizaion of Carbonic Anhydrase Isozymes IV and IX in Rat Salivary Gland / 대한해부학회지

This study presents distribution of carbonic anhydrase (CA) isozymes IV and IX, membrane associated forms, and CA I and II, cytoplasmic forms, in rat parotid and submandibular glands using Western blot analysis and immunohistochemical staining. Western blot analysis demonstrated that CAs I, II and IX were found to be abundantly expressed, but CA IV was weakly expressed in parotid gland. Submandibular gland expressed abundant CAs I and II, weak CA IX, and undetectable level of CA IV. In hematoxylin-eosin staining, parotid gland was entirely composed of serous acini and their ducts while submandibular gland was mixed population of serous and mucous lobules. Most of lobules (submandibular gland proper type) contained mostly serous acini and their ducts with granular convoluted duct. Some lobules (sublingual gland type) contained mostly mucous acini with serous demilune and their ducts without granular convoluted duct. In parotid gland, CAs IV and IX were immunolocalized in duct cells and not in serous acinar cells. Immunoreactivity for CAs I and II was also detectable in duct cells. Serous acinar cells were positive for CA II, and negative for CA I. In submandibular gland, CAs IV and IX were immunolocalized in duct cells but not in acinar cells of both types of lobules. Immunoreactivity for CAs I and II was also detectable in duct cells of both types of lobules. Cells of serous acini and serous demilune were positive for CA II, and negative for CA I. Mucous cells were negative for both CAs I and II. These results demonstrate the distribution of CA isoenzymes in parotid and submandibular glands of the rat, and suggest CAs IV and IX as well as CAs I and II are related to electrolytes metabolism of saliva in duct cells.

Study on the Physico-chemical Properties of Endothelium Corneum Amylase / 中国中医药信息杂志

Objective To search for the optimal processing procedure and physico-chemical properties of Endothelium corneum amylase. Methods After dried under 50 ℃ ,amylase was extracted from Endothelium corneum with phosphate-buffered saline. The amylase activity was examined at different temperature,pH and different metal ions,and amylase isozyme was discerned on PAGE. Results The optimal pH,optimal temperature,and optimal substrate concentration for amylase activity were 8.67,50 ℃ and 1.8 g/mL,respectively. Under the experimental conditions,the amylase Km was 0.92 (starch was used as substrate of amylase). Four metal ions tested,including Ca2+,Fe2+,Zn2+,Mn2+,could inhibit the amylase activity to some extent. Furthermore,it showed one amylase isozyme band using PAGE,suggesting only one amylase isozyme existing in the Endothelium corneum tentatively. Conclusion The effects of metal ions should be considered in the process of Endothelium corneum amylase.

Myeloperoxidase (MPO) Isozymes of Normal Neutrophils from Normal Persons and Reactive Neutrophils from Patients with Neutrophilia; Using 10% Native PAGE and Activity Stain for MPO / 대한혈액학회지

BACKGROUND: MPO is an antimicrobial enzyme in the primary granule of neutrophil. MPO exhibits MPO-I, MPO-II, MPO-III using chromatography in human peripheral leukocytes. we investigated MPO isozymes from human leukocytes of peripheral blood. METHODS: Neutrophils were prepared from 5-8mL of peripheral blood in the group of normal persons (normal group) and the group of patients with neutrophilia (patient group). Their cells were adjusted to 2.4X10(7) cells and sonified. We got finally the crude MPO from which native PAGE was performed and treated with diaminobenzine and H2O2. RESULTS: Normal neutrophil MPO had MPO-1 and MPO-2. The expression rates of MPO-1 and that of MPO-2 were respectively 100%, 67.5% in the normal group and 56.6%, 30.2% in the patient group (P=0.000, P=0.000). The expression rate of combined MPO-1 and MPO-2 and that of MPO-1 without MPO-2 were respectively 67.6%, 32.4% in the normal group and 28.3%, 28.3% in the patient group (P=0.000, P=0.000). The expression of MPO-2 without MPO-1 and that of no MPO isozymes were respectively 0.0%, 0.0% in the normal group and 1.9%, 41.5% in the patient group (P=0.000, P=0.000). CONCLUSION: Normal neutrophil MPO expresses MPO-1 and MPO-2 usig native PAGE. The main MPO isozyme is MPO-1. Expressions of MPO isozymes are variable in the patient group.

The Changes of Superoxide Dismutase Isozymes in the Placenta of Patients with Preeclampsia / 대한산부인과학회잡지

OBJECTIVE: The aim of this article was investigated whether changes of superoxide dismutase isozymes in the placenta of patients with preeclampsia contribute to radical-induced tissue injury. METHODS: The activities of antioxidant enzymes (superoxide dismutase (SOD), catalase, glutathione peroxidase (GSHPx)) and the contents of thiobarbituric acid reactive substance (TBARS) in the erythrocytes and in the placenta were assayed from 35 women with preclampsia and 35 normotensive pregnant women. RESULTS: The superoxide dismutase (SOD) activities were significantly reduced in the erhtyrocytes and the placenta of patients with pre-eclampsia compared with normotensive pregnant women. The activity of catalase was increased in the erythrocytes of patients with preeclampsia but the statistically significant difference of catalase activity in the placenta and GSHPx activity in both erythrocytes and placenta were not observed. The contents of TBARS were increased significantly in the erythrocytes and placenta of patients with preeclampsia. In preeclamptic placenta, copper and zinc containing superoxide dismutase (CuZn-SOD) was decreased (3.9+/-0.5 vs 5.1+/-0.6 U/mg protein) whereas manganeus containing superoxide dismutase (Mn-SOD) was increased (2.0+/-0.3 vs 2.7+/-0.4 U/mg protein). CONCLUSION: In these results, the decreased CuZn-SOD activity may some roles in increment of TBARS contents in pre-eclamptic placenta and decreased CuZn-SOD activity may be more prone to oxidative stress in the placenta.