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Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC 50 , 67 μM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

With the current worldwide pandemic of COVID-19, there is an urgent need to develop effective treatment and prevention methods against SARS-CoV-2 infection. We have previously reported that the proanthocyanidin (PAC) fraction in blueberry (BB) leaves has strong antiviral activity against hepatitis C virus (HCV) and human T-lymphocytic leukemia virus type 1 (HTLV-1). In this study, we used Kunisato 35 Gou (K35) derived from the rabbit eye blueberry (Vaccinium virgatum Aiton), which has a high PAC content in the leaves and stems. The mean of polymerization (mDP) of PAC in K35 was the highest of 7.88 in Fraction 8 (Fr8) from the stems and 12.28 of Fraction 7 (Fr7) in the leaves. The composition of BB-PAC in K35 is that most are B-type bonds with a small number of A-type bonds and cinchonain I as extension units. A strong antiviral effect was observed in Fr7, with a high polymerized PAC content in both the leaves and stems. Furthermore, when we examined the difference in the action of BB-PAC before and after SARS-CoV-2 infection, we found a stronger inhibitory effect in the pre-infection period. Moreover, BB-PAC Fr7 inhibited the activity of angiotensin II converting enzyme (ACE2), although no effect was observed in a neutralization test of pseudotyped SARS-CoV-2. The viral chymotrypsin-like cysteine protease (3CLpro) of SARS-CoV-2 was also inhibited by BB-PAC Fr7 in leaves and stems. These results indicate that BB-PAC has at least two different inhibitory effects, and that it is effective in suppressing SARS-CoV-2 infection regardless of the time of infection., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The progression of chronic kidney disease (CKD) increases the risks of cardiovascular morbidity and end-stage kidney disease. Indoxyl sulfate (IS), which is derived from dietary l-tryptophan by the action of bacterial l-tryptophan indole-lyase (TIL) in the gut, serves as a uremic toxin that exacerbates CKD-related kidney disorder. A mouse model previously showed that inhibition of TIL by 2-aza-l-tyrosine effectively reduced the plasma IS level, causing the recovery of renal damage. In this study, we found that (+)-sesamin and related lignans, which occur abundantly in sesame seeds, inhibit intestinal bacteria TILs. Kinetic studies revealed that (+)-sesamin and sesamol competitively inhibited Escherichia coli TIL (EcTIL) with K i values of 7 μM and 14 μM, respectively. These K i values were smaller than that of 2-aza-l-tyrosine (143 μM). Molecular docking simulation of (+)-sesamin- (or sesamol-)binding to EcTIL predicted that these inhibitors potentially bind near the active site of EcTIL, where the cofactor pyridoxal 5'-phosphate is bound, consistent with the kinetic results. (+)-Sesamin is a phytochemical with a long history of consumption and is generally regarded as safe. Hence, dietary supplementation of (+)-sesamin encapsulated in enteric capsules could be a promising mechanism-based strategy to prevent CKD progression. Moreover, the present findings would provide a new structural basis for designing more potent TIL inhibitors for the development of mechanism-based therapeutic drugs to treat CKD., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Recent research has revealed that glioblastoma (GBM) avoids the immune system via strong expression of indoleamine 2,3-dioxygenase 1 (IDO1). IDO1, an enzyme involved in tryptophan metabolism, is now proposed as a new target in GBM treatment, since several reports have demonstrated that IDO1 expression is related to GBM malignancy. On the other hand, it is well known that glioma stem cells (GSCs) are strongly related to the malignancy of GBM. However, there is as yet no report evaluating the relationship between GSCs and IDO1. We therefore examined the expression levels of IDO1 in GSCs in order to identify a new therapeutic target for GBM based on the immune systems of GSCs. In the present study, we employed human GBM cell lines (U-138MG, U-251MG) and patient-derived GSC model cell lines (0125-GSC, 0222-GSC). GSC model cell lines Rev-U-138MG and Rev-U-251MG were established by culturing U-138MG and U-251MG in serum-free media, while differentiated GBM model cell lines 0125-DGC and 0222-DGC were established by culturing 0125-GSC and 0222-GSC in serum-containing media. The expression levels of stem cell markers (Nanog, Nestin, Oct4 and Sox2) and IDO1 protein and mRNA were determined. Rev-U-138MG and Rev-U-251MG formed spheres and their expression levels of stem cell markers were increased as compared to U-138MG and U-251MG. On the other hand, 0125-DGC and 0222-DGC suffered breakdown of sphere formation, despite the original 0125-GSC and 0222-GSC forming spheres, and their expression levels of the markers were decreased. IDO1 expressions were strongly recognized in Rev-U-138MG, Rev-U-251MG, 0125-GSC and 0222-GSC as compared to U-138MG, U-251MG, 0125-DGC and 0222-DGC. These findings demonstrate that GSCs exhibit treatment resistance with immunosuppression via high expression levels of IDO1, and could represent a novel target for GBM treatment., Competing Interests: Declaration of competing interest We have no potential conflicts of interest to disclosure., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Invariant natural killer T (iNKT) cells exhibit potent antitumor effects upon activation by recognizing a specific glycolipid antigen. We previously performed phase I-II clinical studies to utilize iNKT cells using α-galactosylceramide-pulsed dendritic cells and identified leukotriene B4 12-hydroxydehydrogenase (LTB4DH) as a biomarker highly expressed in T cells derived from non-small cell lung cancer (NSCLC) patients who showed prolonged survival in respond to the iNKT cell immunotherapy. Because LTB4DH expression correlated with prolonged survival of NSCLC patients, we considered LTB4DH to play a role in iNKT cell immunotherapy. We herein demonstrate that the overexpression of LTB4DH in CD4 + or CD8 + T cells increases interferon-γ production and tumoricidal activity in the presence of prostaglandin E 2 . Moreover, the expression of granzyme a, granzyme b, and perforin mRNA was increased in LTB4DH-overexpressing cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The hyperpolarization-activated cyclic nucleotide-gated 2 (HCN2) channel is a major subtype of the HCN channel family expressed in the nervous system that sets the membrane potential, regulates cell excitability and senses changes in the extracellular environment. Neurons express various Gα s -protein-coupled receptors (GPCRs), many of which show ligand-independent constitutive activity. These membrane-bound proteins are expressed in various subcellular compartments of neurons. Therefore, some proportion of HCN2 channels opens in response to the basal cAMP pool size produced by constitutively active GPCRs. Here, we employed an exogenous HEK293 expression system and voltage-clamp patch-clamp recordings to investigate basal HCN2 channel activity in the presence of two GPCRs with diverse basal activities in a single compartment. We utilized the β2-adrenoceptor (β2AR) together with odorant receptors (ORs), as both GPCR families are known to show strong basal activity. Consequently, β2AR alone strongly enhanced the activity of HCN2 channels, and co-expression of ORs further diversified the HCN2 channel activity, which was totally abolished by an adenylate cyclase inhibitor. Thus, we conclude that the dual expression of constitutively active GPCRs establishes the diverse range of the basal cAMP pool size in resting cells through mutual additive or suppressive interactions, even in the absence of external stimulation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Metabolic enzymes, including those involved in flavonoid biosynthesis, are proposed to form weakly bound, ordered protein complexes, called "metabolons". Some hypothetical models of flavonoid biosynthetic metabolons have been proposed, in which metabolic enzymes are believed to anchor to the cytoplasmic surface of the endoplasmic reticulum (ER) via ER-bound cytochrome P450 isozymes (P450s). However, no convincing evidence for the interaction of flavonoid biosynthetic enzymes with P450s has been reported previously. Here, we analyzed binary protein-protein interactions of 2-hydroxyisoflavanone synthase 1 (GmIFS1), a P450 (CYP93C), with cytoplasmic enzymes involved in isoflavone biosynthesis in soybean. We identified binary interactions between GmIFS1 and chalcone synthase 1 (GmCHS1) and between GmIFS1 and chalcone isomerases (GmCHIs) by using a split-ubiquitin membrane yeast two-hybrid system. These binary interactions were confirmed in planta by means of bimolecular fluorescence complementation (BiFC) using tobacco leaf cells. In these BiFC analyses, fluorescence signals that arose from the interaction of these cytoplasmic enzymes with GmIFS1 generated sharp, network-like intracellular patterns, which was very similar to the ER-localized fluorescence patterns of GmIFS1 labeled with a fluorescent protein. These observations provide strong evidence that, in planta, interaction of GmCHS1 and GmCHIs with GmIFS1 takes place on ER on which GmIFS1 is located, and also provide important clues to understand how enzymes and proteins form metabolons to establish efficient metabolic flux of (iso)flavonoid biosynthesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Histone acetyltransferase p300/CBP-associated factor (PCAF) belonging to GCN5 family regulates various epigenetic events for transcriptional regulation through alterations in the chromatin structure. During normal development of B cells, gene expressions of numerous transcription factors are strictly regulated by epigenetic mechanisms including histone acetylation and deacetylation to complete their development pathways. Here, by analyzing PCAF-deficient DT40 mutants, ΔPCAF, we report that PCAF takes part in transcriptional activation of B cell lymphoma-6 (Bcl-6) and Paired box gene 5 (Pax5), which are essential transcription factors for normal development of B cells. PCAF-deficiency caused drastic decrease in mRNA levels of Bcl-6 and Pax5, and remarkable increase in that of B lymphocyte-induced maturation protein-1 (Blimp-1). In addition, chromatin immunoprecipitation assay showed that PCAF-deficiency caused remarkable decrease in acetylation levels of both H3K9 and H3K14 residues within chromatin surrounding the 5'-flanking regions of Bcl-6 and Pax5 genes in vivo, suggesting that their gene expressions may be regulated by PCAF. These results revealed that PCAF is involved in transactivation of Bcl-6 and Pax5 genes, resulting in down-regulation of Blimp-1 gene expression, and plays a key role in epigenetic regulation of B cell development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The endoplasmic reticulum (ER), a complex membrane structure, has important roles in all eukaryotic cells. Catastrophe of its functions would lead to ER stress that causes various diseases such as cancer, neurodegenerative diseases, diabetes and so on. Prolonged ER stress could trigger apoptosis via activation of various signal transduction pathways. To investigate physiological roles of histone acetyltransferase GCN5 in regulation of ER stress, we analyzed responses of homozygous GCN5-deficient DT40 mutants, ΔGCN5, against ER stress. GCN5-deficiency in DT40 caused drastic resistance against apoptosis induced by pharmacological ER stress agents (thapsigargin and tunicamycin). Pharmaceutical analysis using specific Bcl-2 inhibitors showed that the drastic resistance against prolonged ER stress-induced apoptosis is, in part, due to up-regulation of Bcl-2 gene expression in ΔGCN5. These data revealed that GCN5 is involved in regulation of prolonged ER stress-induced apoptosis through controlling Bcl-2 gene expression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

All isoprenoids are derived from a common C5 unit, isopentenyl diphosphate (IPP). In plants, IPP is synthesized via two distinct pathways; the cytosolic mevalonate pathway and the plastidial non-mevalonate (MEP) pathway. In this study, we used a co-expression analysis to identify transcription factors that coordinately regulate the expression of multiple genes encoding enzymes in the IPP biosynthetic pathway. Some candidates showed especially strong correlations with multiple genes encoding MEP-pathway enzymes. We report here that phytochrome-interacting factor 5 (PIF5), a basic-helix-loop-helix type transcription factor, functions as a positive regulator of the MEP pathway. Its overexpression in T87 suspension cultured cells resulted in increased accumulation of chlorophylls and carotenoids. Detailed analyses of carotenoids by HPLC indicated that some carotenoid biosynthetic pathways were concomitantly up-regulated, possibly as a result of enhanced IPP metabolic flow. Our results also revealed other PIF family proteins that play different roles from that of PIF5 in IPP metabolism., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The transcription factor, early B cell factor 1 (EBF1) with an atypical zinc-finger and helix-loop-helix motif, is essential for development and differentiation of lymphocytes. In mice, EBF1 is involved in the generation of pre-pro B cells (the first specified progenitors of B cells) from common lymphoid progenitors (CLPs) and transcription regulations of various genes involved in B cell-development, for instance, mb-1 and Pax5. During B lymphopoiesis, interestingly, EBF1 is detected throughout from CLPs to mature B cells. However, in immature B cells, the physiological role of EBF1 remains to be elucidated. Here, by analyzing EBF1-deficient DT40 cells, EBF1(-/-), generated by us, we show that EBF1-deficiency caused significant increases (to ∼800%) in both mRNA and protein levels of B lymphocyte-induced maturation protein-1 (Blimp-1), the master gene for plasma cell differentiation. In addition, both transcription and protein synthesis of Blimp-1 were remarkably down-regulated (to ∼20%) by re-expression (over-expression) of EBF1. Chromatin immunoprecipitation assay revealed that EBF1 binds to proximal 5'-upstream regions around two putative EBF1 binding motifs of the gene in vivo. These results suggest that EBF1 takes part in transcriptional regulations of the Blimp-1 gene in immature B cells, and may play a key role in B cell differentiation. This is the first report on a novel EBF1 function in immature B cells as a powerful repressor of Blimp-1 gene expression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Histone acetyltransferase(s) (HATs) are involved in the acetylation of core histones, which is an important event for transcription regulation through alterations in the chromatin structure in eukaryotes. General control non-depressible 5 (GCN5) was first identified as a global coactivator and transcription-related HAT. Here we report that GCN5 regulates the activation of phosphatidylinositol 3-kinase (PI3K)/acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) survival pathway in B cells exposed to oxidative stress via controlling gene expressions of spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk). The GCN5-deficiency remarkably caused apoptotic cell death by treatment with exogenous hydrogen peroxide (H(2)O(2)) in chicken DT40 cells. In GCN5-deficient DT40 cells, gene expressions of Syk and Btk, which are involved in activation of PI3K/Akt survival pathway in DT40 cells exposed to exogenous H(2)O(2), were remarkably decreased compared with those in wild type DT40 cells. In addition, phosphorylation of Akt in H(2)O(2)-treated GCN5-deficient cells was remarkably suppressed as compared to that of DT40. Chromatin immunoprecipitation assay revealed that GCN5 binds to proximal 5'-upstream regions of Syk and Btk genes in vivo. These results suggest that GCN5 takes part in transcriptional regulations of the Syk and Btk genes, and plays a key role in epigenetic regulation of PI3K/Akt survival pathway in B cells exposed to reactive oxygen species such as H(2)O(2)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The membrane bound cytochrome b558 composed of large gp91-phox and small p22-phox subunits, and cytosolic proteins p40-, p47- and p67-phox are important components of superoxide (O(2)(-))-generating system in phagocytes and B lymphocytes. A lack of this system in phagocytes is known to cause serious life-threatening infections. Here, we describe that curcumin, a polyphenol responsible for the yellow color of curry spice turmeric, dramatically activates the O(2)(-)-generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and curcumin, the O(2)(-)-generating activity increased more than 4-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and curcumin slightly enhanced gene expressions of the five components compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and curcumin caused remarkable accumulation of protein levels of p47-phox (to 7-fold) and p67-phox (to 4-fold) compared with those of the RA-treatment alone. These results suggested that curcumin dramatically enhances RA-induced O(2)(-)-generating activity via accumulation of cytosolic p47-phox and p67-phox proteins in U937 cells. Therefore, it should have the potential as an effective modifier in therapy of leukemia and/or as an immunopotentiator., (2010 Elsevier Inc. All rights reserved.)

Histone H3 methylation at lysine 4 (K4) is associated with euchromatic regions and is thought to be important for the transcriptional activation of genes during differentiation. In this study, we found that di- and tri-methylation of histone H3 at K4 and acetylation of histones H3 and H4 from the promoter/enhancer to the transcribed region close to the transcription initiation site of the solute carrier family 2, member 5 (SLC2A5) gene, and its expression, were induced by differentiation of intestine-like Caco-2 cells. These effects were accompanied by contact inhibition of cell growth of these cells. Furthermore, these modifications were induced by co-treatment with a synthetic glucocorticoid hormone dexamethasone and a p44/42 mitogen-activated protein kinase inhibitor PD89059. Our results suggest that methylation of histone H3 at K4 and acetylation of histones H3 and H4 are involved in SLC2A5 gene induction associated with intestinal differentiation of Caco-2 cells., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO(2)) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO(2)) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK., (Copyright 2010. Published by Elsevier Inc.)

Angioid streaks (AS) are eye abnormalities caused by breaks in Bruch's membrane. The condition is often associated with pseudoxanthoma elasticum (PXE). The ATP-binding cassette, sub-family C (CFTR/MRP), member 6 (ABCC6) is reported to be the causal gene for PXE, although there have been no reports on whether the ABCC6 gene is the causal gene for AS. The aims of this study are to isolate the causal mutations for AS using a haplotype-based case-control study. We genotyped 54 Japanese AS patients and 150 controls for 5 single-nucleotide polymorphisms (SNPs). A simple association study using each SNP and a haplotype-based case-control study were performed. Twelve patients with special haplotypes for AS were selected, and were then subjected to gene sequencing. Six variants were successfully identified as causal mutations for AS (p.R419Q, p.E422K, c.2542delG, Del_Exon23, c.3774-3775insC and p.E1427K), and 4 of these were novel. This method can be applied to both identifying susceptibility variants of multifactorial diseases and isolating mutations in single-gene diseases.

The preprotachykinin C gene encodes four endokinins, A, B, C, and D. Endokinins A and B and substance P (SP) are typical tachykinin peptides since their carboxyl-terminal regions share an F-F-G-L-M-amide, while endokinins C and D share an F-Q-G-L-L-amide. It is demonstrated that pretreatment with a peptide consisting of a common sequence between endokinins C and D (EKC/D) attenuates the induction of scratching behavior and thermal hyperalgesia by intrathecal administration of SP or EKA/B (the carboxyl-terminal dacapeptide common in endokinins A and B), suggesting that leucine at the carboxyl-terminal of EKC/D may have a crucial role in eliciting these effects. When the effect of [Leu(11)]-SP and [Leu(10)]-EKA/B on SP-induced pain-related behavior was examined, the induction of pain-related behavior was markedly attenuated by pretreatment with these peptides. This indicates that leucine at the carboxyl-terminal of these peptides plays a crucial role in eliciting this antagonistic effect.

Amounts of soluble histones in cells are tightly regulated to ensure supplying them for the newly synthesized DNA and preventing the toxic effect of excess histones. Prior to incorporation into chromatin, newly synthesized histones H3 and H4 are highly acetylated in pre-deposition complex, wherein H4 is di-acetylated at Lys-5 and Lys-12 residues by histone acetyltransferase-1 (Hat1), but their role in histone metabolism is still unclear. Here, using chicken DT 40 cytosolic extracts, we found that histones H3/H4 and their chaperone Asf1, including RbAp48, a regulatory subunit of Hat1 enzyme, were associated with Hat1. Interestingly, in HAT1-deficient cells, cytosolic histones H3/H4 fractions on sucrose gradient centrifugation, having a sedimentation coefficient of 5-6S in DT40 cells, were shifted to lower molecular mass fractions, with Asf1. Further, sucrose gradient fractionation of semi-purified tagged Asf1-complexes showed the presence of Hat1, RbAp48 and histones H3/H4 at 5-6S fractions in the complexes. These findings suggest the possible involvement of Hat1 in regulating cytosolic H3/H4 pool mediated by Asf1-containing cytosolic H3/H4 pre-deposition complex.

Malondialdehyde (MDA), a lipid peroxidation product, reacts with lysine residues in proteins. Human serum albumin (HSA) is a major target of MDA-modification of serum proteins. To identify, the modification sites of HSA by MDA in vitro, MDA-treated HSA was digested with a protease and the resulting peptides were subjected to liquid chromatography-tandem mass spectrometry. We identified six peptides, which contained a N-propenal adduct at Lys136, Lys174, Lys240, Lys281, Lys525, and Lys541, and revealed that Lys525 is the most reactive residue for MDA modification. Analysis of electrostatic surface potential of a 3-D model structure of HSA indicates that Lys525 is located at the center of positively charged grooves. The results of this study indicate that the modification of proteins by lipid-derived aldehydes may be influenced by the electrostatic potential of the protein surface.

F(1)-ATPase is a rotary molecular motor powered by the torque generated by another rotary motor F(0) to synthesize ATP in vivo. Therefore elucidation of the behavior of F(1) under external torque is very important. Here, we applied controlled external torque by electrorotation and investigated the ATP-driven rotation for the first time. The rotation was accelerated by assisting torque and decelerated by hindering torque, but F(1) rarely showed rotations in the ATP synthesis direction. This is consistent with the prediction by models based on the assumption that the rotation is tightly coupled to ATP hydrolysis and synthesis. At low ATP concentrations (2 and 5 microM), 120 degrees stepwise rotation was observed. Due to the temperature rise during experiment, quantitative interpretation of the data is difficult, but we found that the apparent rate constant of ATP binding clearly decreased by hindering torque and increased by assisting torque.

Bone marrow stromal cells (BMSCs) are a mixture of cells differing in differentiation potential including mesenchymal stem cells, and so far no CD antigens were found to be predictable for the differentiation property of each BMSC. Here we attempted to isolate differentiation-associated CD antigens using 100 immortalized human BMSC (ihBMSC) clones. Among 13 CD antigens analyzed, only CD106/Vascular cell adhesion molecule-1 (VCAM-1) showed a clear correlation with the differentiation potential of each clone; CD106-positive ihBMSC clones were less osteogenic and more adipogenic than CD106-negative clones. This association was confirmed in primary BMSCs sorted by CD106, showing that the CD106-positive fraction contained less osteogenic and more adipogenic cells than the CD106-positive fraction. The evaluation of CD106 fraction of BMSC strains in early passages predicted clearly the osteogenic and adipogenic potential after in vitro induction of differentiation, indicating the usefulness of CD106 as a differentiation-predicting marker of BMSC.

The expression of the chondromodulin-I (ChM-I) gene, a cartilage-specific gene, is regulated by the binding of Sp3 to the core promoter region, which is inhibited by the methylation of CpG in the target genome in the osteogenic lineage, osteosarcoma (OS) cells. The histone tails associated with the hypermethylated promoter region of the ChM-I gene were deacetylated by histone deacetylase 2 (HDAC2) in three ChM-I-negative OS cell lines. Treatment with an HDAC inhibitor induced the binding of Sp3 in one cell line, which became ChM-I-positive. This process was associated with acetylation instead of the dimethylation of histone H3 at lysine 9 (H3-K9) and, surprisingly, the demethylation of the core promoter region. The demethylation was transient, and gradually replaced by methylation after a rapid recovery of histone deacetylaion. These results represent an example of the plasticity of differentiation being regulated by the cell-specific plasticity of epigenetic regulation.

Tissue stem cells may serve as progenitors for malignant tumors derived from the same tissue. Here, we report the establishment of immortalized human mesenchymal stem cells (ihMSC) and tested the feasibility of using ihMSC as presarcomatous cells. Immortalization was achieved by introducing the genes for human telomerase reverse transcriptase and Bmi1. ihMSC retained the potential for multi-directional differentiation of the original MSC. To transform ihMSC, we introduced an oncogenic H-ras(Val12) gene, and established the cell line ihMSC-ras. ihMSC-ras had the phenotype of fully transformed cells and retained adipogenic and chondrogenic, but not osteogenic, potential. Interestingly, ihMSC-ras demonstrated morphological features of autophagy, and inhibition of the ERK pathway suppressed the production of autophagosomes, indicating that ras/ERK signaling is responsible for the induction of autophagy. Thus ihMSC will serve as a material with which to analyze the tumorigenic and differentiation-modifying effects of candidate oncogenes involved in the development of sarcomas.

Histone acetyltransferase 1 (HAT1) is implicated for diacetylation of Lys-5 and Lys-12 of newly synthesized histone H4, the biological significance of which remains unclear. To investigate the in vivo role of HAT1, we generated HAT1-deficient DT40 clone (HAT1(-/-)). HAT1(-/-) cells exhibited greatly reduced diacetylation levels of Lys-5 and Lys-12, and acetylation level of Lys-5 of cytosolic and chromatin histones H4, respectively. The in vitro nucleosome assembly assay and in vivo MNase digestion assay revealed that HAT1 and diacetylation of Lys-5 and Lys-12 of histone H4 are dispensable for replication-coupled chromatin assembly. HAT1(-/-) cells had mild growth defect, conferring sensitivities to methyl methanesulfonate and camptothecin that enforce replication blocks creating DNA double strand breaks. Such heightened sensitivities were associated with prolonged late-S/G2 phase. These results indicate that HAT1 participates in recovering replication block-mediated DNA damages, probably through chromatin modulation based on acetylation of Lys-5 and Lys-12 of histone H4.

Platelet adhesion is mediated by von Willebrand factor (VWF) that binds platelet glycoprotein Ib (GPIb). Previous observations suggested that heparin competitively inhibits the binding of VWF to GPIb and may down-regulate platelet adhesion. We performed charged-to-alanine scanning mutagenesis of domain A1 and studied dose-dependent binding to heparin-Sepharose beads. Mutations at Lys1362 and Arg1395, at which the GPIb binding was markedly decreased, showed 41% and 42% binding, respectively. Clustered mutations in the segments 1332KDRKR1336 and 1405KKKK1408, which have been proposed as heparin binding sequences, showed 72% and 52% binding, respectively. However, single alanine substitutions within these clusters showed normal binding. Our findings suggest that heparin may inhibit the binding of VWF to GPIb by interacting with GPIb binding and interpret why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.

A new category of methicillin-resistant Staphylococcus aureus (MRSA), called community-acquired MRSA (CA-MRSA), has emerged worldwide. In contrast to previous MRSA, most CA-MRSA carries the Panton-Valentine leukocidin (PVL) genes (lukPVSF) as a virulence genetic trait. Sequence analysis of the lukPVSF gene of a Japanese isolate demonstrated that the gene has more similarity to methicillin-susceptible S. aureus from France than MRSA from the United States. Based on the sequences, we developed a real-time PCR assay for the three key genes of CA-MRSA; that is, lukPVSF, mecA (for methicillin resistance), and spa (for S. aureus). Dual or triple assay for lukPVSF, mecA, and spa in one test tube became possible. The detection limit of the assay with probe and SYBR Green methods was between 2.7 and 2.7 x 10(1) CFU/ml. The assay detected PVL-positive MRSA in clinical (blood) isolates.

The addition of lecithin molecules to brain-derived neurotrophic factor (BDNF) has been reported to markedly enhance its pharmacological effect in vivo. In the current study, we show that lecithinized BDNF (PC-BDNF) has a higher affinity than BDNF for neural precursor cells. Although BDNF only slightly increased the expression of the genes for Mash-1, p35, 68 kDa neurofilament, and TrkB receptor, PC-BDNF caused a significant increase in their expression. PC-BDNF also increased the level of neurofilament protein and dramatically increased TrkB mRNA gene expression, which was followed by a sustained activation of the p42/p44 extracellular-regulated kinases. Finally, transplantation of PC-BDNF-treated cells was more effective than BDNF-treated cells at improving impaired motor function caused by spinal cord injury. These findings showed that PC-BDNF has a better potential than BDNF for promoting neural differentiation, partly due to a higher cellular affinity. Furthermore, PC-BDNF-treated cells could be useful for transplantation therapy for central nervous system injuries.

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase requires redox co-enzymes, i.e., flavin mononucleotide (FMN) and NAD(P)H, for activity, although it catalyzes a non-redox reaction. Spectrometric studies and enzyme assays under anaerobic conditions indicate that FMN is reduced through the reaction and is sufficient for activity. The sole function of NAD(P)H appears to be the reduction of FMN since it could be replaced by an alternate reducing agent. When the enzyme was reconstructed with a flavin analogue, no activity was detected, suggesting that the isomerase reaction proceeds via a radical transfer mechanism.

In this study, we screened the anti-tumor activity of murine chemokines including CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1, and CX3CL1 by inoculating murine B16BL6, CT26, or OV-HM tumor cells, all of which were transfected with chemokine-expressing fiber-mutant adenovirus vector, into immunocompetent mice. A tumor-suppressive effect was observed in mice inoculated with CCL19/B16BL6 and XCL1/B16BL6, and CCL22/OV-HM showed considerable retardation in tumor growth. In the cured mice inoculated with CCL22/OV-HM, a long-term specific immune protection against parental tumor was developed. However, we were unable to identify the chemokine that had a suppressive activity common to all three tumor models. Furthermore, an experiment using chemokine-transfected B16BL6 cells was also performed on mice sensitized with melanoma-associated antigen. A drastic enhancement of the frequency of complete rejection was observed in mice inoculated with CCL17-, CCL19-, CCL22-, and CCL27-transfected B16BL6. Altogether, our results suggest that the tumor-suppressive activity of chemokine-gene immunotherapy is greatly influenced by the kind of tumor and the activation state of the host's immune system.

We previously reported not only that chicken HIRA, a homolog of Saccharomyces cerevisiae transcriptional corepressors Hir1p and Hir2p, possesses seven WD dipeptide motifs and a LXXLL motif in its N-terminal half and C-terminal half, respectively, but also that the N-terminal and C-terminal halves, respectively, bind to CAF-1p48 and HDAC-1 and -2 in vitro. Seven WD dipeptide motifs in the N-terminal half of HIRA are required for the in vitro interaction with CAF-1p48. The LXXLL motif at positions 993-997 of HIRA is necessary for the in vitro interaction with HDAC-2. Here we revealed not only that the N-terminal and C-terminal halves of HIRA mediate individually transcription repressions but also that even one of the seven WD dipeptide motifs and the LXXLL motif of HIRA are essential for the mediations in vivo. Moreover, the LXXLL motif is essential for the interaction with endogenous or recombinant HDAC-2 in vivo, probably resulting in formation of the active complex, harboring the HDAC activity. Taken together, these results indicate that HIRA should participate differentially in a number of DNA-utilizing processes, including transcription repressions, through interactions of its distinct regions with CAF-1p48 and HDAC-2, respectively.

Examination of the sequence of a hypothetical gene with an unknown function included in the carotenogenic gene cluster in the genome of a thermoacidophilic archaeon Sulfolobus solfataricus led to the prediction that the gene encodes a novel-type lycopene beta-cyclase, whose N- and C-terminal halves are homologous to the subunits of the bacterial heterodimeric enzymes. The recombinant expression of the gene in lycopene-producing Escherichia coli resulted in the accumulation of beta-carotene in the cells, which verifies the function of the gene. Homologues of the archaeal lycopene beta-cyclase from various organisms such as bacteria, archaea, and fungi have been reported. Although their primary structures are clearly specific to the biological taxa, a phylogenetic analysis revealed the unexpected complicity of the evolutional route of these enzymes.

Dextran glucosidases show high sequence identity (50%) to Bacillus sp. SAM1606 alpha-glucosidase, which is more specific for short-chain substrates. Sequence comparison of these enzymes as well as molecular modeling studies predicted that the extension of loop 4 of the (beta/alpha)(8)-barrel fold may be responsible for the narrower specificity of SAM1606 alpha-glucosidase with respect to substrate chain length. Indeed, deletion mutants of SAM1606 alpha-glucosidase that lack this extension showed higher relative activities toward dextran and long-chain isomaltooligosaccharides. Kinetic and thermodynamic analyses of oligosaccharide hydrolysis catalyzed by SAM1606 alpha-glucosidase and its deletion mutants suggested that the loss of such extension(s) in loop 4 should energetically destabilize the Michaelis complexes with long-chain substrates to result in smaller differences between the activation free energies for the enzymatic hydrolyses of isomaltoheptaose and isomaltose than those observed for the wild-type enzyme. This is the reason that dextran glucosidase, whose loop 4 is shorter in length, shows broader substrate chain-length specificity than does SAM1606 alpha-glucosidase.

In a chicken B cell line, DT40, the disruption of HDAC2 (chHDAC2) gene causes an alteration of several gene expressions including chicken IgM light chain (chIgM-L) gene by 2D-PAGE analysis. To investigate the transcriptional function of chHDAC2, we employed the chIgM-L promoter reporter plasmid. We found that chHDAC2 represses activated chIgM-L promoter activity. In transient expression experiments in NIH 3T3 cell, the specific histone deacetylase inhibitor tricostatin A (TSA) increased transactivation of chIgM-L promoter activity mediated by chicken Oct-1 and OBF-1 proteins. In transient coexpression of the three class I chicken histone deacetylases (chHDAC1-3) tested, only chHDAC2 repressed the activated chIgM-L promoter activity. These findings suggest that chHDAC2 might be recruited to the chIgM-L promoter and specifically repress chIgM-L transcription.

The introduction of several sets of amino acid substitutions into the region around a substrate-binding site of a medium-chain (all-E) prenyl diphosphate synthase, hexaprenyl diphosphate synthase from a thermoacidophilic archaeon Sulfolobus solfataricus, to mimic the product determination mechanisms of various kinds of short-chain enzymes revealed that the structure around the region of the medium-chain enzyme resembles those of eukaryotic farnesyl diphosphate synthases but not those of the other short-chain enzymes, reflecting the evolutional relationships among these enzymes.

Prostacyclin inhibits platelet aggregation, smooth muscle cell proliferation, and vasoconstriction. The prostacyclin synthase (PGIS) gene is a candidate gene for cardiovascular disease. The purpose of this study was to locate possible mutations in the PGIS gene related to hypertension and cerebral infarction. Using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method, we discovered a T to C transition at the +2 position of the splicing donor site of intron 9 in patients with essential hypertension (EH). In vitro expression analysis of an allelic minigene consisting of exons 8-10 revealed that the nucleotide transition causes skipping of exon 9. This in turn alters the translational reading frame of exon 10 and introduces a premature stop codon (TGA). A three-dimensional model shows that the splice site mutation produces a truncated protein with a deletion in the heme-binding region. This splice site mutation was found in only one subject in 200 EH patients and 200 healthy controls. Analysis of the patient's family members revealed the mutation in two of the three siblings. The urinary excretion of prostacyclin metabolites in subjects with the mutation was significantly decreased. All subjects displaying the splice site mutation in the PGIS gene were hypertensive. In this study, we report a novel splicing mutation in the PGIS gene, which is associated with hypertension in a family. It is thought that this mechanism may involve in the pathophysiology of their hypertension.

Mesenchymal stem cells (MSCs) are bone marrow stroma-derived cells, which can differentiate into several types of mesenchymal tissues. Although regarded as tissue-specific stem cells, human MSCs (hMSCs) have a low proliferative ability with a finite life span, which is a hurdle to further analysis of their biology. Here we attempted to establish immortalized hMSCs by retrovirus-mediated gene transfer. The gain in telomerase activity obtained on expression of human telomerase reverse transcriptase (hTERT) was found not to be enough to make the cell line immortal. A combination of hTERT with human papillomavirus E6 and E7 successfully immortalized hMSCs without affecting the potential for adipogenic, osteogenic, and chondrogenic differentiation. From the parental immortalized hMSC, 100 single-cell derived clones were established, of which the differentiation properties varied considerably, including tri-, bi-, and uni-directional clones, suggesting that hMSCs are constituted by a group of cells with different differentiation potential. These cell lines, being the first established immortalized clonal cell lines of hMSCs, could provide insights into the mechanisms regulating the early steps of differentiation from undifferentiated MSCs into a specific lineage.

Hsc62 is the third Hsp70 homolog of Escherichia coli, which we found previously. Hsc62 is structurally and biochemically similar to DnaK, but hscC gene encoding Hsc62 did not compensate for the defects in the dnaK-null mutant of E. coli MC4100 strain. We cloned the ybeV gene and purified the gene product named Hsc56, a 55,687-Da protein with a J-domain like sequence. Hsc56 stimulated the ATPase activity of only Hsc62 but not those of the other Hsp70 homologs, DnaK and Hsc66. Hsc56 contains the -His-Pro-Glu- sequence corresponding to the His-Pro-Asp motif in DnaJ, which is indispensable for DnaJ to interact with DnaK. Conversion of -His-Pro-Glu- to -Ala-Ala-Ala- abolished the ability of Hsc56 to stimulate the ATPase activity of Hsc62. GrpE, a nucleotide exchange factor for DnaK, also stimulated the ATPase activity of Hsc62 in the presence of Hsc56. Hsc62-Hsc56-GrpE is probably a new Hsp70 chaperone system of E. coli.

Ets-1 is a transcription factor regulating the expression of matrix-degrading proteinases and is believed to play a critical role in cell migration and tumor invasion. The aim of this study is to investigate the direct induction of ets-1 with consequential upregulation of collagenase-1 (MMP-1) by cell adhesion to extracellular matrix and to identify intracellular signal transduction pathways involved in ets-1 induction in cultured endothelial cells. The expressions of ets-1 mRNA and protein as well as MMP-1 protein were induced by cell adhesion to type I collagen and antisense ets-1 oligonucleotides impaired that MMP-1 expression. In addition, protein tyrosine kinase (PTK) and protein kinase C (PKC) inhibitors abrogated their induction, showing the suppression of focal adhesion kinase phosphorylation. These results suggest that ets-1 induced by cell adhesion to extracellular matrix directly upregulates MMP-1 expression via PTK and PKC activation in cultured endothelial cells., (©2002 Elsevier Science (USA).)

We cloned cDNA encoding the chicken p46 polypeptide, chp46, homologous to the p48 subunit of chicken chromatin assembly factor-1, chCAF-1p48. It comprises 424 amino acids including a putative initiation Met, is a member of the WD protein family, with seven WD repeat motifs, and exhibits 90.3% identity to chCAF-1p48 and 94.3% identity to the human and mouse p46 polypeptides (hup46 and mop46). The in vitro immunoprecipitation experiment established that chp46 interacts with histones H2B and H4 and chicken histone acetyltransferase-1, chHAT-1, whereas hup46 interacts with histones H2A and H4 and chHAT-1 and chCAF-1p48 with histone H4 and chHAT-1. The in vitro immunoprecipitation experiment, involving truncated mutants of chp46, revealed not only that two regions comprising amino acids 33-179 and 375-404 are necessary for its binding to H2B, but also that two regions comprising amino acids 1-32 and 405-424 are necessary for its binding to H4. Furthermore, the GST pulldown affinity assay, involving truncated mutants of chp46, revealed that a region comprising amino acids 359-404 (in fact, 375-404) binds to chHAT-1 in vitro. Taken together, these results indicate not only that chp46 should participate differentially in a number of DNA-utilizing processes through interactions of its distinct regions with chHAT-1 and histones H2B and H4, but also that the proper propeller structure of chp46 is not necessary for its interaction with chHAT-1., (Copyright 2000 Academic Press.)

Indoleamine 2,3-dioxygenase (IDO) is expressed in trophoblasts and defends the conceptus against rejection by reducing the tryptophan level and suppressing the T cell activity. We isolated a cDNA for tryptophan 2,3-dioxygenase (TDO), another key catabolizing enzyme of tryptophan, from a mouse uterus cDNA library enriched with pregnancy-induced genes. Northern blot and in situ hybridization analyses demonstrated that the TDO mRNA was induced in the decidualized stromal cells around the implanted embryo at the time of implantation. The expression was then upregulated and primarily localized at the mesometrial decidua. TDO mRNA was induced by deciduoma formation as well as embryo transfer but not by ovarian steroid hormones. These findings demonstrated that TDO is induced in the endometrial stromal cells concomitant with decidualization and suggested its involvement in the implantation process by regulating the tryptophan level at the implantation site., (Copyright 2000 Academic Press.)

Thirty-nine of the 44 chicken histone genes are located in a major gene cluster of 110 kb, the others being distributed in four separate regions. All 6 H1 genes, which are present in the cluster and encode different variants, are expressed in the DT40 chicken B cell line, at levels ranging from about 5 to 40%. To clarify differences in the natures of these H1 variants, using gene-targeting techniques, we generated a series of DT40 mutants, which are devoid of each of the 5 H1 genes, respectively. Analyses of six H1-deficient mutants, comprising the latter five and a previously generated H1-deficient mutant, revealed that the protein patterns on 2D-PAGE were definitely different from each other, indicating that each H1 variant plays an individual role in the transcription regulation of specific genes in DT40 cells., (Copyright 2000 Academic Press.)

SAS3 was originally isolated as a gene related to SAS2, which encodes a positive regulator of transcriptional silencing in yeast. The Sas3 protein possesses an evolutionally conserved domain that is shared by a group of SAS-like factors. This conserved domain contains an atypical zinc finger motif and a putative acetyl-CoA binding motif. We showed that recombinant Sas3 exhibits histone acetyltransferase (HAT) activity toward acetylate core histones H2A, H3, and H4. This substrate specificity is similar to those of Tip60 and Esa1. Analysis of a series of deletion mutants revealed that the minimum region required for HAT activity is located within amino acid residues 241-577, including the domain conserved in the MYST family proteins. Amino acid substitution mutant analysis showed that both the acetyl-CoA binding motif and the zinc finger motif are required for HAT activity. These results suggest that SAS3 and its family members require the zinc finger motif for their activity., (Copyright 1999 Academic Press.)

p38 mitogen-activated protein kinase (MAP) kinase is activated by various stresses; however, little is known about cold stress which has been shown to cause various inflammatory diseases. In the present study, we examined the effect of cold stimulation on interleukin-8 (IL-8) expression and a role of p38 MAP kinase in IL-8 expression in human bronchial epithelial cells (BEC) in order to clarify the mechanism in hypothermic temperature-induced inflammation. The results showed that cold stimulation induced tyrosine phosphorylation of p38 MAP kinase but not IL-8 expression. IL-8 expression in BEC was induce when the temperature of incubation changed from 1 degree C to 37 degrees C (cooling and rewarming). The specific p38 MAP kinase inhibitor SB 203580 inhibited cooling and rewarming-induced IL-8 expression, indicating that cooling and rewarming-induced IL-8 expression in BEC was mediated through p38 MAP kinase-dependent pathway.

Nucleotide sequences of two mouse cDNAs encoding new sulfotransferase enzymes were determined. Analysis of the deduced amino acid sequences revealed that one represents a novel member of the phenol sulfotransferase family and the other is highly homologous to human SULT2B1 hydroxysteroid sulfotransferases. The recombinant enzymes, transiently expressed in COS-7 cells, were characterized with respect to their substrate specificity using a variety of substrates for different types of sulfotransferases. The tissue-specific expression of these two new mouse sulfotransferases was examined by Northern blot analysis.

We have determined the structural organization of the human natriuretic peptide receptor A (hNPRA) gene without screening the genomic library. Based on the information for the cDNA of hNPRA, we amplified some fragments of the long polymerase chain reaction (PCR) products covering all genomic sequences of the gene and directly sequenced the products after extraction and purification. The hNPRA gene spans approximately 16 kb and contains 22 exons and 21 introns. All of the exon-intron junction sequences coincide with the GT/AG consensus sequence. The sequence encoding the transmembrane domain, one part of the hNPRA topological structure, exists in exon 7. The results of this first study, determination of the hNPRA gene structure, will facilitate further genetic analysis of the hNPRA gene and its related diseases.

Ets-1 is a transcription factor known to control the expression of genes involved in extracellular matrix remodeling. The purpose of this study is to evaluate the expression of Ets-1 in the process of healing of ulceration in the rats. The time-dependent changes and distribution of Ets-1 in the margins of ulcer were examined. Ets-1 did not express in the normal gastric mucosa. In the marginal granulation tissue, fibroblasts and endothelial cells of capillaries were immunopositive for Ets-1. Ets-1 expression was significantly increased at the early phase, and returned to normal levels at the scarred phase. Serial sectioning revealed that fibroblasts and endothelial cells also expressed MMP-1. Protein levels and mRNA expression of Ets-1 were confirmed by Western blotting and RT-PCR. These findings suggest that Ets-1 plays an important role in angiogenesis in the early phase of ulcer healing.