Takeaki Okamoto

Transcriptional regulation of indoleamine 2,3-dioxygenase (IDO) by tryptophan and its analogue: Down-regulation of the indoleamine 2,3-dioxygenase (IDO) transcription by tryptophan and its analogue

Indoleamine 2,3-dioxygenase (IDO; EC 1.13.11.42) is a rate-limiting enzyme involved in the catabolism of tryptophan, which is an essential amino acid. It is induced under pathological conditions, such as the presence of viral infections or tumour cells. This enzyme is induced by IFN-γ in the mouse rectal carcinoma cell line CMT-93. It is known that both 1-methyl-l-tryptophan (1-MT) and methylthiohydantoin-dl-tryptophan (MTH-trp) are tryptophan analogues, and are authentic inhibitors of the enzymatic activity of IDO. In this study, we examined the effects of both 1-MT and MTH-trp on the IFN-γ inducible IDO expression of CMT-93. As a result, the IFN-γ inducible IDO mRNA and the protein levels in CMT-93 were suppressed by 1-MT and MTH-trp, independently. Moreover, tryptophan (Trp), as a substrate of IDO, also suppressed IDO induction by IFN-γ at the transcriptional level. These results suggest that 1-MT and MTH-trp are as inhibitors of IDO enzymatic activity, and Trp suppresses IDO induction by IFN-γ at the transcriptional level.

Monocyte trans-endothelial migration augments subsequent transmigratory activity with increased PECAM-1 and decreased VE-cadherin at endothelial junctions☆

BACKGROUND Although the importance of monocyte trans-endothelial migration in early atherogenesis is well recognized, it is unclear whether and how one transmigration event affects endothelium to facilitate subsequent ones. In this study, we tested the hypothesis that monocyte transmigration alters endothelial junctional organization to facilitate subsequent transmigration. METHODS AND RESULTS When human monocytes were added twice at intervals of ≈30 min to IL-1beta-prestimulated human umbilical vein endothelial cells in vitro, significant augmentation of transmigration was observed at the second addition (≈1.5-fold, analyzed from a total of 231 monocytes in 3 experiments). Endothelial surface expressions of two major junctional molecules, PECAM-1 and VE-cadherin, increased and decreased respectively, in response to monocyte addition, which could facilitate subsequent transmigration. To further investigate spatiotemporal dynamics of the increasing molecule, PECAM-1, we constructed a PECAM-1-GFP expression system and found that monocyte transmigration induced local accumulation of endothelial PECAM-1 around the transmigration spot, which was followed by transmigration of subsequent monocyte around the same location. Detailed analysis revealed that within the defined region around one transmigration event, 50% of later transmigrating monocytes used the same or similar location as the previous one (10 out of 20 transmigrating monocytes in 11 experiments). CONCLUSIONS These findings show that monocyte trans-endothelial migration alters endothelial junctional organization to a more monocyte-permeable state (increased PECAM-1 and decreased VE-cadherin), resulting in the augmented transmigratory activity at a later stage. This positive feedback mechanism is partially associated with monocyte transmigration-induced local accumulation of endothelial PECAM-1, which promotes transmigration of following monocytes at the same location.

Dihydro-alpha-lipoic acid has more potent cytotoxicity than alpha-lipoic acid

Alpha-lipoic acid has been shown to possess cancer-cell-killing activity via activation of the apoptosis pathway. In this study, the cytotoxic activities of alpha-lipoic and dihydro-alpha-lipoic acid were compared in HL-60 cells. The cell-killing activity of dihydro-alpha-lipoic acid was higher than that of alpha-lipoic acid. Both alpha-lipoic and dihydro-alpha-lipoic acid induced caspase-3 cleavage and internucleosomal DNA fragmentation in treated cells. On the other hand, apparent necrotic or late-stage apoptotic cell populations could be detected in dihydro-alpha-lipoic acid cells but not in those treated with alpha-lipoic acid. Moreover, dihydro-alpha-lipoic acid, but not alpha-lipoic acid, induced marked mitochondrial permeability transition. Antioxidants could not prevent dihydro-alpha-lipoic- or alpha-lipoic-acid-induced cell death. In addition, dihydro-alpha-lipoic and alpha-lipoic acid did not up-regulate cellular reactive oxygen level. These results indicated that dihydro-alpha-lipoic acid exerts more potent cytotoxicity than alpha-lipoic acid through different cytotoxic actions.

Immunostimulatory in Vitro and in Vivo Effects of a Water-Soluble Extract from Kale

The water-soluble fraction of kale (Brassica oleracea L. var. acephala DC.) had immunoglobulin (Ig) production stimulating activity in human hybridoma HB4C5 cells and human peripheral blood lymphocytes. The biochemical and physical properties of the main active substance in kale were found to be a heat-stable protein with a molecular weight higher than 50 kDa. The Ig production-stimulating factors were assumed to act on the translational and/or secreting processes of Igs. This Ig production-stimulating effect was also observed in lymphocytes from the mesenteric lymph node and Peyer’s patches of mice that had been administered with the kale extract for 14 d. The partially purified kale extract was analyzed by LC-ESI-MS/MS, the result indicating ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) as an active substance. Rubisco from spinach indeed exhibited Ig production-stimulating activity in HB4C5 cells. These findings provide another beneficial aspect of kale as a health-promoting foodstuff.

Live-cell visualization of the trans-cellular mode of monocyte transmigration across the vascular endothelium, and its relationship with endothelial PECAM-1

In response to atherogenic stimuli, blood monocytes transmigrate across the vascular endothelium not only through endothelial cell–cell junctions (para-cellular) but also through endothelial cells themselves (trans-cellular). The molecular mechanism of the latter is mostly unknown, because it rarely happens, especially in vitro. Although many reports have recognized trans-cellular migration from snapshot images of leukocytes halfway across the endothelium at non-junctional locations, it often produces a false-positive result, because some leukocytes that initiate trans-cellular migration withdraw and return to the apical endothelial surface. Thus, analyzing the entire process is essential. In this study, complete monocyte trans-cellular migration was successfully captured for live cells, with simultaneous visualization of endothelial PECAM-1. We suggest the possible existence of both PECAM-1-related migration at peri-junctional sites and PECAM-1-unrelated migration at sites remote from junctions. This is the first report to describe the entire process of monocyte trans-cellular migration for live cells and its relationship with endothelial PECAM-1.

Immunostimulation Effects of Yellowtail Heart Extracts in Vitro and in Vivo

The immunostimulation effects of yellowtail heart extracts were examined. Screening various parts of the yellowtail viscera, we found that extracts from the yellowtail heart enhanced IgM production by human hybridoma HB4C5 cells. Yellowtail heart extracts heated at 121 °C for 20 min and dialyzed showed the highest IgM production-stimulating activity toward HB4C5 cells. Also, immunoglobulin production by mouse spleen lymphocytes was stimulated by yellowtail heart extracts in vitro, and lymphocytes derived from mice administered the extract for 20 d were activated in vivo. Yellowtail heart extracts were partially purified by anion-exchange chromatography, and fractions containing a 33 kDa-protein exhibited immunostimulating activity. LC-MS/MS analysis revealed that the 33 kDa-protein was most similar to tropomyosin-4 from various fishes. Purified tropomyosin from porcine muscle enhanced IgM production by HB4C5 cells. This means that tropomyosin-4 is one of the immunostimulating substances in the yellowtail heart.

Homocysteine induced SH-SY5Y apoptosis through activation of NADPH oxidase in U251MG cells.

Epidemiological studies have indicated a correlation between homocysteinemia and dementia, including Alzheimers disease. However, the mechanism by which homocysteine (Hcy) induces neuronal cell death remains unknown. We found that micromolar concentrations of Hcy induced neuroblastoma SH-SY5Y cell death only when co-cultured with glioblastoma U251MG cells. In this culture system, cysteine had no effect on SH-SY5Y cell death. There was an increase in TUNEL-positive cells and loss of mitochondrial membrane potential following treatment with 100 μM Hcy. Addition of conditioned medium prepared from U251MG cells in the presence of 100 μM Hcy also reduced SH-SY5Y cell viability, while this effect was prevented when using conditioned medium from U251MG cells exposed to 100 μM Hcy+apocynin, a specific NADPH oxidase inhibitor. Following exposure to 100 μM Hcy in U251MG cells, expression of Rac1, a compartment of NADPH oxidase, was translocated to the plasma membrane, and the active form of Rac1 was increased. There was no change in peroxide concentration in the medium of U251MG cells after addition of Hcy. Overall, these data suggest that Hcy stimulates Rac1 activation and NADPH oxidase, resulting in superoxide anion production that may induce SH-SY5Y cell apoptosis.

Alcohol dehydrogenase-I from horse liver serves as an immunoglobulin production stimulating factor

Abstract Alcohol dehydrogenase-I (ADH-I; EC 1.1.1.1) derived from horse -liver stimulated IgM production by human-human hybridoma, HB4C5 cells and human peripheral blood lymphocytes. The immunoglobulin production stimulating activity of ADH-I was inactivated by chymotrypsin digestion, even though the enzymatic activity was completely preserved. This fact suggests that the immunoglobulin production stimulating effect of ADH-I is irrelevant to its enzymatic function. This also means that the effect is a novel function of this enzyme. ADH-I enhanced IgM production by transcription-suppressed HB4C5 cells treated with actinomycin D. Moreover, the enzyme was effective to accelerate IgM production by translation-suppressed HB4C5 cells treated with sodium fluoride and cycloheximide. Although the secretion of IgM by HB4C5 cells treated with monensin for suppression of the post-transcription process was inhibited, the intracellular IgM content of the cells was obviously increased by the addition of ADH-I. It is supposed from these results that ADH-I accelerates the translation activity to enhance immunoglobulin productivity. In addition, a laser confocal microscopic analysis revealed that ADH-I from horse liver was subsequently incorporated by HB4C5 cells.

Inhibitory Effect of Ellagitannin Metabolites on IgE-Mediated Allergic Responses in RBL-2H3 Cells

The effects of four ellagitannin metabolites (M1-M4) and ellagic acid on immunoglobulin E-mediated allergic responses in rat basophilic leukemia-2H3 cells were investigated. M1-M4 inhibited the antigen-induced degranulation and secretion of interleukin-4 and tumor necrosis factor-α, but ellagic acid only slightly did so under the same experimental conditions. M1 inhibited the activation of the mitogen-activated protein kinases in antigen-stimulated cells.

Effects of DNA on immunoglobulin production stimulating activity of alcohol dehydrogenase

Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and lymphocytes. The IPSF activity of ADH-I was suppressed by coexistence of short DNA whose chain length is less than 200 base pairs (bp) and fibrous DNA in a dose-dependent manner. These DNA preparations completely inhibited the IPSF activity at the concentration of 250 μg/ml and 1.0 mg/ml, respectively. DNA sample termed long DNA whose average chain length is 400–7000 bp slightly stimulated IPSF activity at 0.06 μg/ml. However, long DNA suppressed IPSF activity by half at 1.0 mg/ml. The laser confocal microscopic analysis had revealed that ADH-I was incorporated by HB4C5 cells. The uptake of ADH-I was strongly inhibited by short DNA and fibrous DNA. However, long DNA did not suppress the internalization of ADH-I into HB4C5 cells. These findings indicate that short DNA and fibrous DNA depress IPSF activity of ADH-I by inhibiting the internalization of this enzyme. According to the gel-filtration analysis using HPLC, ADH-I did not directly interact with short DNA. It is expected from these findings that short DNA influences HB4C5 cells to suppress the internalization of ADH-I. Moreover, these facts also strongly suggest that ADH-I acts as IPSF after internalization into the cell.