Potential role of green tea amino acid l-theanine in the activation of innate immune response by enhancing expression of cytochrome b558 responsible for the reactive oxygen species-generating ability of leukocytes.

l-Theanine (N-ethyl- l-glutamine) is an analog of l-glutamine and l-glutamic acid, accounts for up to 50% of all free amino acids in green tea, and elicits an umami taste. As l-theanine also shows various physiological activities including immune response-modifying activities, it is expected to be an excellent health-promoting phytochemical agent. To know the influences of l-theanine on the human innate immune response, we investigated the effect of l-theanine on the superoxide anion (O2 - )-generating system of leukocytes using U937 cells. The O2 - -generating system in leukocytes consists of membrane cytochrome b558 protein (a complex of p22-phox and gp91-phox proteins) and cytosolic p40-phox, p47-phox, and p67-phox proteins. Addition of 500 µM l-theanine caused remarkable enhancement of the all-trans retinoic acid (ATRA)-induced O2 - -generating activity (to ~470% of ATRA-treated cells), but not l-glutamine and l-glutamic acid. Semiquantitative RT-PCR showed that the transcription level of gp91-phox is significantly increased in ATRA and l-theanine-co-treated cells. Chromatin immunoprecipitation revealed that l-theanine enhances acetylations of Lys-9 and Lys-14 residues of histone H3 within the chromatin surrounding the promoter region of the gp91-phox gene. Immunoblotting demonstrated that membrane cytochrome b558 proteins remarkably accumulate in ATRA + l-theanine-treated cells. These results suggested that l-theanine brings about a remarkable accumulation of cytochrome b558 protein via upregulating the transcription of the gp91-phox gene during leukocyte differentiation, resulting in marked augmentation of the O2 - -generating ability, which is one of the most important functions of leukocytes responsible for the innate immune system.

Nafamostat Mesylate is Not Effective in Preventing Post-Endoscopic Retrograde Cholangiopancreatography Pancreatitis.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is associated with complications such as post-ERCP pancreatitis (PEP). Protease inhibitors, including nafamostat mesylate (NM), have been evaluated for prophylaxis against PEP. AIM: We describe the first multicenter randomized controlled trial assessing the prophylactic efficacy of NM against PEP. METHODS: In this multicenter prospective study, we aimed to enroll 800 patients aged ≥ 20 years with a planned ERCP between December 2012 and March 2019. The primary outcome was the incidence and severity of PEP in patients who did not receive NM (non-NM) versus those who did (NM; 20 mg). Secondary outcomes included the incidence of PEP by NM initiation (pre- and post-ERCP), risk factors for PEP, and NM-related adverse events. RESULTS: Only 441 of the planned 800 patients were enrolled (non-NM: n = 149; NM: n = 292 [pre-ERCP NM: n = 144; post-ERCP NM: n = 148]). Patient characteristics were balanced at baseline with no significant differences between groups. PEP occurred in 40/441 (9%) patients (non-NM: n = 15 [10%]; NM: n = 25 [9%]), including 17 (12%) and eight (8%) in the pre-ERCP and post-ERCP NM groups, respectively. In the NM group, the incidence of PEP was lower in the low-risk group than in the high-risk group. Pancreatic injection and double-guidewire technique were independent risk factors for PEP. NM-related adverse events of hyperkalemia occurred in two (0.7%) patients. CONCLUSIONS: We found no evidence for the prophylactic effect of NM against PEP, regardless of the timing of administration; however, further studies are needed.

Chalcone skeleton promotes transcription of gp91-phox gene but inhibits expression of gp91-phox protein, and hydroxyl groups in hydroxychalcones participate in the stable expression of gp91-phox protein.

The effects of chalcone and butein on the induction of the superoxide anion (O2 - )-generating system were studied in U937 cells by all-trans retinoic acid (RA). The chalcone skeleton, a common structural motif in them, significantly enhanced the transcription of gp91-phox in an epigenetic manner. In contrast, chalcone and butein showed opposite effects on the induction of the O2 - -generating activity by RA and the expression of gp91-phox protein. Chalcone inhibited, whereas butein promoted, the induction of O2 - -generating activity by RA and the expression of gp91-phox protein. These data raise the possibility that modification of the chalcone skeleton could produce more effective differentiation-promoting agents.

Resveratrol strongly enhances the retinoic acid-induced superoxide generating activity via up-regulation of gp91-phox gene expression in U937 cells.

The membrane bound cytochrome b558 composed of gp91-phox and p22-phox proteins, and cytosolic proteins p40-, p47-and p67-phox are important components of superoxide (O2-)-generating system in phagocytes. Here, we describe that resveratrol, a pleiotropic phytochemical belonging to the stilbenoids, dramatically activates the O2--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 O2--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 O2--generating activity via up-regulation of gp91-phox gene expression in U937 cells.

Endoscopic ultrasonography diagnosis of subepithelial lesions.

Using endoscopic ultrasonography (EUS), it is practicable to diagnose subepithelial lesions (SEL) with originating layer, echo level, and internal echo pattern etc. Lipoma, lymphangioma, and cyst have characteristic features; therefore, there is no need for endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). Ectopic pancreas and glomus tumors, which originate from the third and fourth layers, are frequently seen in the antrum. However, ectopic pancreas located in the fundus or body is large and originates from the third and fourth layers (thickening of fourth layer). Each subepithelial lesion has characteristic findings. However, imaging differentiation of tumors originating from the fourth layer is very difficult, even if contrast echo is used. Therefore, EUS-FNA should be done in these tumors, but the diagnostic yield for small lesions is not sufficient for clinical demands. Generally, those tumors, including small ones, should be first followed up in 6 months, then yearly follow up in cases of no significant change in size and features. When those tumors become larger than 1-2 cm, EUS-FNA is recommended. Furthermore, unusual SEL and SEL with malignant findings such as nodular, heterogeneous, anechoic area, and ulceration indicate EUS-FNA. Cap-attached forward-viewing echoendoscope is very helpful for EUS-FNA of small SEL.

Lack of GCN5 remarkably enhances the resistance against prolonged endoplasmic reticulum stress-induced apoptosis through up-regulation of Bcl-2 gene expression.

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.

Histone acetyltransferase PCAF is involved in transactivation of Bcl-6 and Pax5 genes in immature B cells.

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.

Paired box gene 5 isoforms A and B have different functions in transcriptional regulation of B cell development-related genes in immature B cells.

The transcription factor paired box gene 5 (Pax5) is essential for B cell development. In this study, complementation analyses in Pax5-deficient DT40 cells showed that three Pax5 isoforms Pax5A, Pax5B and Pax5BΔEx8 (another spliced isoform of Pax5B lacking exon 8) exhibit distinct roles in transcriptional regulation of six B cell development-related genes (activation-induced cytidine deaminase, Aiolos, BTB and CNC homology 2, B cell lymphoma-6, early B cell factor 1, origin binding factor-1 genes), transcriptions of which are remarkably down-regulated by Pax5-deficiency. Moreover, ectopic expression study shows that these Pax5 isoforms may regulate themselves and each other at the transcriptional level.

Histone acetyltransferase p300/CBP-associated factor is an effective suppressor of secretory immunoglobulin synthesis in immature B cells.

The histone acetyltransferase p300/CBP-associated factor (PCAF) catalyzes acetylation of core histones and plays important roles in epigenetics by altering the chromatin structure in vertebrates. In this study, PCAF-deficient DT40 mutants were analyzed and it was found that PCAF participates in regulation of secretory IgM heavy chain (H-chain) synthesis. Remarkably, PCAF-deficiency causes an increase in the amount of secretory IgM H-chain mRNA, but not in that of IgM light chain and membrane-bound IgM H-chain mRNAs, resulting in dramatic up-regulation of the amount of secretory IgM protein. These findings suggest that PCAF regulates soluble antibody production and is thus an effective suppressor of secretory IgM H-chain synthesis.

Down-regulation of Fas-mediated apoptosis by plasma transglutaminase factor XIII that catalyzes fetal-specific cross-link of the Fas molecule.

The Fas antigen, also designated as APO-1 or CD95, is a member of the tumor necrosis factor receptor superfamily and can mediate apoptotic cell death in various cells. We report here that blood coagulation factor XIII (plasma transglutaminase, fibrin stabilizing factor) inhibits apoptosis induced by a cytotoxic anti-Fas monoclonal antibody in Jurkat cells. When cells were treated with the antibody in fetal calf serum-containing media, higher-molecular-weight (180K) polypeptides containing Fas molecule were detected by immunoblotting. Under conditions where the transglutaminase activity was eliminated or suppressed, the cross-link of Fas was not observed, and concurrently cell death was hastened. Moreover, an antibody against factor XIII strongly accelerated the Fas-mediated apoptosis. Furthermore, addition of partially purified factor XIII neutralized the apoptosis-promoting effect of anti-factor XIII antibody, indicating that this enzyme is involved in cross-link of Fas and down-regulates Fas-mediated apoptotic cell death. Significantly, the cross-link of Fas was seen only in fetal calf serum but not in newly-born calf serum, 1-year-old calf serum or adult bovine serum. These data suggest that plasma transglutaminase factor XIII may play a key role in fetal development of vertebrates via cross-link of Fas antigen.

Fixation of an esophageal stent using a novel re-openable endoclip for a tracheoesophageal fistula.

Although esophageal stenting is one treatment option as a palliative treatment for tracheoesophageal fistulas, serious complications are associated with stent migration. Some reports have described stent fixation using various devices to prevent stent migration. However, these have yet to be sufficiently examined. We performed esophageal stent fixation using the MANTIS Clip (Boston Scientific), a novel re-openable endoclip. An 89-year-old man developed a tracheoesophageal fistula after radiotherapy for esophageal squamous cell carcinoma. Esophageal stenting was considered because the patient had difficulty with oral intake. However, the patient had a mild stenosis, which suggested stent migration. Therefore, we performed esophageal stent fixation by grasping the mouth side of the stent and the normal mucosa of the esophagus with the MANTIS Clip after placement of the stent. The esophageal stent closed the fistula, and the patient was able to take food orally. Upper gastrointestinal endoscopy performed 3 weeks after stenting showed residual MANTIS Clip and no evidence of stent migration. Esophageal stent fixation with MANTIS clips for tracheoesophageal fistulas may be an option to prevent stent migration.

GCN5 protects vertebrate cells against UV-irradiation via controlling gene expression of DNA polymerase η.

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 down-regulated 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.

GCN5 regulates the superoxide-generating system in leukocytes via controlling gp91-phox gene expression.

The superoxide anion (O(2)(-))-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 O(2)(-)-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 O(2)(-)-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 O(2)(-)-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 O(2)(-). 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 O(2)(-)-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 O(2)(-)-generating system in leukocytes.

EBF1 acts as a powerful repressor of Blimp-1 gene expression in immature B cells.

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.

Factors affecting the diagnostic accuracy of endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) for upper gastrointestinal submucosal or extraluminal solid mass lesions.

AIM: A number of potential variables are associated with the diagnostic accuracy of endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA). The aim of this study was to evaluate factors affecting the diagnostic accuracy of EUS-FNA for upper gastrointestinal submucosal or extraluminal solid lesions. METHODS: Patients with such lesions who underwent EUS-FNA between January 2009 and December 2010 were studied retrospectively. Needles of 22, 25 and 19 gauge were used. The associations between the EUS-FNA results and factors such as mass location, mass size, needle size, number of needle passes, combined histologic-cytologic analysis and final diagnosis were analyzed. RESULTS: A total of 170 EUS-FNA procedures were performed in 158 patients with upper gastrointestinal submucosal or extraluminal solid lesions. The overall accuracy of EUS-FNA was 86.5% (147/170). The diagnostic accuracy with three or more needle passes was higher than with less than 3.0 needle passes (90.0%, 108/120 vs 78.0%, 39/50; P < 0.05). Mass location, mass size, and final diagnosis were not associated with EUS-FNA accuracy. Combined cytologic-histologic analysis had significantly higher diagnostic accuracy than either cytologic or histologic analysis alone (P < 0.001). In a subgroup of 90 patients, both 22 and 25 gauge needles were used for EUS-FNA. The overall diagnostic accuracy was similar for 25 gauge needles and 22 gauge needles (80.0% vs 78.9% P = 1.000) in this subgroup. CONCLUSION: Overall, 25 and 22 gauge needles have a similar diagnostic accuracy. Our results suggest that 3.0 or more needle passes and combined cytologic-histologic analysis enhance the diagnostic accuracy of EUS-FNA.

GCN5 regulates the activation of PI3K/Akt survival pathway in B cells exposed to oxidative stress via controlling gene expressions of Syk and Btk.

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).

Ellagic acid and its fermentative derivative urolithin A show reverse effects on the gp91-phox gene expression, resulting in opposite alterations in all-trans retinoic acid-induced superoxide generating activity of U937 cells.

Ellagitannins (esters composed of glucose and ellagic acid) are hydrolyzed to generate ellagic acid in gut followed by conversion of ellagic acid to urolithins such as urolithin A by intestinal bacteria. Since urolithins are absorbed by gut easier than ellagitannins and ellagic acid, and show various physiological activities (e.g. anti-cancer, anti-cardiovascular disease, anti-diabetes mellitus, anti-obesity and anti-Alzheimer disease activities), they are expected as excellent health-promoting phytochemicals. Here, using human monoblast U937 cells, we investigated the effect of ellagic acid and urolithin A on the superoxide anion (O2 -)-generating system of phagocytes, which is consisted of five specific protein factors (membrane proteins: p22-phox and gp91-phox, cytosolic proteins: p40-phox, p47-phox and p67-phox). Twenty micromolar of urolithin A enhanced the all-trans retinoic acid (ATRA)-induced O2 --generating activity (to ~175%) while 20 µM ellagic acid inhibited the ATRA-induced O2 --generating activity (to ~70%). Semiquantitative RT-PCR showed that transcription level of gp91-phox was certainly decreased (to ~70%) in ATRA plus ellagic acid-treated cells, while that of gp91-phox was significantly increased (to ~160%) in ATRA plus urolithin A-treated cells. Chromatin immunoprecipitation assay suggested that urolithin A enhanced acetylations of Lys-9 residues of histone H3 within chromatin surrounding the promoter region of gp91-phox gene, but ellagic acid suppressed the acetylations. Immunoblotting also revealed that ATRA plus urolithin A-treatment up-regulated protein levels of p22-phox (to ~160%) and gp91-phox (to ~170%) although ATRA plus ellagic acid-treatment down-regulated protein levels of p22-phox (to ~70%) and gp91-phox (to ~60%). These results suggested that conversion of ellagic acid to urolithin A in gut may bring about reverse effects on the gp91-phox gene expression, resulting in opposite alterations in O2 --generating activity of intestinal macrophages.

Lacking of Aiolos accelerates pre-mature B cell apoptosis mediated by BCR signaling through elevation in cytochrome c release.

Antigen binding to B cell receptor (BCR) of pre-mature B lymphocytes leads to their apoptosis, while binding to BCR of mature B lymphocytes induces their activation and proliferation. The former binding is believed to be a mechanism so as to exclude B cell clones leading to protection from auto-immune diseases. Cross-linking of BCR of pre-mature B cells, including chicken DT40 cells, with anti-immunoglobulin antibody induces their apoptosis. The PMA/ionomycin treatments, which mimic BCR stimulation, are used to study intracellular signal transduction of B lymphocytes. Here, by analyzing the Aiolos-deficient DT40 cell line, Aiolos(-/-), we reveal that the lack of Aiolos accelerates apoptosis of DT40 cells mediated by BCR signaling. Moreover, the Aiolos-deficiency and BCR signaling cooperatively control this apoptosis through dramatically elevated cytochrome c release from mitochondria to cytosol and elevated caspase (caspase-3, 8 and 9) activities, resulting in drastically diminished amounts of ICAD followed by increased DNA fragmentation. Re-expression study reveals that the shorter isoform of Aiolos (Aio-2) controls PMA/ionomycin-mediated apoptosis via up-regulation and down-regulation of the PKCdelta and bak genes, respectively. These findings could be a powerful trigger to resolve molecular mechanisms of negative selection of B lymphocytes and also auto-immune diseases.

Curcumin dramatically enhances retinoic acid-induced superoxide generating activity via accumulation of p47-phox and p67-phox proteins in U937 cells.

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.