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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. [ABSTRACT FROM AUTHOR]

Highlights: [•] Treatment with a cytotoxic anti-Fas antibody caused cross-link of Fas antigen. [•] Inhibition of factor XIII caused disappearance of the cross-link of Fas. [•] Apoptotic cell death was concurrently hastened by inhibition of factor XIII. [•] The cross-link of Fas was seen only in fetal serum but not in other sera tested. [•] Factor XIII may play a key role in fetal development via cross-link of Fas. [ABSTRACT FROM AUTHOR]

By UV-irradiation, cells are subjected to DNA damage followed by mutation, cell death and/or carcinogenesis. DNA repair systems such as nucleotide excision repair (NER) and translesion DNA synthesis (TLS) protect cells against UV-irradiation. To understand the role of histone acetyltransferase GCN5 in regulation of DNA repair, we studied the sensitivity of GCN5-deficient DT40, GCN5-/-, to various DNA-damaging agents including UV-irradiation, and effects of GCN5-deficiency on the expression of NER- and TLS-related genes. After UV-irradiation, cell death and DNA fragmentation of GCN5-/- were appreciably accelerated as compared with those of DT40. Interestingly, GCN5-/- showed a remarkable sensitivity to only UV-irradiation but not to other DNA-damaging agents tested. Semiquantitative RT-PCR showed that transcription of DNA polymerase η (POLH) gene whose deficiency is responsible for a variant form of xeroderma pigmentosum was drastically downregulated in GCN5-/- (to ∼25%). In addition, ectopic expression of human POLH in GCN5-/- dramatically reversed the sensitivity to UV-irradiation of GCN5-/-to almost the same level of wild type DT40. Moreover, chromatin immunoprecipitation assay revealed that GCN5 binds to the chicken POLH gene 5′-flanking region that contains a typical CpG island and acetylates Lys-9 of histone H3, but not Lys-14 in vivo. These data suggest that GCN5 takes part in transcription regulation of POLH gene through alterations in the chromatin structure by direct interaction with its 5′-flanking region, and protects vertebrate cells against UV-induced DNA damage via controlling POLH gene expression. [ABSTRACT FROM AUTHOR]

The superoxide anion (O2-)-generating system is an important mechanism of innate immune response against microbial infection in phagocytes and is involved in signal transduction mediated by various physiological and pathological signals in phagocytes and other cells, including B lymphocytes. The O2--generating system is composed of five specific proteins: p22-phox, gp91-phox, p40-phox, p47-phox, p67-phox, and a small G protein, Rac. Little is known regarding epigenetic regulation of the genes constituting the O2--generating system. In this study, by analyzing the GCN5 (one of most important histone acetyltransferases)-deficient DT40 cell line, we show that GCN5 deficiency causes loss of the O2--generating activity. Interestingly, transcription of the gp91-phox gene was drastically downregulated (to ~4%) in GCN5-deficient cells. To further study the involvement of GCN5 in transcriptional regulation of gp91-phox, we used in vitro differentiation system of U937 cells. When human monoblastic U937 cells were cultured in the presence of IFN-γ, transcription of gp91-phox was remarkably upregulated, and the cells were differentiated to macrophage-like cells that can produce O2-. Chromatin immunoprecipitation assay using the U937 cells during cultivation with IFN-γ revealed not only that association of GCN5 with the gp91-phox gene promoter was significantly accelerated, but also that GCN5 preferentially elevated acetylation levels of H2BK16 and H3K9 surrounding the promoter. These results suggested that GCN5 regulates the O2--generating system in leukocytes via controlling the gp91-phox gene expression as a supervisor. Our findings obtained in this study should be useful in understanding the molecular mechanisms involved in epigenetic regulation of the O2--generating system in leukocytes. [ABSTRACT FROM AUTHOR]

Abstract: 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 (H2O2) 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 H2O2, were remarkably decreased compared with those in wild type DT40 cells. In addition, phosphorylation of Akt in H2O2-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 H2O2. [Copyright &y& Elsevier]

Abstract: 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 ()-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 -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 -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 -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. [Copyright &y& Elsevier]

Lucigenin-enhanced chemiluminescence (LECL) is widely used for the detection of reactive oxygen species released from various cells and mitochondria. However, the LECL response varies depending on cell species and assay conditions at least in part by unknown factors. Here we report that cell adhesion is an important factor for increasing LECL of tetradecanoylphorbol acetate (TPA)-stimulated human neutrophils. More than 90% LECL remained even after complete removal of the cell suspension 10 min after TPA stimulation, and ~22.5% of neutrophils were adhered to the reaction tube. These results indicate that LECL by an adhering neutrophil is ~45× higher than that by a non-adhering neutrophil. LECL by leukocyte adhesion deficiency neutrophils was one-fifth of that by normal neutrophils and completely disappeared when the cell suspension was removed, confirming that LECL depends highly on cell adhesion. The oxidase activity of adhering neutrophils measured after permeabilization with Renex 30 together with NADPH addition was similar to that of non-adhering neutrophils, indicating that lucigenin and cell adhesion do not enhance the oxidase activity. Based on these findings, we propose that a mixture of adhering and non-adhering neutrophils can be used for simultaneous screenings of adhering activity and the oxidase activity of neutrophils. [ABSTRACT FROM AUTHOR]

Activated NADPH oxidase in neutrophils produces superoxide. We investigated the role of p38 MAP kinase in activating NADPH oxidase stimulated by the bacteria-derived peptide fMLP. fMLP-stimulated superoxide production was completely abolished by SB203580, a p38 MAP kinase inhibitor, whereas anisomycin, a p38 MAP kinase activator, did not induce superoxide production, indicating that p38 MAP kinase was essential, but not sufficient, for NADPH oxidase activation. Anisomycin pretreatment strongly activated p38 MAP kinase in fMLP-stimulated cells, accompanied by greatly increased superoxide production, suggesting that p38 MAP kinase determines the extent of the fMLP-stimulated NADPH oxidase activity. Furthermore, superoxide production was remarkably reactivated by cytochalasin B addition after fMLP-stimulated production had disappeared, and this was correlated with highly activated p38 MAP kinase. These results suggest that p38 MAP kinase is involved not only in activating NADPH oxidase stimulated by fMLP but also in determining the extent of its activity. [ABSTRACT FROM PUBLISHER]

Protein-phosphoinositide interaction participates in targeting proteins to membranes where they function correctly and is often modulated by phosphorylation of lipids. Here we show that protein phosphorylation of p47[sup phox], a cytoplasmic activator of the microbicidal phagocyte oxidase (phox), elicits interaction of p47[sup phox] with phosphoinositides. Although the isolated phox homology (PX) domain of p47[sup phox] can interact directly with phosphoinositides, the lipid-binding activity of this protein is normally suppressed by intramolecular interaction of the PX domain with the C-terminal Src homology 3 (SH3) domain, and hence the wild-type full-length p47[sup phox] is incapable of binding to the lipids. The W263R substitution in this SH3 domain, abrogating the interaction with the PX domain, leads to a binding of p47[sup phox] to phosphoinositides. The findings indicate that disruption of the intramolecular interaction renders the PX domain accessible to the lipids. This conformational change is likely induced by phosphorylation of p47[sup phox], because protein kinase C treatment of the wild-type p47[sup phox] but not of a mutant protein with the S303/ 304/328A substitution culminates in an interaction with phosphoinositides. Furthermore, although the wild-type p47[sup phox] translocates upon cell stimulation to membranes to activate the oxidase, neither the kinase-insensitive p47[sup phox] nor lipid-bindingdefective proteins, one lacking the PX domain and the other carrying the R90K substitution in this domain, migrates. Thus the protein phosphorylation-driven conformational change of p47[sup phox] enables its PX domain to bind to phosphoinositides, the interaction of which plays a crucial role in recruitment of p47[sup phox] from the cytoplasm to membranes and subsequent activation of the phagocyte oxidase. [ABSTRACT FROM AUTHOR]

Reactive oxygen intermediates (ROIs), including superoxide anions and hydrogen peroxide, are generated by phagocytes in invertebrates, as well as in vertebrates. To understand the molecular mechanisms underlying the generation of ROIs by hemocytes of the solitary ascidian Halocynthia roretzi, we established a method of measuring ROIs using luminol-dependent chemiluminescence (LDCL). LDCL analyses revealed that both zymosan and phorbol myristate acetate (PMA), but not lipopolysaccharide, β1,3-glucan, or formylpeptide, induced the generation of ROIs by H. roretzi hemocytes. The zymosan-induced LDCL was markedly inhibited by the addition of superoxide dismutase (SOD) or H. roretzi plasma. A calcium-chelating reagent, BAPTA-AM, completely inhibited the zymosan-induced LDCL. On the other hand, the PMA-induced LDCL was only slightly inhibited by the addition of SOD or BAPTA-AM. Spectroscopic analysis at a low temperature revealed that H. roretzi hemocytes had absorption spectra specific for type b cytochrome, a component of the NADPH oxidase complex in mammalian phagocytes. These results strongly suggest that H. roretzi hemocytes generate superoxide anions upon phagocytosis and that intracellular calcium ions and possibly an NADPH oxidase complex are involved in their generation by H. roretzi hemocytes. [Copyright &y& Elsevier]

Activation of the superoxide-producing phagocyte NADPH oxidase, crucial in host defense, requires the cytosolic proteins p67phox and p47phox. They translocate to the membrane upon cell stimulation and activate flavocytochrome b558, the membrane-integrated catalytic core of this enzyme system. The activators p67phox and p47phox form a ternary complex together with p40phox, an adaptor protein with unknown function, comprising the PX/PB2, SH3 and PC motif-containing domains: p40phox associates with p67phox via binding of the p40phox PC motif to the p67phox PB1 domain, while p47phox directly interacts with p67phox but not with p40phox. Here we show that p40phox enhances membrane translocation of p67phox and p47phox in stimulated cells, which leads to facilitated production of superoxide. The enhancement cannot be elicited by a mutant p40phox carrying the D289A substitution in PC or a p67phox with the K355A substitution in PB1, each being defective in binding to its respective partner. Thus p40phox participates in activation of the phagocyte oxidase by regulating membrane recruitment of p67phox and p47phox via the PB1-PC interaction with p67phox. [ABSTRACT FROM AUTHOR]

The membrane bound cytochrome b 558 composed of gp91-phox and p22-phox proteins, and cytosolic proteins p40-, p47-and p67-phox are important components of superoxide (O 2 − )-generating system in phagocytes. Here, we describe that resveratrol, a pleiotropic phytochemical belonging to the stilbenoids, 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 resveratrol, the O 2 − -generating activity increased more than 5-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and resveratrol strongly enhanced transcription of the gp91-phox compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and resveratrol caused remarkable accumulation of protein levels of gp91-phox (to 4-fold), p22-phox (to 5-fold) and p47-phox (to 4-fold) compared with those of the RA-treatment alone. In addition, ChIP assay suggested that resveratrol participates in enhancing the gene expression of gp91-phox via promoting acetylation of Lys-9 residues and Lys-14 residues of histone H3 within chromatin around the promoter regions of the gene. These results suggested that resveratrol strongly enhances the RA-induced O 2 − -generating activity via up-regulation of gp91-phox gene expression in U937 cells. [ABSTRACT FROM AUTHOR]

Coronavirus disease 2019 (COVID-19) pandemic poses a threat to human beings and numerous cases of infection as well as millions of victims have been reported. The binding of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor binding domain (RBD) to human angiotensin converting enzyme 2 (hACE2) is known to promote the engulfment of the virus by host cells. Employment of flavor/fragrance compositions to prevent SARS-CoV-2 infection by inhibiting the binding of viral RBD (vRBD) to hACE2 might serve as a favorable, simple, and easy method for inexpensively preventing COVID-19, as flavor/fragrance compositions are known to directly interact with the mucosa in the respiratory and digestive systems and have a long history of use and safety assessment. Herein we report the results of screening of flavor/fragrance compositions that inhibit the binding of vRBD to hACE2. We found that the inhibitory effect was observed with not only the conventional vRBD, but also variant vRBDs, such as L452R, E484K, and N501Y single-residue variants, and the K417N+E484K+N501Y triple-residue variant. Most of the examined flavor/fragrance compositions are not known to have anti-viral effects. Cinnamyl alcohol and Helional inhibited the binding of vRBD to VeroE6 cells, a monkey kidney cell line expressing ACE2. We termed the composition with inhibitory effect on vRBD-hACE2 binding as "the molecularly targeted flavor/fragrance compositions". COVID-19 development could be prevented by using these compositions with reasonable administration methods such as inhalation, oral administration, and epidermal application. [ABSTRACT FROM AUTHOR]

Bladder cancer is an often widely disseminated and deadly cancer. To block the malignant outgrowth of bladder cancer, we must elucidate the molecular-level characteristics of not only bladder cancer cells but also their surrounding milieu. As part of this effort, we have long been studying extracellular S100A8/A9, which is elevated by the inflammation associated with certain cancers. Extracellularly enriched S100A8/A9 can hasten a shift to metastatic transition in multiple types of cancer cells. Intriguingly, high-level S100A8/A9 has been detected in the urine of bladder-cancer patients, and the level increases with the stage of malignancy. Nonetheless, S100A8/A9 has been investigated mainly as a potential biomarker of bladder cancers, and there have been no investigations of its role in bladder-cancer growth and metastasis. We herein report that extracellular S100A8/A9 induces upregulation of growth, migration and invasion in bladder cancer cells through its binding with cell-surface Toll-like receptor 4 (TLR4). Our molecular analysis revealed the TLR4 downstream signal that accelerates such cancer cell events. Tumor progression locus 2 (TPL2) was a key factor facilitating the aggressiveness of cancer cells. Upon binding of S100A8/A9 with TLR4, TPL2 activation was enhanced by an action with a TLR4 adaptor molecule, TIR domain-containing adaptor protein (TIRAP), which in turn led to activation of the mitogen-activated protein kinase (MAPK) cascade of TPL2. Finally, we showed that sustained inhibition of TLR4 in cancer cells effectively dampened cancer survival in vivo. Collectively, our results indicate that the S100A8/A9-TLR4-TPL2 axis influences the growth, survival, and invasive motility of bladder cancer cells. • TLR4 is upregulated in bladder cancer cells. • S100A8/A9-TLR4 axis plays a crucial role in bladder cancer progression. • TPL2 is required for bladder cancer progression under TLR4 upon S100A8/A9 binding. • TPL2 requires TIRAP adaptor protein for its activation. [ABSTRACT FROM AUTHOR]

The dissection of the complex multistep process of metastasis exposes vulnerabilities that could be exploited to prevent metastasis. To search for possible factors that favor metastatic outgrowth, we have been focusing on secretory S100A8/A9. A heterodimer complex of the S100A8 and S100A9 proteins, S100A8/A9 functions as a strong chemoattractant, growth factor, and immune suppressor, both promoting the cancer milieu at the cancer-onset site and cultivating remote, premetastatic cancer sites. We previously reported that melanoma cells show lung-tropic metastasis owing to the abundant expression of S100A8/A9 in the lung. In the present study, we addressed the question of why melanoma cells are not metastasized into the brain at significant levels in mice despite the marked induction of S100A8/A9 in the brain. We discovered the presence of plasma histidine-rich glycoprotein (HRG), a brain-metastasis suppression factor against S100A8/A9. Using S100A8/A9 as an affinity ligand, we searched for and purified the binding plasma proteins of S100A8/A9 and identified HRG as the major protein on mass spectrometric analysis. HRG prevents the binding of S100A8/A9 to the B16-BL6 melanoma cell surface via the formation of the S100A8/A9 complex. HRG also inhibited the S100A8/A9-induced migration and invasion of A375 melanoma cells. When we knocked down HRG in mice bearing skin melanoma, metastasis to both the brain and lungs was significantly enhanced. The clinical examination of plasma S100A8/A9 and HRG levels showed that lung cancer patients with brain metastasis had higher S100A8/A9 and lower HRG levels than nonmetastatic patients. These results suggest that the plasma protein HRG strongly protects the brain and lungs from the threat of melanoma metastasis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Chronic granulomatous disease (CGD) is a primary immunodeficiency disease that is characterized by susceptibility to bacterial and fungal infections. Various mutations in CYBB encoding the gp91 subunit of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase impair the respiratory burst of all types of phagocytic cells and result in X-linked CGD (X-CGD). Purpose: We here sought to evaluate the underlying cause in an attenuated phenotype in an X-CGD patient. The patient is a 31-year-old male who had been diagnosed as having X-CGD based on the absence of nitroblue tetrazolium reduction and the presence of a CYBB mutation at the age of 1 year. He has been in good health after overcoming recurrent bacterial infections in infancy. Methods: We investigated genomic DNA analysis of CYBB gene, residual activity of NADPH oxidase, and expression of gp91 in both polymorphonuclear leukocytes (PMNs) and monocytes/macrophages in the present patient. Results: Although his underlying germline mutation, c.1016C>A (p.P339H) in the CYBB gene, was identified in both PMNs and monocytes, the expression and functional activity of gp91 retained in monocytes/macrophages, in stark contrast to markedly reduced PMNs. Conclusions: Our results indicate that residual reactive oxygen intermediates (ROI) production in PMNs plays an important role in infantile stage in X-CGD, but thereafter retained function of monocytes/macrophages might compensate for the function of NADPH oxidase deficient PMNs and might be an important parameter for predicting the prognosis of X-CGD patients. [ABSTRACT FROM AUTHOR]

Abstract: GCN5 is involved in the acetylation of core histones, which is an important epigenetic event for transcriptional regulation through alterations in the chromatin structure in eukaryotes. To investigate physiological roles of GCN5, we have systematically analyzed phenotypes of homozygous GCN5-deficient DT40 mutants. Here, we report participation of GCN5 in regulation of IgM heavy chain (H-chain) gene expression. GCN5-deficiency down-regulates gene expressions of IgM H-chain (as whole, membrane-bound and secreted forms of its mRNA) but not light chain (L-chain), causing decreases in membrane-bound and secreted forms of IgM proteins. Chromatin immnoprecipitation assay revealed that GCN5 binds to the chicken IgM H-chain gene around its constant region but not L-chain gene, and acetylate Lys-9 residues of histone H3 within chromatin surrounding the constant region. These results suggest that GCN5 takes part in transcriptional regulation of the IgM H-chain gene via histone acetylation resulting in formation of relaxed chromatin arrangement around its coding region and plays a key role in epigenetic regulation of B cell functions. [Copyright &y& Elsevier]

Highlights: [•] GCN5-deficiency in DT40 caused drastic down-regulation of transcription of PKCθ. [•] EBF1-deficiency brought about remarkable up-regulation of transcription of PKCθ. [•] Re-expression of EBF1 dramatically suppressed transcription of PKCθ. [•] GCN5 binds to the 5′-flanking region of the PKCθ gene and acetylates histone H3. [•] EBF1 binds to the 5′-flanking regions of the PKCθ gene containing EBF1 binding motifs. [Copyright &y& Elsevier]

Abstract: To compare the responses of individual neutrophils to chemoattractants, migration pathway data were obtained using TAXIScan, an optically accessible/horizontal apparatus in which a concentration gradient is established reproducibly for a given stimulus. The observed linear-mode trajectory pattern of neutrophils toward N-formyl-methionyl-leucyl-phenylalanine (fMLP) or Interleukin (IL)-8/CXCL8 was distinguished from random migration patterns toward leukotriene (LT) B4 or platelet activating factor (PAF). The median values of velocity and directionality calculated for individual cells toward fMLP and IL-8 were both relatively similar and high, whereas the values toward LTB4 and PAF were widely dispersed over a lower range of directionality and from low to high ranges of velocity. The different patterns between the groups may be explained by unique morphology with single polarity toward fMLP and IL-8, and unstable morphology with multiple polarities toward LTB4 and PAF. Unique morphologies toward fMLP and IL-8 were not affected by coexisting LTB4 or PAF. On the other hand, the addition of suboptimum concentrations of fMLP or IL-8 to LTB4 or PAF induced a nearly maximum chemotactic response in most cells. These data suggest that exogenous formyl peptides and endogenous chemokines augment neutrophil accumulation at inflammation sites, whereas lipid mediators may play a role in supporting activation of the inflammatory cells for recruitment. [Copyright &y& Elsevier]

Abstract: Activation of the phagocyte NADPH oxidase requires the regulatory proteins p47 phox and p67 phox , each harboring two SH3 domains. p67 phox interacts with p47 phox via simultaneous binding of the p67 phox C-terminal SH3 domain to both the proline-rich region (PRR) of amino acid residues 360–369 and its C-terminally flanking region of p47 phox ; the role of the interaction in oxidase regulation has not been fully understood. Here we show that the p47 phox –p67 phox interaction is disrupted not only by deletion of the PRR but also by substitution for basic residues in the extra-PRR (K383E/K385E). The substitution impaired oxidase activation partially in vitro and much more profoundly in vivo, indicating the significance of the p47 phox extra-PRR. Replacement of Ser-379 in the extra-PRR, a residue known to undergo phosphorylation in stimulated cells, by aspartate attenuates the interaction and thus results in a defective superoxide production, suggesting that phosphorylation of Ser-379 is involved in oxidase regulation. [Copyright &y& Elsevier]

The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacteriumAnaplasma phagocytophiluminhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated.bfl-1mRNA levels in uninfected neutrophils after 12 h in culture were reduced to≈ 5–25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance ofbfl-1mRNA levels byA. phagocytophilum. Differences inmcl-1,bax,bcl-w,badorbakmRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10–12 h incubation, whereasA. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner inA. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest thatA. phagocytophiluminhibits human neutrophil apoptosis via transcriptional upregulation ofbfl-1and inhibition of mitochondria-mediated activation of caspase 3. [ABSTRACT FROM AUTHOR]

An interaction between acute myeloid leukemia (AML) cells and endothelial cells in the bone marrow seems to play a critical role in chemosensitivity on leukemia treatment. The endothelial niche reportedly enhances the paracrine action of the soluble secretory proteins responsible for chemoresistance in a vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway–dependent manner. To further investigate the contribution of VEGF-A/VEGFR-2 signaling to the chemoresistance of AML cells, a biochemical assay system in which the AML cells were cocultured with human endothelial EA.hy926 cells in a monolayer was developed. By coculture with EA.hy926 cells, this study revealed that the AML cells resisted apoptosis induced by the anticancer drug cytarabine. SU4312, a VEGFR-2 inhibitor, attenuated VEGFR-2 phosphorylation and VEGF-A/VEGFR-2 signaling–dependent endothelial cell migration; thus, this inhibitor was observed to block VEGF-A/VEGFR-2 signaling. Interestingly, this inhibitor did not reverse the chemoresistance. When VEGFR-2 was knocked out in EA.hy926 cells using the CRISPR–Cas9 system, the cytarabine-induced apoptosis of AML cells did not significantly change compared with that of wild-type cells. Thus, coculture-induced chemoresistance appears to be independent of VEGF-A/VEGFR-2 signaling. When the transwell, a coculturing device, separated the AML cells from the EA.hy926 cells in a monolayer, the coculture-induced chemoresistance was inhibited. Given that the migration of VEGF-A/VEGFR-2 signaling–dependent endothelial cells is necessary for the endothelial niche formation in the bone marrow, VEGF-A/VEGFR-2 signaling contributes to chemoresistance by mediating the niche formation process, but not to the chemoresistance of AML cells in the niche. • Coculture with endothelial cells inhibits the drug-induced apoptosis of AML cells. • The chemoresistance is not reversed by the inhibition or knockout of VEGFR-2. • The VEGFR-2 pathway is dispensable for endothelial cells-induced AML chemoresistance. [ABSTRACT FROM AUTHOR]

NADPH oxidase (Nox) 4 produces H2O2 by forming a heterodimer with p22phox and is involved in hemangioendothelioma development through monocyte chemoattractant protein-1 (MCP-1) upregulation. Here, we show that Nox4 protein levels were maintained by p22phox in hemangioendothelioma cells and Nox4 protein stability was dependent on p22phox coexpression. Conversely, the degradation of Nox4 monomer was enhanced by p22phox knockdown. Under hypoxic conditions in hemangioendothelioma cells, p22phox was downregulated at the mRNA and protein levels. Downregulation of p22phox protein resulted in the enhanced degradation of Nox4 protein in hypoxia-treated hemangioendothelioma cells. In contrast, Nox2, a Nox isoform, was not altered at the protein level under hypoxic conditions. Nox2 exhibited a higher affinity for p22phox compared with Nox4, suggesting that when coexpressed with Nox4 in the same cells, Nox2 acts as a competitor. Nox2 knockdown restored Nox4 protein levels partially reduced by hypoxic treatment. Thus, Nox4 protein levels were attenuated in hypoxia-treated cells resulting from p22phox depletion. MCP-1 secretion was decreased concurrently with hypoxia-induced Nox4 downregulation compared with that under normoxia. [ABSTRACT FROM AUTHOR]

Autophagy induced in cancer cells during chemotherapy is classified into two types, which differ depending on the kind of cells or anticancer drugs. The first type of autophagy contributes to the death of cells treated with drugs. In contrast, the second type plays a crucial role in preventing anticancer drug-induced cell damages; the use of an autophagy inhibitor is considered effective in improving the efficacy of chemotherapy. Thus, it is important to determine which type of autophagy is induced during chemotherapy. Here, we showed that a novel inhibitor of RNA polymerase I, suppresses growth, induces cell cycle arrest and promotes apoptosis in leukemia cell lines. The number of apoptotic cells induced by co-treatment with CX-5461 and chloroquine, an autophagy inhibitor, increased compared with CX-5461 alone. Thus, the autophagy which may be induced by CX-5461 was the second type. The number of apoptotic cells induced by co-treatment with CX-5461 and CQ increased compared with CX-5461 alone. [ABSTRACT FROM AUTHOR]

Superoxide producing NADPH oxidase 1 (Nox1), abundantly expressed in the colon epithelium, plays a crucial role in mucosal host defenses. In this study, we found that pre-treatment of cells with edaravone, a free radical scavenger, inhibited Nox1 constitutive activity even after washout without affecting Nox1 trafficking to the plasma membrane and membrane recruitment of the cytosolic regulators Noxo1 and Noxa1. These results suggest that a Nox1-derived product is involved in the step that initiates the electron transfer reaction after the formation of the Nox1–Noxo1–Noxa1 complex. Furthermore, we show that the mean migration directionality and velocity of epithelial cells were significantly enhanced by the inhibition of constitutive Nox1 activity. Thus, the constitutive Nox1 activity limits undesired cell migration in resting cells while participating in a positive feedback loop toward its own oxidase activity. [ABSTRACT FROM AUTHOR]

Background: Migration of cancer cell correlates with distant metastasis and local invasion, which are good targets for cancer treatment. An optically accessible device "TAXIScan" was developed, which provides considerably more information regarding the cellular dynamics and less quantity of samples than do the existing methods. Here, we report the establishment of a system to analyze the nature of pancreatic cancer cells using TAXIScan and we evaluated lysophosphatidic acid (LPA)-elicited pancreatic cell migration.Methods: Pancreatic cancer cell lines, BxPC3, PANC-1, AsPC1, and MIAPaCa-2, were analyzed for adhesion as well as migration towards LPA by TAXIScan using parameters such as velocity and directionality or for the number of migrated cells by the Boyden chamber methods. To confirm that the migration was initiated by LPA, the expression of LPA receptors and activation of intracellular signal transductions were examined by quantitative reverse transcriptase polymerase reaction and western blotting.Results: Scaffold coating was necessary for the adhesion of pancreatic cancer cells, and collagen I and Matrigel were found to be good scaffolds. BxPC3 and PANC-1 cells clearly migrated towards the concentration gradient formed by injecting 1 μL LPA, which was abrogated by pre-treatment with LPA inhibitor, Ki16425 (IC50 for the directionality ≈ 1.86 μM). The LPA dependent migration was further confirmed by mRNA and protein expression of LPA receptors as well as phosphorylation of signaling molecules. LPA1 mRNA was highest among the 6 receptors, and LPA1, LPA2 and LPA3 proteins were detected in BxPC3 and PANC-1 cells. Phosphorylation of Akt (Thr308 and Ser473) and p42/44MAPK in BxPC3 and PANC-1 cells was observed after LPA stimulation, which was clearly inhibited by pre-treatment with a compound Ki16425.Conclusions: We established a novel pancreatic cancer cell migration assay system using TAXIScan. This assay device provides multiple information on migrating cells simultaneously, such as their morphology, directionality, and velocity, with a small volume of sample and can be a powerful tool for analyzing the nature of cancer cells and for identifying new factors that affect cell functions. [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Superoxide-producing NADPH oxidase, gp91 phox /NOX2, in phagocytes plays a critical role in the host defenses against pathogens. Moreover, gp91 phox /NOX2 contributes to the oxidative stress in endothelial cells. Therefore, investigating the level of gp91 phox /NOX2 with immunoblotting is important for estimating the amount of superoxide produced. Here, we showed that the epitopes in human gp91 phox /NOX2 recognized by monoclonal antibodies (mAbs) CL-5 and 48 were in amino acids 132–147 and 136–144, respectively. Although the epitopes were close to the N -glycosylation sites, N -glycan maturation did not affect mAbs recognition. When Pro-136 was substituted with Arg, the corresponding mouse residue, human gp91 phox /NOX2 was not recognized by mAbs CL-5 and 48; however, the substitution did not affect gp91 phox /NOX2-based oxidase activity. This finding explains why these mAbs specifically recognize the human but not mouse gp91 phox /NOX2. Hence, these mAbs are useful for investigating the level of gp91 phox /NOX2 without amino acid substitutions in the epitopes. • The epitope in human gp91 phox /NOX2 recognized by mAb CL-5 is in amino acids 132–147. • The epitope in human gp91 phox /NOX2 recognized by mAb 48 is in amino acids 136–144. • Pro-136 is responsible for the recognition by both mAbs CL-5 and 48. • N -glycans are unnecessary for the recognition by mAbs CL-5 and 48. [ABSTRACT FROM AUTHOR]

The superoxide-producing NAD(P)H oxidase Nox4 was initially identified as an enzyme that is highly expressed in the kidney and is possibly involved in oxygen sensing and cellular senescence. Although the oxidase is also abundant in vascular endothelial cells, its role remains to be elucidated. Here we show that Nox4 preferentially localizes to the nucleus of human umbilical vein endothelial cells (HUVECs), by immunocytochemistry and immunoelectron microscopy using three kinds of affinity-purified antibodies raised against distinct immunogens from human Nox4. Silencing of Nox4 by RNA interference (RNAi) abrogates nuclear signals given with the antibodies, confirming the nuclear localization of Nox4. The nuclear fraction of HUVECs exhibits an NAD(P)H-dependent superoxide-producing activity in a manner dependent on Nox4, which activity can be enhanced upon cell stimulation with phorbol 12-myristate 13-acetate. This stimulant also facilitates gene expression as estimated in the present transfection assay of HUVECs using a reporter regulated by the Maf-recognition element MARE, a DNA sequence that constitutes a part of oxidative stress response. Both basal and stimulated transcriptional activities are impaired by RNAi-mediated Nox4 silencing. Thus Nox4 appears to produce superoxide in the nucleus of HUVECs, thereby regulating gene expression via a mechanism for oxidative stress response. [ABSTRACT FROM AUTHOR]

A 60-year-old asymptomatic woman was referred to our hospital because of an abnormal chest roentgenogram during a routine medical checkup. The patient had no history of memorable infectious diseases, except a liver abscess caused by Serratia marcescens at age 46 years. Her son was diagnosed with chronic granulomatous disease at the age of 1 year. She had never smoked cigarettes and drank only occasionally. [ABSTRACT FROM AUTHOR]