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1.
Int J Biol Macromol ; 196: 163-171, 2022 Jan 31.
Article in English | MEDLINE | ID: mdl-34920069

ABSTRACT

For safe preservation and consumption of fish, freshness monitoring and antimicrobial control is crucial. Edible films comprising natural antimicrobial and spoilage indicator agents represent a convenient method for such preservation. Edible chitosan-based films were prepared using red cabbage (RC) and clove bud oil (CBO)-loaded chitosan/carrageenan capsules as spoilage indicator and antimicrobial agents, respectively. CBO-loaded capsules were prepared by the ionic gelation of chitosan and carrageenan. Films containing CBO capsules exhibited significantly higher antimicrobial activity than films containing non-encapsulated free CBO, as confirmed by minimum inhibitory concentration and time-kill assays. The highest antimicrobial activity was observed in the largest capsules (1.7 µm). After incubation for 48 h, the pH of fish peptone agar containing Pseudomonas fluorescens increased from approximately 6.0 to 9.0, and a color change from purple to deep blue was clearly observed during the growth of fish-spoiling bacteria. Thus, our results suggested that edible films containing CBO-loaded capsules and RC showed the potential to inhibit microbial growth in fish and to visibly indicate fish freshness.


Subject(s)
Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Brassica/chemistry , Chitosan/chemistry , Clove Oil/chemistry , Edible Films , Food Preservation , Seafood , Animals , Chemical Phenomena , Fishes , Food Preservation/methods , Microbial Sensitivity Tests
2.
J Biomol Screen ; 21(8): 786-94, 2016 Sep.
Article in English | MEDLINE | ID: mdl-27369108

ABSTRACT

SETD8 is the methyltransferase responsible for monomethylation of lysine at position 20 of the N-terminus of histone H4 (H4K20). This activity has been implicated in both DNA damage and cell cycle progression. Existing biochemical assays have utilized truncated enzymes containing the SET domain of SETD8 and peptide substrates. In this report, we present the development of a mechanistically balanced biochemical assay using full-length SETD8 and a recombinant nucleosome substrate. This improves the binding of SAM, SAH, and sinefungin by up to 10,000-fold. A small collection of inhibitors structurally related to SAM were screened and 40 compounds were identified that only inhibit SETD8 when a nucleosome substrate is used.


Subject(s)
High-Throughput Screening Assays/methods , Histone-Lysine N-Methyltransferase/metabolism , Nucleosomes/genetics , Peptides/isolation & purification , Adenosine/analogs & derivatives , Adenosine/metabolism , DNA Damage/genetics , Histone-Lysine N-Methyltransferase/genetics , Histones/genetics , Humans , Lysine/genetics , Nucleosomes/metabolism , PR-SET Domains/genetics , Peptides/genetics , Protein Binding , Structure-Activity Relationship , Substrate Specificity
3.
Mol Cell Biol ; 26(8): 3048-59, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16581780

ABSTRACT

Hepatitis C virus (HCV) NS5B protein is a membrane-associated phosphoprotein that possesses an RNA-dependent RNA polymerase activity. We recently reported that NS5A protein interacts with TRAF2 and modulates tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB and Jun N-terminal protein kinase (JNK). Since NS5A and NS5B are the essential components of the HCV replication complex, we examined whether NS5B could modulate TNF-alpha-induced NF-kappaB and JNK activation. In this study, we have demonstrated that TNF-alpha-induced NF-kappaB activation is inhibited by NS5B protein in HEK293 and hepatic cells. Furthermore, NS5B protein inhibited both TRAF2- and IKK-induced NF-kappaB activation. Using coimmunoprecipitation assays, we show that NS5B interacts with IKKalpha. Most importantly, NS5B protein in HCV subgenomic replicon cells interacted with endogenous IKKalpha, and then TNF-alpha-mediated IKKalpha kinase activation was significantly decreased by NS5B. Using in vitro kinase assay, we have further found that NS5B protein synergistically activated TNF-alpha-mediated JNK activity in HEK293 and hepatic cells. These data suggest that NS5B protein modulates TNF-alpha signaling pathways and may contribute to HCV pathogenesis.


Subject(s)
Hepacivirus/metabolism , I-kappa B Kinase/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/metabolism , Viral Nonstructural Proteins/physiology , Animals , COS Cells , Carcinoma, Hepatocellular/pathology , Cell Line , Cell Line, Tumor , Chlorocebus aethiops , Enzyme Activation/drug effects , Genes, Reporter , Hepacivirus/genetics , Humans , I-kappa B Kinase/analysis , JNK Mitogen-Activated Protein Kinases/analysis , JNK Mitogen-Activated Protein Kinases/metabolism , Liver Neoplasms/pathology , Luciferases/metabolism , MAP Kinase Kinase 4/metabolism , Precipitin Tests , Protein Binding , Tumor Necrosis Factor-alpha/pharmacology , Viral Nonstructural Proteins/genetics
4.
J Biol Chem ; 281(10): 6699-706, 2006 Mar 10.
Article in English | MEDLINE | ID: mdl-16407216

ABSTRACT

The IkappaB kinase (IKK) complex consists of the catalytic subunits IKKalpha and IKKbeta and a regulatory subunit, IKKgamma/NEMO. Even though IKKalpha and IKKbeta share significant sequence similarity, they have distinct biological roles. It has been demonstrated that IKKs are involved in regulating the proliferation of both normal and tumor cells, although the mechanisms by which they function in this process remain to be better defined. In this study, we demonstrate that IKKalpha, but not IKKbeta, is important for estrogen-induced cell cycle progression by regulating the transcription of the E2F1 gene as well as other E2F1-responsive genes, including thymidine kinase 1, proliferating cell nuclear antigen, cyclin E, and cdc25A. The role of IKKalpha in regulating E2F1 was not the result of reduced levels of cyclin D1, as overexpression of this gene could not overcome the effects of IKKalpha knock-down. Furthermore, estrogen treatment increased the association of endogenous IKKalpha and E2F1, and this interaction occurred on promoters bound by E2F1. IKKalpha also potentiated the ability of p300/CBP-associated factor to acetylate E2F1. Taken together, these data suggest a novel mechanism by which IKKalpha can influence estrogen-mediated cell cycle progression through its regulation of E2F1.


Subject(s)
Cell Cycle/physiology , E2F1 Transcription Factor/biosynthesis , Estrogens/physiology , Gene Expression Regulation, Neoplastic/physiology , I-kappa B Kinase/physiology , Animals , Cell Cycle/genetics , Cell Line, Tumor , E2F1 Transcription Factor/genetics , E2F1 Transcription Factor/physiology , Humans , Mice , Promoter Regions, Genetic , Thymidine Kinase/genetics
5.
World J Gastroenterol ; 11(25): 3887-92, 2005 Jul 07.
Article in English | MEDLINE | ID: mdl-15991288

ABSTRACT

AIM: We have previously demonstrated that hepatitis C virus (HCV) core protein is efficiently released into the culture medium in insect cells. The objective of this study is to characterize the HCV core secretion in insect cells. METHODS: We constructed recombinant baculoviruses expressing various-length of mutant core proteins, expressed these proteins in insect cells, and examined core protein secretion in insect cells. RESULTS: Only wild type core was efficiently released into the culture medium, although the protein expression level of wild type core was lower than those of other mutant core proteins. We found that the shorter form of the core construct expressed the higher level of protein. However, if more than 18 amino acids of the core were truncated at the C-terminus, core proteins were no longer secreted into the culture medium. Membrane flotation data show that the secreted core proteins are associated with the cellular membrane protein, indicating that HCV core is secreted as a membrane complex. CONCLUSION: The C-terminal 18 amino acids of HCV core were crucial for core secretion into the culture media. Since HCV replication occurs on lipid raft membrane structure, these results suggest that HCV may utilize a unique core release mechanism to escape immune surveillance, thereby potentially representing the feature of HCV morphogenesis.


Subject(s)
Protein Structure, Tertiary/physiology , Viral Core Proteins/genetics , Viral Core Proteins/metabolism , Animals , Baculoviridae/genetics , Gene Transfer Techniques , Genetic Vectors , Recombination, Genetic , Spodoptera/cytology
6.
Mol Cell ; 18(1): 71-82, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15808510

ABSTRACT

The IkappaB kinases IKKalpha and IKKbeta regulate distinct cytoplasmic and nuclear events that are critical for cytokine-mediated activation of the NF-kappaB pathway. Because the IKKs have previously been demonstrated to associate with the nuclear hormone receptor coactivator AIB1/SRC-3, the question of whether either IKKalpha or IKKbeta may be involved in increasing the expression of hormone-responsive genes was addressed. We demonstrated that IKKalpha, in conjunction with ERalpha and AIB1/SRC-3, is important in activating the transcription of estrogen-responsive genes, including cyclin D1 and c-myc, to result in the enhanced proliferation of breast cancer cells. Estrogen treatment facilitated the association of IKKalpha, ERalpha, and AIB1/SRC-3 to estrogen-responsive promoters and increased IKKalpha phosphorylation of ERalpha, AIB1/SRC-3, and histone H3. These results suggest that IKKalpha plays a major role in regulating the biological effects of estrogen via its promoter association and modification of components of the transcription complex.


Subject(s)
Estradiol/pharmacology , Gene Expression Regulation, Neoplastic/genetics , Gene Expression Regulation , Protein Serine-Threonine Kinases/metabolism , Receptors, Estrogen/physiology , Apoptosis , Base Sequence , Cell Division , Cell Line, Tumor , Cyclin D1/genetics , DNA Primers , Estrogen Receptor alpha/metabolism , Humans , I-kappa B Kinase , Promoter Regions, Genetic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Transcriptional Activation , Transfection
7.
J Biochem Mol Biol ; 37(6): 735-40, 2004 Nov 30.
Article in English | MEDLINE | ID: mdl-15607034

ABSTRACT

Hepatitis C virus (HCV) is a causal agent of the chronic liver infection. To understand HCV morphogenesis, we studied the assembly of HCV structural proteins in insect cells. We constructed recombinant baculovirus expression vectors consisting of either HCV core alone, core-E1, or core-E1-E2. These structural proteins were expressed in insect cells and were examined to assemble into particles. Neither core-E1 nor core-E1-E2 was capable of assembling into virus-like particles (VLPs). It was surprising that the core protein alone was assembled into core-like particles. These particles were released into the culture medium as early as 2 days after infection. In our system, HCV structural proteins including envelope proteins did not assemble into VLPs. Instead, the core protein itself has the intrinsic capacity to assemble into amorphous core-like particles. Furthermore, released core particles were associated with HCV RNA, indicating that core proteins were assembled into nucleocapsids. These results suggest that HCV may utilize a unique core release mechanism to evade the hosts defense mechanism, thus contributing to the persistence of HCV infection.


Subject(s)
Culture Media/chemistry , Hepacivirus/metabolism , Insecta , Viral Core Proteins/metabolism , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Cells, Cultured , Hepacivirus/pathogenicity , Humans , Viral Core Proteins/chemistry , Viral Core Proteins/genetics
8.
J Biol Chem ; 278(33): 30711-8, 2003 Aug 15.
Article in English | MEDLINE | ID: mdl-12796506

ABSTRACT

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) is a phosphoprotein possessing various functions. We have previously reported that the HCV NS5A protein interacts with tumor necrosis factor (TNF) receptor-associated factor (TRAF) domain of TRAF2 (Park, K.-J., Choi, S.-H., Lee, S. Y., Hwang, S. B., and Lai, M. M. C. (2002) J. Biol. Chem. 277, 13122-13128). Both TNF-alpha- and TRAF2-mediated nuclear factor-kappaB (NF-kappaB) activations were inhibited by NS5A-TRAF2 interaction. Because TRAF2 is required for the activation of both NF-kappaB and c-Jun N-terminal kinase (JNK), we investigated HCV NS5A protein for its potential capacity to modulate TRAF2-mediated JNK activity. Using in vitro kinase assay, we have found that NS5A protein synergistically activated both TNF-alpha- and TRAF2-mediated JNK in human embryonic kidney 293T cells. Furthermore, synergism of NS5A-mediated JNK activation was inhibited by dominant-negative form of MEK kinase 1. Our in vivo binding data show that NS5A does not inhibit interaction between TNF receptor-associated death domain and TRAF2 protein, indicating that NS5A and TRAF2 may form a ternary complex with TNF receptor-associated death domain. These results indicate that HCV NS5A protein modulates TNF signaling of the host cells and may play a role in HCV pathogenesis.


Subject(s)
Hepacivirus/enzymology , Hepatitis C/virology , MAP Kinase Kinase Kinase 1 , Mitogen-Activated Protein Kinases/metabolism , Proteins/metabolism , Viral Nonstructural Proteins/metabolism , Cell Line , Gene Expression , Genes, Dominant , Hepacivirus/pathogenicity , Hepatitis C/metabolism , Humans , JNK Mitogen-Activated Protein Kinases , Kidney/cytology , Mutation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/physiology , TNF Receptor-Associated Factor 1 , TNF Receptor-Associated Factor 2 , Tumor Necrosis Factor-alpha/metabolism , Viral Nonstructural Proteins/genetics , Virulence
9.
J Biol Chem ; 278(37): 35272-8, 2003 Sep 12.
Article in English | MEDLINE | ID: mdl-12829720

ABSTRACT

Heat shock protein 27 (Hsp27) is a ubiquitously expressed member of the heat shock protein family that has been implicated in various biological functions including the response to heat shock, oxidative stress, and cytokine treatment. Previous studies have demonstrated that heat shock proteins are involved in regulating signal transduction pathways including the NF-kappa B pathway. In this study, we demonstrated that Hsp27 associates with the I kappa B kinase (IKK) complex and that this interaction was stimulated by tumor necrosis factor alpha treatment. Phosphorylation of Hsp27 by the kinase mitogen-activated protein kinase-activated protein kinase 2, a downstream substrate of the mitogen-activated protein kinase p38, enhanced the association of Hsp27 with IKK beta to result in decreased IKK activity. Consistent with these observations, treatment of cells with a p38 inhibitor reduced the association of Hsp27 with IKK beta and thus resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in down-regulating IKK signaling by reducing its activity following tumor necrosis factor alpha stimulation.


Subject(s)
Heat-Shock Proteins , NF-kappa B/metabolism , Neoplasm Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Base Sequence , DNA Primers , HSP27 Heat-Shock Proteins , HeLa Cells , Humans , I-kappa B Kinase , Kinetics , Molecular Chaperones , NF-kappa B/drug effects , Neoplasm Proteins/genetics , Phosphorylation , RNA, Messenger/genetics , RNA, Small Nuclear/metabolism , Recombinant Proteins/metabolism , Transfection
10.
Mol Cell ; 11(4): 1055-66, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12718890

ABSTRACT

SPT5 and its binding partner SPT4 function in both positively and negatively regulating transcriptional elongation. The demonstration that SPT5 and RNA polymerase II are targets for phosphorylation by CDK9/cyclin T1 indicates that posttranslational modifications of these factors are important in regulating the elongation process. In this study, we utilized a biochemical approach to demonstrate that SPT5 was specifically associated with the protein arginine methyltransferases PRMT1 and PRMT5 and that SPT5 methylation regulated its interaction with RNA polymerase II. Specific arginine residues in SPT5 that are methylated by these enzymes were identified and demonstrated to be important in regulating its promoter association and subsequent effects on transcriptional elongation. These results suggest that methylation of SPT5 is an important posttranslational modification that is involved in regulating its transcriptional elongation properties in response to viral and cellular factors.


Subject(s)
Chromosomal Proteins, Non-Histone , Eukaryotic Cells/enzymology , Protein Processing, Post-Translational/genetics , RNA Polymerase II/metabolism , Transcription, Genetic/genetics , Transcriptional Elongation Factors/metabolism , Arginine/genetics , Arginine/metabolism , Cytokines/genetics , Cytokines/metabolism , Cytokines/pharmacology , Gene Expression Regulation, Viral/genetics , HIV-1/genetics , HIV-1/metabolism , HeLa Cells , Humans , Methylation , Mutation/genetics , Promoter Regions, Genetic/genetics , Protein Methyltransferases/genetics , Protein Methyltransferases/metabolism , Protein-Arginine N-Methyltransferases/genetics , Protein-Arginine N-Methyltransferases/metabolism , RNA Polymerase II/genetics , Transcriptional Elongation Factors/genetics
11.
J Mol Biol ; 326(1): 105-15, 2003 Feb 07.
Article in English | MEDLINE | ID: mdl-12547194

ABSTRACT

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.


Subject(s)
MAP Kinase Kinase Kinases/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Enzyme Activation/drug effects , Gene Expression , HeLa Cells , Humans , I-kappa B Kinase , Interleukin-1/pharmacology , MAP Kinase Kinase Kinases/genetics , Protein Serine-Threonine Kinases/genetics , Proteins/genetics , Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal Transduction/drug effects , TNF Receptor-Associated Factor 2 , Transfection , Tumor Necrosis Factor-alpha/pharmacology
12.
Intervirology ; 45(3): 142-9, 2002.
Article in English | MEDLINE | ID: mdl-12403918

ABSTRACT

Hepatitis delta antigen (HDAg) consists of two species, small HDAg (SHDAg) and large HDAg (LHDAg), which are identical in sequence with the exception that the large form contains an additional 19 amino acids at the C-terminus. Both HDAgs are nuclear phosphoproteins. However, LHDAg is hyperphosphorylated, i.e. it is at least 10 times more phosphorylated than SHDAg. To determine the phosphorylation site(s) of the LHDAg, we mutated all the conserved serine residues and expressed these mutant proteins using a recombinant baculovirus expression system. By labeling insect cells in vivo with (32)P-orthophosphate and immunoprecipitation, we showed that LHDAg is phosphorylated at multiple serine residues. Although LHDAg contains two additional serines at its 19-amino acid extension, mutations of these two amino acids did not affect the overall phosphorylation level. Most importantly, the phosphorylation level of middle domain-deleted LHDAg (M75del) was significantly higher than that of wild-type LHDAg. We conclude that phosphorylation of the LHDAg occurs at multiple sites and that hyperphosphorylation is associated with alteration of protein conformation.


Subject(s)
Hepatitis Delta Virus/immunology , Hepatitis delta Antigens/chemistry , Hepatitis delta Antigens/metabolism , Animals , Cells, Cultured , Genetic Vectors , Hepatitis Delta Virus/chemistry , Hepatitis Delta Virus/genetics , Hepatitis delta Antigens/genetics , Mutagenesis, Site-Directed , Mutation , Nucleopolyhedroviruses/genetics , Phosphorylation , Precipitin Tests , Protein Conformation , Spodoptera/virology
13.
J Biol Chem ; 277(15): 13122-8, 2002 Apr 12.
Article in English | MEDLINE | ID: mdl-11821416

ABSTRACT

The hepatitis C virus nonstructural protein 5A (NS5A) is a multifunctional phosphoprotein that leads to pleiotropic responses, in part by regulating cell growth and cellular signaling pathways. Here we show that overexpression of NS5A inhibits tumor necrosis factor (TNF)-alpha-induced nuclear factor kappaB (NF-kappaB) activation in HEK293 cells, as determined by luciferase reporter gene expression and by electrophoretic mobility shift assay. When overexpressed, NS5A cannot inhibit the recruitment of TNF receptor-associated factor 2 (TRAF2) and IkappaB kinase (IKK)beta into the TNF receptor 1-TNF receptor-associated death domain complex. In contrast, NS5A is a part of the TNF receptor 1 signaling complex. NF-kappaB activation by TNF receptor-associated death domain and TRAF2 was inhibited by NS5A, whereas MEKK1 and IKKbeta-dependent NF-kappaB activation was not affected, suggesting that NS5A may inhibit NF-kappaB activation signaled by TRAF2. Coimmunoprecipitation and colocalization of NS5A and TRAF2 expressed in vivo provide compelling evidence that NS5A directly interacts with TRAF2. This interaction was mapped to the middle one-third (amino acids 148-301) of NS5A and the TRAF domain of TRAF2. Our findings suggest a possible molecular mechanism that could explain the ability of NS5A to negatively regulate TNF-alpha-induced NF-kappaB activation.


Subject(s)
MAP Kinase Kinase Kinase 1 , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/physiology , Viral Nonstructural Proteins/physiology , Animals , Base Sequence , Cell Line , DNA Primers , Electrophoretic Mobility Shift Assay , Humans , I-kappa B Kinase , Microscopy, Confocal , Protein Serine-Threonine Kinases/metabolism , Proteins/antagonists & inhibitors , TNF Receptor-Associated Factor 2
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