Migratory activation of parasitized dendritic cells by the protozoan Toxoplasma gondii 14‐3‐3 protein

The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon infection, parasitized dendritic cells (DCs) and microglia exhibit a hypermigratory phenotype in vitro that has been associated with enhancing parasite dissemination in vivo in mice. One unresolved question is how parasites commandeer parasitized cells to achieve systemic dissemination by a ‘Trojan‐horse’ mechanism. By chromatography and mass spectrometry analyses, we identified an orthologue of the 14‐3‐3 protein family, T. gondii 14‐3‐3 (Tg14‐3‐3), as mediator of DC hypermotility. We demonstrate that parasite‐derived polypeptide fractions enriched for Tg14‐3‐3 or recombinant Tg14‐3‐3 are sufficient to induce the hypermotile phenotype when introduced by protein transfection into murine DCs, human DCs or microglia. Further, gene transfer of Tg14‐3‐3 by lentiviral transduction induced hypermotility in primary human DCs. In parasites expressing Tg14‐3‐3 in a ligand‐regulatable fashion, overexpression of Tg14‐3‐3 was correlated with induction of hypermotility in parasitized DCs. Localization studies in infected DCs identified Tg14‐3‐3 within the parasitophorous vacuolar space and a rapid recruitment of host cell 14‐3‐3 to the parasitophorous vacuole membrane. The present work identifies a determinant role for Tg14‐3‐3 in the induction of the migratory activation of immune cells by T. gondii. Collectively, the findings reveal Tg14‐3‐3 as a novel target for an intracellular pathogen that acts by hijacking the host cell's migratory properties to disseminate.     

Accumulation of glycinebetaine in rice plants that overexpress choline monooxygenase from spinach and evaluation of their tolerance to abiotic stress

BACKGROUND AND AIMS: Glycinebetaine (GB), a quaternary ammonium compound, is a very effective compatible solute. In higher plants, GB is synthesized from choline (Cho) via betaine aldehyde (BA). The first and second steps in the biosynthesis of GB are catalysed by choline monooxygenase (CMO) and by betaine aldehyde dehydrogenase (BADH), respectively. Rice (Oryza sativa), which has two genes for BADH, does not accumulate GB because it lacks a functional gene for CMO. Rice plants accumulate GB in the presence of exogenously applied BA, which leads to the development of a significant tolerance to salt, cold and heat stress. The goal in this study was to evaluate and to discuss the effects of endogenously accumulated GB in rice. METHODS: Transgenic rice plants that overexpressed a gene for CMO from spinach (Spinacia oleracea) were produced by Agrobacterium-mediated transformation. After Southern and western blotting analysis, GB in rice leaves was quantified by (1)H-NMR spectroscopy and the tolerance of GB-accumulating plants to abiotic stress was investigated. KEY RESULTS: Transgenic plants that had a single copy of the transgene and expressed spinach CMO accumulated GB at the level of 0.29-0.43 micromol g(-1) d. wt and had enhanced tolerance to salt stress and temperature stress in the seedling stage. CONCLUSIONS: In the CMO-expressing rice plants, the localization of spinach CMO and of endogenous BADHs might be different and/or the catalytic activity of spinach CMO in rice plants might be lower than it is in spinach. These possibilities might explain the low levels of GB in the transgenic rice plants. It was concluded that CMO-expressing rice plants were not effective for accumulation of GB and improvement of productivity.     

Identification of a Novel Subpopulation of Tumor-Initiating Cells from Gemcitabine-Resistant Pancreatic Ductal Adenocarcinoma Patients

Pancreatic ductal adenocarcinoma is highly resistant to systemic chemotherapy. Although there are many reports using pancreatic cancer cells derived from patients who did not receive chemotherapy, characteristics of pancreatic cancer cells from chemotherapy-resistant patients remain unclear. In this study, we set out to establish a cancer cell line in disseminated cancer cells derived from gemcitabine-resistant pancreatic ductal adenocarcinoma patients. By use of in vitro co-culture system with stromal cells, we established a novel pancreatic tumor-initiating cell line. The cell line required its direct interaction with stromal cells for its in vitro clonogenic growth and passaging. Their direct interaction induced basal lamina-like extracellular matrix formation that maintained colony formation. The cell line expressed CD133 protein, which expression level changed autonomously and by culture conditions. These results demonstrated that there were novel pancreatic tumor-initiating cells that required direct interactions with stromal cells for their in vitro cultivation in gemcitabine-resistant pancreatic ductal adenocarcinoma. This cell line would help to develop novel therapies that enhance effects of gemcitabine or novel anti-cancer drugs.     

Molecular targeting of hepatocyte growth factor by an antagonist,NK4, in the treatment of rheumatoid arthritis

Introduction

Hepatocyte growth factor (HGF) is a potent proangiogenic molecule that induces neovascularization. The HGF antagonist, NK4, competitively antagonizes HGF binding to its receptor. In the present study, we determined the inhibitory effect of NK4 in a rheumatoid arthritis (RA) model using SKG mice.

Methods

Arthritis was induced in SKG mice by a single intraperitoneal injection of β-glucan. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was also injected intravenously at the time of or 1 month after β-glucan injection. Ankle bone destruction was examined radiographically. The histopathologic features of joints were examined using hematoxylin and eosin and immunohistochemical staining. Enzyme-linked immunosorbent assays were used to determine the serum levels of HGF, interferon γ (IFN-γ, interleukin 4 (IL-4) and IL-17 production by CD4⁺ T cells stimulated with allogeneic spleen cells.

Results

The intravenous injection of AdCMV.NK4 into SKG mice suppressed the progression of β-glucan-induced arthritis. Bone destruction was also inhibited by NK4 treatment. The histopathologic findings of the ankles revealed that angiogenesis, inflammatory cytokines and RANKL expression in synovial tissues were significantly inhibited by NK4 treatment. Recombinant NK4 (rNK4) proteins inhibited IFN-γ, IL-4 and IL-17 production by CD4⁺ T cells stimulated with allogeneic spleen cells.

Conclusions

These results indicate that NK4 inhibits arthritis by inhibition of angiogenesis and inflammatory cytokine production by CD4⁺ T cells. Therefore, molecular targeting of angiogenic inducers by NK4 can potentially be used as a novel therapeutic approach for the treatment of RA.     

Neuronal Fos-like immunoreactivity associated with dexamethasone-induced hypertension in rats and effects of glucagon-like peptide-2

Aims

Dexamethasone-induced hypertension models have been used to study the mechanisms of glucocorticoid-induced hypertension, but the role of glucocorticoids in central cardiovascular regulation is not clearly understood. In the present study, we investigated the sites associated with dexamethasone-induced hypertension in the central nervous system in rats. We further investigated whether glucagon-like peptide-2 (GLP-2) was effective for dexamethasone-induced hypertension.

Main methods

Male Sprague–Dawley rats were treated with saline or dexamethasone (0.03 mg/kg/day, s.c) for 10 days. GLP-2 (60 μg/kg, i.v.) was given to rats after dexamethasone treatment. We measured systolic blood pressure by a tail-cuff method in conscious rats, and arterial blood pressure in anesthetized rats. Immunohistochemical techniques were used to detection of the c-fos protein (Fos).

Key findings

Fos-immunoreactivity (Fos-IR) in the dorsomedial hypothalamic nucleus (DMH) was higher in dexamethasone-treated rats than in saline-treated rats. However, Fos-IR in the infralimbic cortex, amygdala, and hippocampus was similar in saline-treated and dexamethasone-treated rats. Peripheral administration of GLP-2 reduced mean arterial blood pressure by 26%. After the peripheral administration of GLP-2, Fos-IR in the caudal ventrolateral medulla (CVLM) increased in dexamethasone-treated rats.

Significance

Chronic dexamethasone treatment induced Fos-IR in the DMH. Peripheral administration of GLP-2 suppressed dexamethasone-induced hypertension in rats by enhancing inhibitory neuronal activity.     

Rapid cytoskeleton remodelling in dendritic cells following invasion by Toxoplasma gondii coincides with the onset of a hypermigratory phenotype

Host cell manipulation is an important feature of the obligate intracellular parasite Toxoplasma gondii. Recent reports have shown that the tachyzoite stages subvert dendritic cells (DC) as a conduit for dissemination (Trojan horse) during acute infection. To examine the cellular basis of these processes, we performed a detailed analysis of the early events following tachyzoite invasion of human monocyte‐derived DC. We demonstrate that within minutes after tachyzoite penetration, profound morphological changes take place in DC that coincide with a migratory activation. Active parasite invasion of DC led to cytoskeletal actin redistribution with loss of adhesive podosome structures and redistribution of integrins (CD18 and CD11c), that concurred with the onset of DC hypermotility in vitro. Inhibition of parasite rhoptry secretion and invasion, but not inhibition of parasite or host cell protein synthesis, abrogated the onset of morphological changes and hypermotility in DC dose‐dependently. Also, infected DC, but not by‐stander DC, exhibited upregulation of C‐C chemokine receptor 7 (CCR7). Yet, the onset of parasite‐induced DC hypermotility preceded chemotactic migratory responsesin vitro. Collectively, present data reveal that invasion of DC by T. gondii initiates a series of regulated events, including rapid cytoskeleton rearrangements, hypermotility and chemotaxis, that promote the migratory activation of DC.     

High-Yield Purification of Glucokinase from Rat Liver

A rapid and reliable method for the purification of rat liver glucokinase was developed. The procedure consists of DEAE-cellulose ion-exchange chromatography, Phenyl-Sepharose hydrophobic interaction chromatography, DEAE-Affi Gel Blue dye-ligand chromatography, and duplicate steps of glucosamine-Sepharose affinity chromatography. Glucokinase was purified to a specific activity of 290 units/mg protein in a yield of 55% in 6 days. The final enzyme preparations were completely homogeneous in most experiments as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The estimated molecular weight (51.000) and sigmoidal saturation function for glucose of purified glucokinase were in good agreement with published data.     

Triterpenoid saponins from Echinopsis macrogona (Cactaceae)

Triterpene saponins, pachanosides C1, E1, F1 and G1 (1-4), and bridgesides A1, C1, C2, D1, D2, E1 and E2 (5-11) were isolated from Echinopsis macrogona. Compounds 1-4 were saponins with pachanane type triterpene saponins, while the others (5-11) were oleanane type triterpene saponins. While the aglycones of 2-4 and 8-11 were hitherto unknown, the structure of pachanol C was revised in this paper. Their structures were elucidated on the basis of chemical and physicochemical evidence.