Alpha-Zein

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Brian A Larkins - One of the best experts on this subject based on the ideXlab platform.

  • The maize Mucronate mutation is a deletion in the 16-kDa gamma-zein gene that induces the unfolded protein response.
    Plant Journal, 2006
    Co-Authors: Bryan C. Gibbon, Brian A Larkins, Jeffrey W. Gillikin, Rebecca S. Boston, Rudolf Jung
    Abstract:

    : Mucronate (Mc) was identified as a dominant maize (Zea mays L.) opaque kernel mutation that alters zein storage protein synthesis. Zein protein bodies in Mc endosperm are misshapen and are associated with increased levels of ER Lumenal Binding Protein (BiP). Using GeneCalling to profile endosperm RNA transcripts, we identified an aberrant RNA in Mc that encodes the 16-kDa gamma-zein protein. The transcript contains a 38-bp deletion (nucleotides 406-444 after the initiation codon) that creates a frame-shift mutation and an abnormal sequence for the last 63 amino acids. Genetic mapping revealed the Mc mutation is linked with the locus encoding the 16-kDa gamma-zein, and two-dimensional gel electrophoresis confirmed the 16-kDa gamma-zein protein is altered in Mc. The mutant protein exhibited changes in solubility properties and co-immunoprecipitated with the molecular chaperone, BiP. Transgenic maize plants expressing the Mc 16-kDa gamma-zein manifested an opaque kernel phenotype with enhanced levels of BiP in the endosperm, similar to the Mc mutant. Unlike the wild-type protein, the Mc 16-kDa gamma-zein interacted only weakly with the 22-kDa Alpha-Zein when expressed in the yeast two-hybrid system. These results indicate that the Mc phenotype results from a frame-shift mutation in the gene encoding the 16-kDa gamma-zein protein, leading to the unfolded protein response in developing endosperm.

  • Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183 B139
    2003
    Co-Authors: Brian A Larkins
    Abstract:

    Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183 Final Technical Report and Patent Summary Dr. Brian A. Larkins, Department of Plant Sciences, University of Arizona, Tucson, AZ 85721 Endosperm texture is an important quality trait in maize, as it influences the shipping characteristics of the grain, its susceptibility to insects, the yield of grits from dry milling, energy costs during wet milling, and the baking and digestibility properties of the flour. There appears to be a causal relationship between kernel hardness and the formation of zein-containing protein bodies, as mutations affecting protein body number and structure are associated with a soft, starchy kernel. In this project we used a variety of approaches to better understand this relationship and investigate the molecular and biochemical changes associated with starchy endosperm mutants. We characterized the distribution of zein mRNAs on endosperm rough endoplasmic reticulum (RER) membranes and the interactions between zein proteins, as each of these could influence the structure of protein bodies. Based on in situ hybridization, mRNAs encoding the 22-kD alpha- and 27-kD gamma-zeins are randomly distributed on RER; hence, mRNA targeting does not appear to influence the formation of protein bodies. Investigation of the interactionsmore » between zein proteins (alpha, beta, gamma, delta) with the yeast two-hybrid system showed that interactions between the 19- and 22-Alpha-Zeins are relatively weak, although each of them interacted strongly with the 10-kD delta-zein. Strong interactions were detected between the alpha- and delta-zeins and the 16-kD gamma- and 15-kD beta-zeins; however, the 50-kD and 27-kD gamma-zeins did not interact detectably with the alpha- and delta-zein proteins. The NH2- and COOH-terminal domains of the 22-kD Alpha-Zein were found to interact most strongly with the 15-kD beta- and 16-kD gamma-zeins, suggesting the 16-kD and 15-kD proteins bind and assemble Alpha-Zeins in protein bodies. Additional evidence supporting this hypothesis was obtained by showing that the starchy endosperm mutant, Mucuronate, appears to result from a defective 16-kD gamma-zein protein. By deletion mutagenesis, we identified domains within an Alpha-Zein that cause it to interact with other zein proteins, particularly gamma-zeins. This allowed us to develop a minimal Alpha-Zein gene construct that can be used as a vector to target heterologous proteins, such as green fluorescent protein, into protein bodies. We characterized the nature of storage proteins synthesized in the endosperm using a genomics analysis of endosperm ESTs. This study identified several new storage proteins and demonstrated the existence of novel protein storage vacuoles. We used mRNA transcript profiling of eight different starchy endosperm (opaque) mutants (o1, o2, o5, o9, o11, Mucronate, Defective endosperm B30, and floury2) to identify patterns of gene expression that are consistently altered in all of them, or that are unique to each one of them. These mutants fall into two subgroups: one systematically manifests an ''unfolded protein'' response (fl2, Mc, DeB30) and the other (o1, o2, o5, o9, o11) does not. Genes encoding cytoskeletal proteins are generally up-regulated in all the mutants, and this may be associated with higher lysine contents in several of them.« less

  • A Defective Signal Peptide Tethers the floury-2 Zein to the Endoplasmic Reticulum Membrane
    Plant Physiology, 1997
    Co-Authors: Jeffrey W. Gillikin, Brian A Larkins, Craig E. Coleman, F. Zhang, Hank W. Bass, Rebecca S. Boston
    Abstract:

    The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD [alpha]-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD [alpha]-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this [alpha]-zein reveals a putative mutation alaine->valine (Ala->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 [alpha]-zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 [alpha]-zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 [alpha]-zein and the fl2 [alpha]-zein (Val->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 [alpha]-zein (Val->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 [alpha]-zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.

  • The maize gamma-zein sequesters Alpha-Zein and stabilizes its accumulation in protein bodies of transgenic tobacco endosperm.
    The Plant Cell, 1996
    Co-Authors: Craig E. Coleman, Eliot M. Herman, Koichi Takasaki, Brian A Larkins
    Abstract:

    Zeins are seed storage proteins that form accretions called protein bodies in the rough endoplasmic reticulum of maize endosperm cells. Four types of zeins, alpha, beta, gamma, and delta, aggregate in a distinctive spatial pattern within the protein body. We created transgenic tobacco plants expressing Alpha-Zein, gamma-zein, or both to examine the interactions between these proteins leading to the formation of protein bodies in the endosperm. Whereas gamma-zein accumulated in seeds of these plants, stable accumulation of Alpha-Zein required simultaneous synthesis of gamma-zein. The zein proteins formed accretions in the endoplasmic reticulum similar to those in maize endosperm. Protein bodies were also found in protein storage vacuoles. The accumulation of both types of zeins peaked early in development and declined during maturation. Even in the presence of gamma-zein, there was a turnover of Alpha-Zein, suggesting that the interaction between the two proteins might be transitory. We suggest that gamma-zein plays an important role in protein body formation and demonstrate the utility of tobacco for studying interactions between different zeins.

  • A defective signal peptide in the maize high-lysine mutant floury 2.
    Proceedings of the National Academy of Sciences of the United States of America, 1995
    Co-Authors: Craig E. Coleman, Mauricio A. Lopes, Jeffrey W. Gillikin, Rebecca S. Boston, Brian A Larkins
    Abstract:

    Abstract The maize floury 2 (fl2) mutation enhances the lysine content of the grain, but the soft texture of the endosperm makes it unsuitable for commercial production. The mutant phenotype is linked with the appearance of a 24-kDa Alpha-Zein protein and increased synthesis of binding protein, both of which are associated with irregularly shaped protein bodies. We have cloned the gene encoding the 24-kDa protein and show that it is expressed as a 22-kDa Alpha-Zein with an uncleaved signal peptide. Comparison of the deduced N-terminal amino acid sequence of the 24-kDa Alpha-Zein protein with other Alpha-Zeins revealed an alanine to valine substitution at the C-terminal position of the signal peptide, a histidine insertion within the seventh alpha-helical repeat, and an alanine to threonine substitution with the same alpha-helical repeat of the protein. Structural defects associated with this Alpha-Zein explain many of the phenotypic effects of the fl2 mutation.

Joachim Messing - One of the best experts on this subject based on the ideXlab platform.

  • Tissue-specificity of storage protein genes has evolved with younger gene copies.
    Maydica, 2020
    Co-Authors: Y. Wu, Joachim Messing
    Abstract:

    Seed storage proteins are critical for nitro- gen storage in seeds and the nutrition of livestock and humans. In maize synthesis begin 10-12 days after polli- nation (DAP) and peaks about 18-24 DAP. The major frac- tion of storage proteins in maize is classified as pro- lamins, which are rich in the amino acids proline and glu- tamine. Although storage proteins can accumulate in em- bryo tissue, the maize prolamins, also called zeins, accu- mulate in the triploid endosperm. Localization in either embryo or endosperm, however, requires the onset of the synthesis of tissue-specific trans-acting factors in a tempo- ral fashion. It was therefore unexpected that one of the zein genes is strongly expressed in tissue culture. When a chimeric gene controlling expression of the green fluores- cence protein (GFP) was introduced into immature cul- tured embryos by Agrobacterium-mediated transforma- tion, the GFP gave rise to green callus, indicating that the 27-kDa promoter of the gamma zein gene was active. Subsequent analysis of the endogenous 27-kDa gamma- zein gene showed that 27-kDa gamma-zein protein was made as well, indicating that expression of this gene was not due to the transformation procedure itself. This could be further demonstrated by the fact that the general trans- acting factor of prolamin genes, the prolamin-box-binding factor (PBF), was also expressed in the green callus. However, the O2 transcription factor that is required for the expression of a subset of alpha zein genes was not expressed, suggesting that tissue-specificity evolved into the combinatorial action of different trans-acting factors along with younger target genes arising from gene dupli- cations.

  • Differential Gene Expression and Epiregulation of Alpha Zein Gene Copies in Maize Haplotypes
    PLOS Genetics, 2011
    Co-Authors: Mihai Miclaus, Jian-hong Xu, Joachim Messing
    Abstract:

    Multigenic traits are very common in plants and cause diversity. Nutritional quality is such a trait, and one of its factors is the composition and relative expression of storage protein genes. In maize, they represent a medium-size gene family distributed over several chromosomes and unlinked locations. Two inbreds, B73 and BSSS53, both from the Iowa Stiff Stock Synthetic collection, have been selected to analyze allelic and non-allelic variability in these regions that span between 80–500 kb of chromosomal DNA. Genes were copied to unlinked sites before and after allotetraploidization of maize, but before transposition enlarged intergenic regions in a haplotype-specific manner. Once genes are copied, expression of donor genes is reduced relative to new copies. Epigenetic regulation seems to contribute to silencing older copies, because some of them can be reactivated when endosperm is maintained as cultured cells, indicating that copy number variation might contribute to a reserve of gene copies. Bisulfite sequencing of the promoter region also shows different methylation patterns among gene clusters as well as differences between tissues, suggesting a possible position effect on regulatory mechanisms as a result of inserting copies at unlinked locations. The observations offer a potential paradigm for how different gene families evolve and the impact this has on their expression and regulation of their members.

  • Organization of the prolamin gene family provides insight into the evolution of the maize genome and gene duplications in grass species
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Jian-hong Xu, Joachim Messing
    Abstract:

    Zea mays, commonly known as corn, is perhaps the most greatly produced crop in terms of tonnage and a major food, feed, and biofuel resource. Here we analyzed its prolamin gene family, encoding the major seed storage proteins, as a model for gene evolution by syntenic alignments with sorghum and rice, two genomes that have been sequenced recently. Because a high-density gene map has been constructed for maize inbred B73, all prolamin gene copies can be identified in their chromosomal context. Alignment of respective chromosomal regions of these species via conserved genes allow us to identify the pedigree of prolamin gene copies in space and time. Its youngest and largest gene family, the alpha prolamins, arose about 22-26 million years ago (Mya) after the split of the Panicoideae (including maize, sorghum, and millet) from the Pooideae (including wheat, barley, and oats) and Oryzoideae (rice). The first dispersal of alpha prolamin gene copies occurred before the split of the progenitors of maize and sorghum about 11.9 Mya. One of the two progenitors of maize gained a new alpha zein locus, absent in the other lineage, to form a nonduplicated locus in maize after allotetraplodization about 4.8 Mya. But dispersed copies gave rise to tandem duplications through uneven expansion and gene silencing of this gene family in maize and sorghum, possibly because of maize's greater recombination and mutation rates resulting from its diploidization process. Interestingly, new gene loci in maize represent junctions of ancestral chromosome fragments and sites of new centromeres in sorghum and rice.

  • Asymmetric localization of seed storage protein RNAs to distinct subdomains of the endoplasmic reticulum in developing maize endosperm cells
    Plant and Cell Physiology, 2004
    Co-Authors: Haruhiko Washida, Aya Sugino, Asim Esen, Joachim Messing, Thomas W. Okita
    Abstract:

    : Plant storage proteins are synthesized and stored in different compartments of the plant endomembrane system. Developing maize seeds synthesize and accumulate prolamin (zein) and 11S globulin (legumin-1) type proteins, which are sequestered in the endoplasmic reticulum (ER) lumen and storage vacuoles, respectively. Immunofluorescence studies showed that the lumenal chaperone BiP was not randomly distributed within the ER in developing maize endosperm but concentrated within the zein-containing protein bodies. Analysis of the spatial distribution of RNAs in maize endosperm sections by in situ RT-PCR showed that, contrary to the conclusions made in an earlier study [Kim et al. (2002) Plant Cell 14: 655-672], the zein and legumin-1 RNAs are not symmetrically distributed on the ER but, instead, targeted to specific ER subdomains. RNAs coding for 22 kDa Alpha-Zein, 15 kDa beta-zein, 27 kDa gamma-zein and 10 kDa delta-zein were localized to ER-bounded zein protein bodies, whereas 51 kDa legumin-1 RNAs were distributed on adjacent cisternal ER proximal to the zein protein bodies. These results indicate that the maize storage protein RNAs are targeted to specific ER subdomains in developing maize endosperm and that RNA localization may be a prevalent mechanism to sort proteins within plant cells.

  • A new opaque variant of maize by a single dominant RNA-interference-inducing transgene.
    Genetics, 2003
    Co-Authors: Gregorio Segal, Rentao Song, Joachim Messing
    Abstract:

    In maize, Alpha-Zeins, the main protein components of seed stores, are major determinants of nutritional imbalance when maize is used as the sole food source. Mutations like opaque-2 (o2) are used in breeding varieties with improved nutritional quality. However, o2 works in a recessive fashion by affecting the expression of a subset of 22-kD Alpha-Zeins, as well as additional endosperm gene functions. Thus, we sought a dominant mutation that could suppress the storage protein genes without interrupting O2 synthesis. We found that maize transformed with RNA interference (RNAi) constructs derived from a 22-kD zein gene could produce a dominant opaque phenotype. This phenotype segregates in a normal Mendelian fashion and eliminates 22-kD zeins without affecting the accumulation of other zein proteins. A system for regulated transgene expression generating antisense RNA also reduced the expression of 22-kD zein genes, but failed to give an opaque phenotype. Therefore, it appears that small interfering RNAs not only may play an important regulatory role during plant development, but also are effective genetic tools for dissecting the function of gene families. Since the dominant phenotype is also correlated with increased lysine content, the new mutant illustrates an approach for creating more nutritious crop plants.

Barbara Ballmer-weber - One of the best experts on this subject based on the ideXlab platform.

  • Maize food allergy: lipid-transfer proteins, endochitinases, and Alpha-Zein precursor are relevant maize allergens in double-blind placebo-controlled maize-challenge-positive patients
    Analytical and Bioanalytical Chemistry, 2009
    Co-Authors: Elide A. Pastorello, Laura Farioli, Valerio Pravettoni, Joseph Scibilia, Amedeo Conti, Donatella Fortunato, Linda Borgonovo, Simona Bonomi, Laura Primavesi, Barbara Ballmer-weber
    Abstract:

    Italian patients with maize anaphylaxis have been shown to have IgE toward two major maize allergens: an alpha-amylase inhibitor and a 9-kDa LTP. A complete study on maize food allergens in patients with positive maize double-blind, placebo-controlled food challenge (DBPCFC) is lacking. The objective was to utilize the three maize protein fractions to identify and characterize the most relevant IgE-binding proteins recognized by the sera of Italian and Swiss patients with either a positive maize-DBPCFC or a history of maize-induced anaphylaxis. Osborne’s protein fractions of maize were extracted to obtain water-soluble, total zein, and total protein fractions. Protein IgE-binding capacity was investigated by SDS-PAGE immunoblotting using the sera from DBPCFC-positive patients and from patients with maize-induced anaphylaxis. Purified maize LTP was used to inhibit the IgE immunoblotting of the three protein fractions. IgE immunoblotting demonstrated that the 9-kDa LTP was recognized by all the Italian patients and by none of the Swiss patients. Other allergens were: 14-kDa α-amylase inhibitor, 30-kDa endochitinases A and -B, 19 kDa zein-β precursor, and 26   kDa zein-α precursor; a newly described allergen, the globulin-2 precursor, identified in the total protein fraction. It is noteworthy that maize LTP and endochitinase were cross-reactive with grape LTP and one grape endochitinase. LTP was found to be the only major allergen in Italian patients with either positive maize challenge or a history of maize-induced anaphylaxis. We have identified other maize allergens in subjects with maize food allergy, as grape cross-reactive endochitinase, however, the clinical significance of these proteins needs to be investigated in larger groups of patients with allergy to these food items.

Hui Chai - One of the best experts on this subject based on the ideXlab platform.

  • ginsenoside rb1 inhibits tumor necrosis factor α induced vascular cell adhesion molecule 1 expression in human endothelial cells
    Biological & Pharmaceutical Bulletin, 2008
    Co-Authors: Hui Chai, Li Feng Huang, Qiu Yan Wang, Yan Fu
    Abstract:

    : We investigated whether ginsenoside Rb1 (Rb1) could block tumor necrosis factor-alpha (TNF-alpha)-induced over-expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVECs-L). Cells were treated with various concentrations of TNF-alpha with or without Rb1 pre-treatment for 16 h. The mRNA and protein levels of VCAM-1 were determined with real-time polymerase chain reaction (PCR) and flow cytometry, respectively. Human monocytic THP-1 cells labeled with fluorescent dye (Calcein-AM) was used for the adhesion assay on HUVEC monolayers. Dihydroethidium (DHE) was used to demonstrate in situ levels of superoxide production. JC-1 dye was used to measure changes in mitochondrial membrane potential. Activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) was determined by Bio-Plex immunoassay. TNF-alpha treatment significantly increased the mRNA and protein levels of VCAM-1 in HUVECs in a dose dependent manner. Rb1 pre-treatment effectively blocked the TNF-alpha-induced expression of VCAM-1 mRNA or protein by 80% and 43%, respectively (p<0.01). THP-1 adhesion was also blocked. Furthermore, Rb1 reduced the TNF-alpha-induced increase of superoxide anion production by 41% and inhibited the TNF-alpha-induced decrease of mitochondrial membrane potential by 44% in HUVECs. Rb1 also effectively blocked TNF-alpha-induced activation of p38, c-Jun N-terminal protein kinase, extracellular signal-regulated kinase 1/2 and IkappaBalpha. In conclusion, Rb1 effectively blocked the TNF-alpha-induced over-expression of VCAM-1, increased THP-1 adhesion and over-production of superoxide anion. Furthermore, Rb1 inhibited TNF-alpha-induced MAPKs and NF-kappaB activation. These data suggested that Rb1 might have potential therapeutic effects in controlling inflammation in vascular diseases.

  • WITHDRAWN_Ginsenoside Rb1 inhibits tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression in human endothelial cells
    Biological and Pharmaceutical Bulletin, 2008
    Co-Authors: Hui Chai, Li Feng Huang, Qiu Yan Wang, Tian Xie, Yan Fu
    Abstract:

    We investigated whether ginsenoside Rb1 (Rb1) could block tumor necrosis factor-alpha (TNF-alpha)-induced over-expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVECs-L). Cells were treated with various concentrations of TNF-alpha with or without Rb1 pre-treatment for 16 h. The mRNA and protein levels of VCAM-1 were determined with real-time polymerase chain reaction (PCR) and flow cytometry, respectively. Human monocytic THP-1 cells labeled with fluorescent dye (Calcein-AM) was used for the adhesion assay on HUVEC monolayers. Dihydroethidium (DHE) was used to demonstrate in situ levels of superoxide production. JC-1 dye was used to measure changes in mitochondrial membrane potential. Activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) was determined by Bio-Plex immunoassay. TNF-alpha treatment significantly increased the mRNA and protein levels of VCAM-1 in HUVECs in a dose dependent manner. Rb1 pre-treatment effectively blocked the TNF-alpha-induced expression of VCAM-1 mRNA or protein by 80% and 43%, respectively (p

  • Ginsenoside Rb1 Inhibits Tumor Necrosis Factor-alpha-Induced Vascular Cell Adhesion Molecule-1 Expression in Human Endothelial Cells
    Biological & Pharmaceutical Bulletin, 2008
    Co-Authors: Hui Chai, Li Feng Huang, Qiu Yan Wang, Tian Xie, Yongping Fu
    Abstract:

    We investigated whether ginsenoside Rb1 (Rb1) could block tumor necrosis factor-alpha (TNF-alpha)-induced over-expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVECs-L). Cells were treated with various concentrations of TNF-alpha with or without Rb1 pre-treatment for 16 h. The mRNA and protein levels of VCAM-1 were determined with real-time polymerase chain reaction (PCR) and flow cytometry, respectively. Human monocytic THP-1 cells labeled with fluorescent dye (Calcein-AM) was used for the adhesion assay on HUVEC monolayers. Dihydroethidium (DHE) was used to demonstrate in situ levels of superoxide production. JC-1 dye was used to measure changes in mitochondrial membrane potential. Activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-kappa B) was determined by Bio-Plex immunoassay. TNF-alpha treatment significantly increased the mRNA and protein levels of VCAM-1 in HUVECs in a dose dependent manner. Rb1 pre-treatment effectively blocked the TNF-alpha-induced expression of VCAM-1 mRNA or protein by 80% and 43%, respectively (p

Yan Fu - One of the best experts on this subject based on the ideXlab platform.

  • ginsenoside rb1 inhibits tumor necrosis factor α induced vascular cell adhesion molecule 1 expression in human endothelial cells
    Biological & Pharmaceutical Bulletin, 2008
    Co-Authors: Hui Chai, Li Feng Huang, Qiu Yan Wang, Yan Fu
    Abstract:

    : We investigated whether ginsenoside Rb1 (Rb1) could block tumor necrosis factor-alpha (TNF-alpha)-induced over-expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVECs-L). Cells were treated with various concentrations of TNF-alpha with or without Rb1 pre-treatment for 16 h. The mRNA and protein levels of VCAM-1 were determined with real-time polymerase chain reaction (PCR) and flow cytometry, respectively. Human monocytic THP-1 cells labeled with fluorescent dye (Calcein-AM) was used for the adhesion assay on HUVEC monolayers. Dihydroethidium (DHE) was used to demonstrate in situ levels of superoxide production. JC-1 dye was used to measure changes in mitochondrial membrane potential. Activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) was determined by Bio-Plex immunoassay. TNF-alpha treatment significantly increased the mRNA and protein levels of VCAM-1 in HUVECs in a dose dependent manner. Rb1 pre-treatment effectively blocked the TNF-alpha-induced expression of VCAM-1 mRNA or protein by 80% and 43%, respectively (p<0.01). THP-1 adhesion was also blocked. Furthermore, Rb1 reduced the TNF-alpha-induced increase of superoxide anion production by 41% and inhibited the TNF-alpha-induced decrease of mitochondrial membrane potential by 44% in HUVECs. Rb1 also effectively blocked TNF-alpha-induced activation of p38, c-Jun N-terminal protein kinase, extracellular signal-regulated kinase 1/2 and IkappaBalpha. In conclusion, Rb1 effectively blocked the TNF-alpha-induced over-expression of VCAM-1, increased THP-1 adhesion and over-production of superoxide anion. Furthermore, Rb1 inhibited TNF-alpha-induced MAPKs and NF-kappaB activation. These data suggested that Rb1 might have potential therapeutic effects in controlling inflammation in vascular diseases.

  • WITHDRAWN_Ginsenoside Rb1 inhibits tumor necrosis factor-alpha-induced vascular cell adhesion molecule-1 expression in human endothelial cells
    Biological and Pharmaceutical Bulletin, 2008
    Co-Authors: Hui Chai, Li Feng Huang, Qiu Yan Wang, Tian Xie, Yan Fu
    Abstract:

    We investigated whether ginsenoside Rb1 (Rb1) could block tumor necrosis factor-alpha (TNF-alpha)-induced over-expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVECs-L). Cells were treated with various concentrations of TNF-alpha with or without Rb1 pre-treatment for 16 h. The mRNA and protein levels of VCAM-1 were determined with real-time polymerase chain reaction (PCR) and flow cytometry, respectively. Human monocytic THP-1 cells labeled with fluorescent dye (Calcein-AM) was used for the adhesion assay on HUVEC monolayers. Dihydroethidium (DHE) was used to demonstrate in situ levels of superoxide production. JC-1 dye was used to measure changes in mitochondrial membrane potential. Activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) was determined by Bio-Plex immunoassay. TNF-alpha treatment significantly increased the mRNA and protein levels of VCAM-1 in HUVECs in a dose dependent manner. Rb1 pre-treatment effectively blocked the TNF-alpha-induced expression of VCAM-1 mRNA or protein by 80% and 43%, respectively (p