Anthocyanin synthesis potential in betalain-producing Caryophyllales plants.

Although anthocyanins are widely distributed in higher plants, betalains have replaced anthocyanins in most species of the order Caryophyllales. The accumulation of flavonols in Caryophyllales plants implies that the late step of anthocyanin biosynthesis from dihydroflavonols to anthocyanins may be blocked in Caryophyllales. The isolation and characterization of functional dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) from Caryophyllales plants has indicated a lack of anthocyanins due to suppression of DFR and ANS. In this study, we demonstrated that overexpression of DFR and ANS from Spinacia oleracea (SoDFR and SoANS, respectively) with PhAN9, which encodes glutathione S-transferase (required for anthocyanin sequestration) from Petunia induces ectopic anthocyanin accumulation in yellow tepals of the cactus Astrophytum myriostigma. A promoter assay of SoANS showed that the Arabidopsis MYB transcription factor PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) activated the SoANS promoter in Arabidopsis leaves. The overexpression of Arabidopsis transcription factors with PhAN9 also induced ectopic anthocyanin accumulation in yellow cactus tepals. PAP homologs from betalain-producing Caryophyllales did not activate the promoter of ANS. In-depth characterization of Caryophyllales PAPs and site-directed mutagenesis in the R2R3-MYB domains identified the amino acid residues affecting transactivation of Caryophyllales PAPs. The substitution of amino acid residues recovered the transactivation ability of Caryophyllales PAPs. Therefore, loss of function in MYB transcription factors may suppress expression of genes involved in the late stage of anthocyanin synthesis, resulting in a lack of anthocyanin in betalain-producing Caryophyllales plants.

HO-1 inhibits IL-13-induced goblet cell hyperplasia associated with CLCA1 suppression in normal human bronchial epithelial cells.

Mucus hypersecretion and goblet cell hyperplasia are common features that characterize asthma. IL-13 increases mucin (MUC) 5AC, the major component of airway mucus, in airway epithelial cells. According to the literature, IL-13 receptor activation leads to STAT6 activation and consequent induction of chloride channel accessory 1 (CLCA1) gene expression, associated with the induction of MUC5AC. Heme oxygenase-1 (HO-1) is an enzyme that catalyzes oxidation of heme to biliverdin, and has anti-inflammatory and anti-oxidant properties. We examined the effects of HO-1 on mucin production and goblet cell hyperplasia induced by IL-13. Moreover, we assessed the cell signaling intermediates that appear to be responsible for mucin production. Normal human bronchial epithelial (NHBE) cells were grown at air liquid interface (ALI) in the presence or absence of IL-13 and hemin, a HO-1 inducer, for 14 days. Protein concentration was analyzed using ELISA, and mRNA expression was examined by real-time PCR. Histochemical analysis was performed using HE staining, andWestern blotting was performed to evaluate signaling transduction pathway. Hemin (4 µM) significantly increased HO-1 protein expression (p b 0.01) and HO-1 mRNA expression (p b 0.001). IL-13 significantly increased goblet cells, MUC5AC protein secretion (p b 0.01) and MUC5AC mRNA (p b 0.001), and these were decreased by hemin by way of HO-1. Tin protoporphyrin (SnPP)-IX, a HO-1 inhibitor, blocked the effect of hemin restoring MUC5AC protein secretion (p b 0.05) and goblet cell hyperplasia. Hemin decreased the expression of CLCA1 mRNA (p b 0.05) and it was reversed by SnPP-IX, but could not suppress IL-13-induced phosphorylation of STAT6 or SAM pointed domain-containing ETS transcription factor (SPDEF) and Forkhead box A2 (FOXA2) mRNA expression. In summary, HO-1 overexpression suppressed IL-13-induced goblet cell hyperplasia and MUC5AC production, and involvement of CLCA1 in the mechanism was suggested.

High expression of 67-kDa laminin receptor relates to the proliferation of leukemia cells and increases expression of GM-CSF receptor.

OBJECTIVE: The 67-kDa laminin receptor (LR) is a nonintegrin receptor for laminin, a major component of the extracellular matrix. To elucidate the role of LR in leukemia cells, we studied the relationship between the phenotype of leukemia cells and LR expression. MATERIALS AND METHODS: The relationship between clinical features of acute myeloid leukemia and expression of LR was examined. LR was overexpressed or suppressed by the introduction of complementary DNA or small interfering RNA for LR in a human leukemia cell line to test the effect of LR on the phenotype of leukemia. Expression of granulocyte-macrophage colony-stimulating factor receptors (GM-CSFR) was also tested in leukemia cells, including clinical samples. RESULTS: Expression of LR was significantly related to elevation of white blood cell count, lactate dehydrogenase, and survival among acute myeloid leukemia patients. Forced expression of LR enhanced proliferation, cell-cycle progression, and antiapoptosis of leukemia cells associated with phosphorylation of a transcription factor, signal transducer and activator of transcription 5, in the absence of stimulation by laminin. On the other hand, suppression of LR expression had the opposite effects. The number of GM-CSFR increased in leukemia cells overexpressing LR, and there was a significant relationship between the expression of LR and GM-CSFR in acute myeloid leukemia samples. CONCLUSIONS: These results suggest that LR expression influenced the characteristics of leukemia cells toward an aggressive phenotype and increased the number of GM-CSFR. These changes might be partly related to enhanced GM-CSF signaling.

Long-term efficacy of imatinib in a practical setting is correlated with imatinib trough concentration that is influenced by body size: a report by the Nagasaki CML Study Group.

Imatinib has dramatically improved long-term survival of chronic myelogenous leukemia (CML) patients. To analyze its efficacy in a practical setting, we registered most of CML patients in Nagasaki Prefecture of Japan. Of these, 73 patients received imatinib as an initial therapy. The overall survival rate of these patients was 88.7% at 6 years, and the cumulative complete cytogenetic response rate was 82.5% at 18 months. These results are comparable with the data of other reports including the IRIS study; however, the administered imatinib dose was smaller in our study than that in other reports. To address these discrepancies, we measured the trough concentration of imatinib among 35 patients. Although 39% of the patients were administered less than 400 mg/day, the trough level was comparable to those of previous reports. The trough level of imatinib showed a significant relationship with its efficacy, and was clearly related to dose of imatinib administrated and dose of imatinib divided by body surface area (BSA). Considering the smaller BSA of Japanese patients as compared to those of foreign origin, the results suggest that a lower dose of imatinib could maintain enough trough level and provided excellent results for the treatment of CML in our registry.

Preparation of two-dimensionally ordered microbeads structure dispensed with an ink-jet and its application to ELISA.

In order to the increase sensitivity of enzyme-linked immunosorbent assay (ELISA) using micro-droplet reaction system formed by ink-jet, we employed small dots of two-dimensionally well-ordered structure of polystyrene (PS) microbeads (710 +/- 25 microm in diameter) formed on the surface of polydimethyl siloxane (PDMS). An aqueous suspension of polystyrene microbeads (5 microm) was ejected on the PDMS plate with ink-jet. The PS beads were automatically assembled by capillary force accompanied with solvent evaporation. The evaporation rate was controlled by ambient relative humidity. The assembled beads were solidly immobilized on the surface of PDMS surface. The dots of well-ordered structure were stable against vigorous washing. ELISA using the structure as a reaction location was three times more sensitive than the material without the structure due to the increase of the surface area and consequent increase of the amount of antibody.

Imatinib provides durable molecular and cytogenetic responses in a practical setting for both newly diagnosed and previously treated chronic myelogenous leukemia: a study in nagasaki prefecture, Japan.

To evaluate the efficacy of imatinib in a practical setting, we registered 43 patients with newly diagnosed chronic myelogenous leukemia (CML) (group I) and 56 patients with previously diagnosed CML (group II) at 11 hematology centers in Nagasaki prefecture, Japan, from December 2001 to July 2005 and analyzed the molecular responses. Cytopenia, fluid retention, and skin rash were major adverse events, along with elevation in creatine phosphokinase levels. With a follow-up of approximately 3.5 years, imatinib treatment led to 88.7% overall survival (OS) and 85.2% progression-free survival (PFS) rates for group I, and 79.8% OS and 76.6% PFS rates for group II; the rates were not significantly different despite a lower average imatinib dose in group II. The rates of complete cytogenetic response at 30 months and major molecular response at 24 months were 86.1% and 62.5%, respectively, in group I, and 77.9% and 58.3% in group II; the rates were not significantly different. As has been reported by other groups, these results demonstrate that imatinib treatment can provide excellent clinical and molecular effects for not only newly diagnosed but also previously treated CML patients in practical settings that cover a wider variety of patients than clinical trials.

Chemokine expression in human erythroid leukemia cell line AS-E2: macrophage inflammatory protein-3alpha/CCL20 is induced by inflammatory cytokines.

OBJECTIVE: Normal and malignant hematopoietic cells are shown to express and secrete various cytokines and chemokines, some of which are believed to play an important role in normal and abnormal hematopoiesis in an autocrine/paracrine manner. To explore the possibility of a cytokine/chemokine network participating in the pathophysiology of anemic disorders, we evaluated the ability of inflammatory cytokines to induce chemokine expression using erythroid progenitor cells. METHODS: Erythropoietin-dependent human leukemia cell line AS-E2 was used as a model of erythroid colony-forming unit (CFU-E) cells. The expression of mRNA of 8 chemokines was examined using RT-PCR, before and after TNF-alpha, IFN-gamma, and IL-1beta stimulation. For MIP-3alpha, the promoter activity was analyzed by luciferase assay and secretion was confirmed by ELISA. The expression of CCR6, the specific receptor for MIP-3alpha, was analyzed by RT-PCR and flow cytometry. RESULTS: Unstimulated AS-E2 cells constitutively expressed transcripts for MCP-4, IP-10, PF-4, IL-8, and MIP-3alpha. Stimulation with TNF-alpha, IFN-gamma, and IL-1beta upregulated MIP-3alpha mRNA expression and induced its protein secretion. Luciferase assay revealed that these cytokines could upregulate promoter activity of the MIP-3alpha gene, possibly through the NF-kappaB pathway. CCR6 mRNA was detected and its intracellular expression was confirmed. CONCLUSION: These data suggest that inflammatory cytokine-stimulated erythroid progenitors secrete MIP-3alpha, which may function in an autocrine/paracrine manner. Furthermore, the existence of intracellular CCR6 suggests the involvement in cytokine signaling of a MIP-3alpha-dependent internal autocrine mechanism. These mechanisms may play a role in pathophysiology of anemic disorders, such as secondary anemia and bone marrow failure syndromes.

Anthocyanidin synthase in non-anthocyanin-producing Caryophyllales species.

Red colors in flowers are mainly produced by two types of pigments: anthocyanins and betacyanins. Although anthocyanins are widely distributed in higher plants, betacyanins have replaced anthocyanins in the Caryophyllales. There has been no report so far to find anthocyanins and betacyanins existing together within the same plant. This curious phenomenon has been examined from genetic and evolutionary perspectives, however nothing is known at the molecular level about the mutual exclusion of anthocyanins and betacyanins in higher plants. Here, we show that spinach (Spinacia oleracea) and pokeweed (Phytolacca americana), which are both members of the Caryophyllales, have functional anthocyanidin synthases (ANSs). The ability of ANSs of the Caryophyllales to oxidize trans-leucocyanidin to cyanidin is comparable to that of ANSs in anthocyanin-producing plants. Expression profiles reveal that, in spinach, dihydroflavonol 4-reductase (DFR) and ANS are not expressed in most tissues and organs, except seeds, in which ANS may contribute to proanthocyanidin synthesis. One possible explanation for the lack of anthocyanins in the Caryophyllales is the suppression or limited expression of the DFR and ANS.

Durable remission by sobuzoxane in an HIV-seronegative patient with human herpesvirus 8-negative primary effusion lymphoma.

Primary effusion lymphoma (PEL) is recognized as a unique clinicopathological entity associated with human herpesvirus 8 (HHV-8), and it occurs almost exclusively in human immunodeficiency virus (HIV)-infected individuals. In the majority of PEL cases, Epstein-Barr virus (EBV) has been found in the tumor cells as well. We describe here an elderly HIV seronegative female patient with PEL in the pleura and pericardium not associated with HHV-8 or EBV. Cytologic examinations of the pleural effusion revealed large lymphoma cells with immunophenotypes positive for CD8, CD10, CD19, CD20, CD22, CD24, CD45, and HLA-DR but negative for CD30 and surface immunoglobulin. Chromosome analysis showed complicated abnormalities including add(3)(q27). Immunoglobulin gene rearrangement was detected by Southern blotting; however, c-myc, Bcl-2, and Bcl-6 genes were not rearranged. The patient was treated with a modified CHOP (cyclophosphamide, hydroxydoxorubicin, oncovine, and prednisolone) regimen, and achieved remission. Recurrence of PEL in the pericardium as effusion lymphoma was found 3 months after the discontinuation of CHOP. After approximately 1 year of intermittent multiagent salvage therapy for pericardial recurrences, a treatment that resulted in a partial response, 3 cycles of monotherapy with sobuzoxane were administered. At the time of this report the patient had been free from PEL for more than 18 months without chemotherapy.