NF-κB participates in hepcidin up-regulation induced by iron overload in HH4 hepatocytes / 中国病理生理杂志

[ ABSTRACT] AIM:To study the effects of nuclear factor kappa B ( NF-κB) on human hepcidin expression in fer-ric ammonium citrate ( FAC)-induced HH4 hepatocytes.METHODS:Non-transformed HH4 cells were exposed to FAC at concentrations of 0.1, 1, 5 and 10 mmol/L for 48 h.The expression of iron regulatory gene hepcidin was determined by semi-quantitative RT-PCR.The effects of NF-κB on hepcidin transcriptional activity were detected using chromatin immuno-precipitation (ChIP), electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay system, combined with the inhibition experiments of intracellular NF-κB activity.RESULTS: FAC at concentrations of 5 mmol/L and 10 mmol/L significantly enhanced the expression of hepcidin.The results of ChIP and EMSA showed the binding of NF-κB to the upstream of hepcidin promoter.Treatment with NF-κB inhibitor BAY 11-7082 attenuated hepcidin expression.The lucif-erase activity in the cells transfected with recombinant luciferase reporter plasmid was obviously higher than that in control group.CONCLUSION:NF-κB is the transcription factor that contributes to hepcidin expression in iron overload-induced HH4 cells.

Expression and characterization of ArgR, an arginine regulatory protein in Corynebacterium crenatum / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Corynebacterium crenatum MT, a mutant from C. crenatum AS 1.542 with a lethal argR gene, exhibits high arginine production. To confirm the effect of ArgR on arginine biosynthesis in C. crenatum, an intact argR gene from wild-type AS 1.542 was introduced into C. crenatum MT, resulting in C. crenatum MT. sp, and the changes of transcriptional levels of the arginine biosynthetic genes and arginine production were compared between the mutant strain and the recombinant strain.</p><p><b>METHODS</b>Quantitative real-time polymerase chain reaction was employed to analyze the changes of the related genes at the transcriptional level, electrophoretic mobility shift assays were used to determine ArgR binding with the argCJBDF, argGH, and carAB promoter regions, and arginine production was determined with an automated amino acid analyzer.</p><p><b>RESULTS</b>Arginine production assays showed a 69.9% reduction in arginine from 9.01 ± 0.22 mg/mL in C. crenatum MT to 2.71 ± 0.13 mg/mL (P<0.05) in C. crenatum MT. sp. The argC, argB, argD, argF, argJ, argG, and carA genes were down-regulated significantly in C. crenatum MT. sp compared with those in its parental C. crenatum MT strain. The electrophoretic mobility shift assays showed that the promoter regions were directly bound to the ArgR protein.</p><p><b>CONCLUSION</b>The arginine biosynthetic genes in C. crenatum are clearly controlled by the negative regulator ArgR, and intact ArgR in C. crenatum MT results in a significant descrease in arginine production.</p>

CYP1A2 polymorphism and theophylline clearance in Korean non-smoking asthmatics

BACKGROUND: Theophylline is mainly metabolized by cytochrome P450 (CYP) 1A2 and CYP2E1 which show inter-individual variations. However, the underlying mechanism remains unknown in humans. We investigated the relationship between differences in theophylline clearance and genetic polymorphisms in the CYP1A2 and CYP2E1 gene in 89 Korean asthmatic patients. METHODS: Polymerase chain reaction (PCR) was performed on the 5'-flanking region of those genes. PCR products were directly sequenced and confirmed using the SNaP shot method. We determined whether the detected SNPs affected gene transcription using electrophoretic mobility shift assay (EMSA). Theophylline clearance (mL/kg/h) was assessed by using a Bayesian approach.

Deficiencia de glucosa-6-fosfato deshidrogenasa eritrocitaria en Rosario / Erythrocyte glucose-6-phosphate dehydrogenase deficiency in Rosario / Deficiência de glicose-6-fosfato desidrogenase eritrocitária em Rosario

Se estudió la actividad enzimática (AE) de la enzima glucosa-6-fosfato deshidrogenasa eritrocitaria (G6PD) y la movilidad electroforética (ME) en una población de hombres y mujeres de la ciudad de Rosario (provincia de Santa Fe), Argentina y zona de influencia. Para la determinación de AE se utilizó la técnica cinética de Glock y McLean y para la electroforesis de la enzima, la técnica de M.C. Rattazzi y L.C. Bernini en acetato de celulosa. Los valores normales de actividad enzimática (AE) para hombres y mujeres adultos fueron de 8,1 ± 1,4 UI G6PD/g Hb. Se demostró que los valores de AE son independientes de la edad, sexo y concentración de hemoglobina. En todos los grupos etarios estudiados no se observaron diferencias significativas de AE con respecto a los adultos normales a excepción de los neonatos que presentaron un significativo aumento de la misma, lo cual está directamente relacionado con las características fisiológicas de los eritrocitos del recién nacido. Entre los 686 individuos estudiados se detectaron 2 pacientes deficientes de G6PD, lo que dio una prevalencia de 0,3% y el patrón electroforético correspondiente a esta población fue 98% (n: 672) para G6PD B y 2% (n: 14) para G6PD con movilidad rápida tipo A.

Enzymatic activity (EA) of erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and electrophoretic mobility (EM) have been studied in a population of males and females in the city of Rosario and its area of influence. To determine EA, the Glock and McLean kinetic technique was used. Electrophoretic mobility assay was performed by M.C. Rattazzi and L.C. Bernini technique in cellulose acetate gel. Results demonstrated that the EA values in normal individual are independent of age, sex and hemoglobin values. The normal values of EA were: 8.1±1.4 IU of G6PD/g Hb. There were no significant differences in different age groups studied regarding healthy adults, except for neonatal group that yielded a significant EA increase which is directly related to the physiological characteristics of newborn erythrocytes. Two patients out of 686 individuals bearing G6PD deficiency were detected, corresponding to 0.3% prevalence. The electrophorectic mobility pattern was 98% (n: 672) for G6PD B, and 2% (n: 14) for G6PD A fast mobility variant.

Foi estudada a atividade enzimática (AE) da enzima glicose-6-fosfato desidrogenase eritrocitária (G6PD) e a mobilidade eletroforética (ME) numa população de homens e mulheres da cidade de Rosario, província de Santa Fe, Argentina e zona de influência. Para a determinação da AE foi utilizada a técnica cinética de GlocK e Mc Lean e para a eletroforese da enzima a técnica de M.C. Rattazzi e L.C. Bernini em acetato de celulose. Os valores normais de atividade enzimática (AE) para homens e mulheres adultos foram de 8,1 ± 1,4 UI G6PD/g Hb. Foi demonstrado que os valores da AE são independentes da idade, sexo e concentração de hemoglobina. Em nenhum dos grupos etários estudados foram observadas diferenças significativas de AE no que diz respeito aos adultos normais, com exceção dos neonatos que apresentaram um significativo aumento da mesma, o qual está diretamente relacionado com as características fisiológicas dos eritrócitos do recém-nascido. Entre os 686 indivíduos estudados foram detectados 2 pacientes deficientes de G6PD, o que deu uma prevalência de 0,3% e o padrão eletroforético correspondente a esta população foi de 98% (n: 672) para a G6PD B e 2% (n: 14) para G6PD com mobilidade rápida tipo A.

Arginase isoforms in frog and lizard tissues.

Isoforms of arginase in the liver and kidney tissues of the ureotelic frog (Rana tigerina) and uricotelic lizard (Calotes versicolor) were fractionated by DEAE-cellulose chromatography (pH 8.3). Four molecular forms, designated as A’1, A2, A3 and A4 based on the KCl concentration required for their elution from the ion-exchange column, were detected in lizard liver, while only two forms were found in lizard kidney (A3 and A4) and frog liver and kidney (A2 and A3). No major differences were found in the pH optimum, substrate affinity and molecular weight of the isoenzymes. The isoforms in lizard tissues were either totally unaffected or only partially immunoprecipitated by antibodies raised against rat liver and beef liver arginases, but those in frog tissues were significantly activated by the two antibodies. While the physiological importance of the presence of four isoforms in lizard liver remains enigmatic, different sets of isoenzymes were present in the liver of the two ureotelic vertebrates, rat and frog. Hence, it appeared that a given mode of nitrotelism was not associated with a specific set of isoenzymes. Also, the data were not consistent with the generally held view that a basic isoform of arginase served as a component of the urea cycle in liver and a neutral/slightly acidic form functions in the synthesis of proline, glutamate and polyamines in extra-hepatic tissues. The isoforms appeared to show considerable functional overlap.

Gel mobility shift scanning of pectin-inducible promoter from Penicillium griseoroseum reveals the involvement of a CCAAT element in the expression of a polygalacturonase gene

Previous reports have described pgg2, a polygalacturonase-encoding gene of Penicillium griseoroseum, as an attractive model for transcriptional regulation studies, due to its high expression throughout several in vitro growth conditions, even in the presence of non-inducing sugars such as sucrose. A search for regulatory motifs in the 5' upstream regulatory sequence of pgg2 identified a putative CCAAT box that could justify this expression profile. This element, located 270 bp upstream of the translational start codon, was tested as binding target for regulatory proteins. Analysis of a 170 bp promoter fragment by electrophoretic mobility shift assay (EMSA) with nuclear extracts prepared from mycelia grown in pectin-containing culture medium revealed a high mobility complex that was subsequently confirmed by analyzing it with a double-stranded oligonucleotide spanning the CCAAT motif. A substitution in the core sequence for GTAGG partially abolished the formation of specific complexes, showing the involvement of the CCAAT box in the regulation of the polygalacturonase gene studied.

Determination of nuclear transcription factor activity using a modified electrophoretic mobility shift assay

In this work, several major procedures of the electrophoretic mobility shift assay (EMSA) were modified including swift extraction of the nucleic protein, labeling of the probe and radioautography. The modified assay required shorter time, simplified the nucleic protein extraction, increased the radioactivity of the labeling probe, skipped the tedious process of gel drying, and produced clear images. Its results were comparable, reproducible and stable. It thus has merited for wide application.

A determinação da alteração na mobilidade eletroforética (EMSA), o método de mais ampla utilização para o estudo das interações proteínaácidos nucléicos, é tediosa e difícil de dominar. De acordo com os protocolos dacumentados e com base em nossa prática, nós modificamos os diversos processos principais dessa determinação incluindo no que diz respeito a extração de proteiínas nucleicas, marcação das provas e radioautografia. A determinação modificada requer menor tempo, simplifica a extração de ácidos nucleicos, eleva a radioatividade da prova marcada, evita o processo tedioso de secagem do gel e produz claras imagens. Seus resultados são comparáveis, reproduzíveis e estáveis, merecendo, desse modo, ampla aplicação.

The relation between Escherichia coli surface functional groups' composition and their physicochemical properties

Escherichia coli surface characteristics including hydrophobicity, electrophoretic mobility and surface functional groups' composition were investigated. These characteristics were determined respectively by water contact angle measurements, microelectrophoresis and x-ray photoelectron spectroscopy (XPS). The relation between the physicochemical properties and functional groups' composition was also examined. The electrophoretic mobility at pH 7 appeared to be governed on the cell surface by the (O=C) functional groups. The cell surface's hydrophilicity was associated with high levels of (C-(O.N)) and (OH-(C-O-C)) functional groups, whereas the cell surface's hydrophobicity was associated with (C-(C,H)) functional groups.

Características de superfície de Escherichia coli, tais como hidrofobicidade, mobilidade eletroforética e composição dos grupos funcionais de superfície, foram estudadas. Essas características foram determinadas por medidas de angulo de contato com água, microeletroforese e espectroscopia fotoeletrônica de raio-X (XPS), respectivamente. A relação entre as propriedades fisicoquímicas e a composição dos grupos funcionais foi também examinada. Aparentemente, a mobilidade eletroforética em pH 7 é controlada na superfície celular pelos grupos funcionais (O=C). A hidrofilicidade da superfície celular estava associada com altos níveis dos grupos funcionais [C-(O.N)] e [OH-(C-O-C)], enquanto a hidrofobicidade estava associada com os grupos funcionais [C-(C,H)].

Activation of Transcription Factor NF-kappaB and c-Jun/AP-1 by Various Compositions of Particles in Osteoclast Precursor Cells

PURPOSE: This study was performed in order to investigate the effects of various particle preparations on NF-kappaB and c-Jun/AP-1 activity in osteoclast precursor cells. MATERIALS AND METHODS: Osteoclast precursor cells isolated from C57BL mice were treated with PMMA (polymethylmethacrylate) spheres, polystyrene, titanium particles, and retrieved metal particles from failed cementless total hip replacements. NF-kappaB and c-Jun/AP-1 DNA binding activities were analyzed using electrophoretic mobility shift assays (EMSA). RESULTS: Commercially available PMMA and polystyrene spheres routinely showed negativity on endotoxin assays, but titanium particles and retrieved metal particles consistently showed positivity. PMMA spheres, with a maximal response noted at 30 minutes with an optimal concentration of 0.6 mg/ml, were potent stimulator of NF-kappaB and c-Jun/AP-1 activity in osteoclast precursor cells. Other particles (polystyrene, titanium, metal retrievals) also activated transcription factor NF-kappaB and c-Jun/AP-1 compared to controls. Endotoxin removal from retrieved metal particles diminished the biologic effect by approximately 40%. CONCLUSION: Particles of various compositions and sizes (PMMA, polystyrene, titanium, and retrieved metal particles) activated the NF-kappaB and c-Jun/AP-1 signaling pathways. This suggests that NF-kappaB and c-Jun/AP-1 may have important roles in the pathogenesis of periprosthetic osteolysis.

Prokaryotic Expression,Purification and DNA Binding Activity of DEK Protein's Carboxy-terminal DNA-binding Region / 中国生物工程杂志

DEK protein's carboxy-terminal DNA-binding region(CBD)is a newly found DNA-binding domain of DEK,which contains several phosphorylation sites and has a close correlation with DEK protein's function in vivo and in vitro.Using prokaryotic expression system,the peptide of DEK protein's carboxy-terminal DNA-binding region(CDB)was expressed and purified.In detail,the CDB DNA fragment was constructed into pET-30a(+)vector,and E.coli BL21(DE3)competent cells were used as host cells.The fusion protein His-CBD was expressed by induction of IPTG and purified by Ni-NTA agarose.The result of SDS-PAGE showed that the molecular weight of the purified protein was about 10.7kDa.Electrophoretic mobility shift assay(EMSA)indicated that DEK-CDB prefered to bind to supercoiled form of DNA in vitro,it had similar character to the binding of whole length DEK protein with DNA.This suggested that the carboxy-terminal DNA-binding region of DEK protein might function on the binding of DEK protein to DNA partly.

Prokaryotic Expression and Purification of a Cotton Dehydration Responsive Element Binding Protein GhDBP1 and Its DNA Binding Activity / 生物化学与生物物理进展

Cotton (Gossypium hirsutum) is one of the most important economic crops in the world. Its growth and productivity were affected by environment stresses such as drought, cold and high salinity. Thus, the enhanced stress tolerance in this plant is of great importance. As the dehydration responsive element (DRE) binding protein (DBP) plays an important role in the regulation of plant resistance to environmental stresses and is quite useful for generating transgenic plants tolerant to these stresses, isolation and functional analysis of DBPs in cotton are important to cotton production. In the previous work, a DBP gene from cotton, named as GhDBP1, was isolated and its expression patterns in cotton plants was demonstrated at the transcriptional level. Here, the expression,purification and DNA binding activity of GhDBP1 were reported. The entire coding region of the GhDBP1 gene was inserted into an expression vector, pET28a, and transformed into Escherichia coli BL21 (DE3). The fusion protein was successfully expressed under IPTG induction and the purified recombinant protein was obtained by Ni-NTA affinity chromatography. Non-radioactive electrophoretic mobility shift assay revealed that the purified GhDBP1 protein was able to form a specific complex with the previously characterized DRE element. In addition, the computer modeling of the DNA-binding domain of GhDBP1 were performed using SWISS-MODEL software. The main-chain structures and the folding patterns of the DNA-binding domain of GhDBP1 were similar to the known structure of the DNA-binding domain of the Arabidopsis thaliana GCC box-binding protein AtERF1. These results indicate that GhDBP1 is a DRE-binding transcription factor and might use the structure similar to that of AtERF1 to interact with DRE sequence.

OPG Inhibits PMMA Induced Osteoclastogenesis and NF-kappaB Activation in Osteoclast Precursor Cells / 대한정형외과연구학회지

PURPOSE: We investigate the effect of osteoprotegerin (OPG) on activation of osteoclastogenesis and NF-kappaB activation by PMMA (Polymethyl methacrylate) particles in osteoclast precursor cells. MATERIALS AND METHODS: Osteoclast precursor cells (CSF-1 dependent) were obtained from whole bone marrow of C57BL mouse. Four experiments included 1) different dose of RANKL (Receptor Activator of NF-kappaB ligand) treatment (0, 1, 10, 40 ng/ml) 2) PMMA treatment +/- RANKL 3) PMMA treatment with different dose of RANKL 4) PMMA treatment +/- OPG. After treatments, cultured cells were stained with TRAP (Tartrate resistant alkaline phosphatase). The activity of NF-kappaB DNA nuclear translocation was detected by EMSA (electrophoretic mobility shift assay). RESULTS: The experiments with RANKL on osteoclast precursors differentiation demonstrated a dose-dependent stimulation of osteoclastogenesis (p<0.05). Control cultures without RANKL had no osteoclasts, while maintenance in 1 ng/ml of RANKL results in low level osteoclast formation. PMMA particles activated osteoclastogenesis in RANKL-primed osteoclast precursor cells. And the effect of particles on osteoclastogenesis were dependent on RANKL concentration (p<0.03). OPG treatment significantly decreased osteoclast formation and NF-kappaB DNA binding activity by PMMA particles in osteoclast precursor cells. CONCLUSION: OPG inhibits activation of osteoclast formation and NF-kappaB DNA binding activity by PMMA particles through RANK-RANKL pathway.

Effect of pyrrolidine dithiocarbamate on expression of E-selectin and oxidative stress related enzymes in rats with severe acute pancreatitis induced by L-arginine / 第三军医大学学报

Objective To explore the effect of pyrrolidine dithiocarbamate(PDTC) on the expression of E-selectin and the activities of enzymes in oxidative stress in the pancreas of rats with severe acute pancreatitis(SAP).Methods Rat model of ASP was build by intraperitoneal injection of L-arginine(totally 5 g/kg,twice in an interval of 1 h).Thirty-six SD rats were assigned to 6 groups randomly and equally,that is,normal control,SAP,PDTC pretreatment group 1(1 mg/kg) and 2(100 mg/kg),and PDTC treatment group 1(1 mg/kg) and 2(100 mg/kg).PDTC pretreatment was preformed by subcutaneous injection of PDTC 1 h before first injection of L-arginine.For the animals in treatment groups,PDTC was given 3 h after first injection.Normal control was given intraperitoneal injection of normal saline.In 24 h after the first injection of L-arginine,the activity of myeloperoxidase(MPO),lipid peroxide(LPO) and nitric oxide synthase(NOS) were detected.Expressions of E-selectin was detected through inmmunohistochemical method,and activity of NF-?B-DNA binding were detected by electrophoretic mobility shift assay(EMSA).Results In SAP groups and PDTC pretreatment and treatment groups,the expression of E-selectin,activities of NF-?B,MPO,LPO,and inducible NOS(iNOS) were higher,but the activity of constitutive NOS(cNOS) was lower than those in the normal control(P0.05).Conclusion PDTC of 1 mg/kg can alleviate the inflammation in SAP by blocking the translocation of NF-?B,however,the 100 mg/kg PDTC shows little protection in this process.

Association of Polymorphic Regulatory Region of TIGR Gene with Glaucoma

PURPOSE: To identify the polymorphism in the regulatory region of trabecular meshwork inducible glucocorticoid response(TIGR) gene and evaluate the association of it with glaucoma. METHODS: 5'regulatory region of TIGR gene of 101 normal persons and 91 unrelated glaucoma patients were analyzed by DNA sequencing and restriction enzyme digestion. To know the possible effects of the polymorphism on the transcription rate of TIGR gene, electrophoretic mobility shift assay and luciferase reporter gene assay were performed with cultured cells, and their extracts of trabecular meshwork and ciliary body in which the gene was expressed. RESULTS: Of the 480 bp examined, G to A transition(G-241A) located at 241 bp upstream from transcription start site was identified and its frequency of occurrence was proved to be higher in steroid induced glaucoma patients(18.9%) compared with that in normal population(8.9%), POAG(8.3%) and normal tension glaucoma patients(6.7%, P<0.05). In mobility shift assay, the G-241A probe was proved to have affinity to some DNA-binding proteins and its affinity was revealed to be two times stronger than that of normal sequence. The luciferase activities, however, were observed to be similar in cells transfected with vectors having normal promoter sequence or G-241A containing one. CONCLUSION: The result suggest that G-241A itself is not a cause of steroid-induced glaucoma but is in linkage disequilibrium with the actual causes of the disease.

Nuclear protein binding patterns in the 5'-upstream regulatory elements of HLA class I genes

The expression of MHC class I genes has been thought to be regulated by two major cis-acting regulatory elements. The first region, enhancer A (Enh A) spanning from positions -210 to -165 contains perfect palindrome (PP), TGGGGATTCCCCA. The PP is well-conserved both in mouse and human MHC class I genes, even though the PP is disrupted by 2 bp substitutions (TGAGGATTCTCCA) in HLA-C genes. Three proteins binding to the Enh A of HLA-A and -B locus genes, but very weakly or nearly not to the Enh A of HLA-C locus gene have been identified. To determine functional importance of the PP for binding of trans-acting protein, mutant DNA probes were made by site-directed in vitro mutagenesis and then electrophoretic mobility shift assay was performed. HLA-A mutant DNA probe, in which the PP is disrupted, shows the same nuclear protein binding pattern as that of the HLA-C gene, and HLA-C mutant DNA probe, in which the PP is introduced, shows the same nuclear protein binding pattern as that of the wild type HLA-A gene. These data suggest that the perfect palindrome and its cognate DNA binding nuclear protein play an important role in the HLA class I gene regulation, and thus the lower expression of HLA-C antigen may be ascribed to no or very weak factor binding to the nonpalindromic sequences of HLA-C upstream DNA.

Electrokinetic studies on concanavalin A as a tool to probe the surface characteristics of differentiated lymphoid cells.

The redistribution of surface receptors induced by the binding of concanavalin A to different types of lymphoid cells was studied by the techniques of cell electrophoresis and fluorescence microscopy. The cells studied included, splenic lymphocytes from normal healthy as well as terminally leukaemic mice, thymocytes from mice of varying ages from newborns to adults and antigen sensitised or educated lymphocytes. These cells were in different stages of growth and/or differentiation. The nature and especially the behaviour of surface receptors in response to treatment with concanavalin A under capping conditions differed markedly but appeared to be dependent on the differentiational status of the cells. On this basis, the adult thymocytes were found to consist of two sub-populations differing in their proliferative and differentiational status. The proportions of these varied during their ontogenic development. Lymphocytes specifically committed to an antigen bound concanavalin A but were found to be incapable of bringing about the redistribution of the surface receptor-ligand complexes.

Regulation of rat liver NADP+ -isocitrate dehydrogenase during aging.

In an attempt to understand the mechanism of aging in relation to the differences in enzyme regulation, the induction and kinetic properties of NADP+ -isocitrate dehydrogenase of the liver of immature (6 weeks), mature (13 weeks), adult (33 weeks) and old (85 weeks) female rats were studied. The specific activity of the cytoplasmic and mitochondrial NADP+ -isocitrate dehydrogenase increased up to the adult age (33 weeks) and decreased in the old rats (85 weeks). Overiectomy decreased and estradiol administration induced activity of both the mitochondrial and eytoplasmic enzyme in the liver ol immature, mature and adult rats but had no significant effect in old rats. However, the activity of mitochondrial NADP+ -isocitrate dehydrogenase decreased and eytoplasmic NADP+ -isocitrate dehydrogenase increased following ovariectomy in old rats (85 weeks). Hormone-mediated induction of enzyme activity was actinomycin D sensitive. The Km for isocitrate and NADP, Ki value for oxalomalate, heat stability and electrophoretic mobility of the purified enzyme from the cytosol fraction of the liver of immature and old rats were similar. It can he concluded that the enzyme does not change structurally with age.