Epstein-Barr virus nuclear antigen 3C (EBNA3C) interacts with the metabolism sensing C-terminal binding protein (CtBP) repressor to upregulate host genes

Epstein-Barr virus (EBV) infection is associated with the development of specific types of lymphoma and some epithelial cancers. EBV infection of resting B-lymphocytes in vitro drives them to proliferate as lymphoblastoid cell lines (LCLs) and serves as a model for studying EBV lymphomagenesis. EBV nuclear antigen 3C (EBNA3C) is one of the genes required for LCL growth and previous work has suggested that suppression of the CDKN2A encoded tumor suppressor p16^INK4A and possibly p14^ARF is central to EBNA3C’s role in this growth transformation. To directly assess whether loss of p16 and/or p14 was sufficient to explain EBNA3C growth effects, we used CRISPR/Cas9 to disrupt specific CDKN2A exons in EBV transformed LCLs. Disruption of p16 specific exon 1α and the p16/p14 shared exon 2 were each sufficient to restore growth in the absence of EBNA3C. Using EBNA3C conditional LCLs knocked out for either exon 1α or 2, we identified EBNA3C induced and repressed genes. By trans-complementing with EBNA3C mutants, we determined specific genes that require EBNA3C interaction with RBPJ or CtBP for their regulation. Unexpectedly, interaction with the CtBP repressor was required not only for repression, but also for EBNA3C induction of many host genes. Contrary to previously proposed models, we found that EBNA3C does not recruit CtBP to the promoters of these genes. Instead, our results suggest that CtBP is bound to these promoters in the absence of EBNA3C and that EBNA3C interaction with CtBP interferes with the repressive function of CtBP, leading to EBNA3C mediated upregulation.     

Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

The adaptor protein-2 (AP-2) complex is a heterotetramer involved in clathrin-mediated endocytosis of cargo proteins from the plasma membrane in animal cells. The homologous genes of AP-2 subunits are present in the genomes of plants; however, their identities and roles in endocytic pathways are not clearly defined in plants. Here, we reveal the molecular composition of the AP-2 complex of Arabidopsis thaliana and its dynamics on the plasma membrane. We identified all of the α-, β-, σ-, and μ-subunits of the AP-2 complex and detected a weak interaction of the AP-2 complex with clathrin heavy chain. The μ-subunit protein fused to green fluorescent protein (AP2M-GFP) was localized to the plasma membrane and to the cytoplasm. Live-cell imaging using a variable-angle epifluorescence microscope revealed that AP2M-GFP transiently forms punctate structures on the plasma membrane. Homozygous ap2m mutant plants exhibited abnormal floral structures, including reduced stamen elongation and delayed anther dehiscence, which led to a failure of pollination and a subsequent reduction of fertility. Our study provides a molecular basis for understanding AP-2–dependent endocytic pathways in plants and their roles in floral organ development and plant reproduction.     

Redox state of albumin affects its lipid mediator binding characteristics

Depending on its redox status, albumin is known to exist as two forms: reduced albumin or human mercaptalbumin (HMA); and oxidised albumin or human nonmercaptalbumin (HNA). The ratio of HNA to HMA is reportedly elevated in several diseases. Since lipid mediators, such as eicosanoids and lysophospholipids, are typically bound to albumin, we examined the possible preferences of lipid mediators for HNA or HMA. We observed that DHA-derived and EPA-derived eicosanoids preferred to be bound to HMA, while the levels of lysophospholipid mediators, such as lysophosphatidic acids and sphingosine 1-phosphate, were higher in the HNA fraction. Considering the bioactivities reported in previous basic studies, these results suggest that proatherosclerotic lipid mediators might generally prefer HNA, while antiatherosclerotic ones might prefer HMA. Oxidative stress affects the redox status of albumin, which might modulate the dynamism of lipid mediators. This pathway might be partly involved in the association between oxidation and atherosclerosis.     

Diagnostic Tests for Primary Renal Hypouricemia

Primary renal hypouricemia is a genetic disorder characterized by defective renal uric acid (UA) reabsorption with complications such as nephrolithiasis and exercise-induced acute renal failure. The known causes are: defects in the SLC22A12 gene, encoding the human urate transporter 1 (hURAT1), and also impairment of voltage urate transporter (URATv1), encoded by SLC2A9 (GLUT9) gene. Diagnosis is based on hypouricemia (<119 μmol/L) and increased fractional excretion of UA (>10%). To date, the cases with mutations in hURAT1 gene have been reported in East Asia only. More than 100 Japanese patients have been described. Hypouricemia is sometimes overlooked; therefore, we have set up the flowchart for this disorder. The patients were selected for molecular analysis from 620 Czech hypouricemic patients. Secondary causes of hyperuricosuric hypouricemia were excluded. The estimations of (1) serum UA, (2) excretion fraction of UA, and (3) analysis of hURAT1 and URATv1 genes follow. Three transitions and one deletion (four times) in SLC22A12 gene and one nucleotide insertion in SLC2A9 gene in seven Czech patients were found. Three patients had acute renal failure and urate nephrolithiasis. In addition, five nonsynonymous sequence variants and three nonsynonymous sequence variants in SLC2A9 gene were found in two UK patients suffering from acute renal failure. Our finding of the defects in SLC22A12 and SLC2A9 genes gives further evidence of the causative genes of primary renal hypouricemia and supports their important role in regulation of serum urate levels in humans.     

Taxonomy of two host specialized Phakopsora populations on Meliosma in Japan

Phakopsora meliosmae, a macrocyclic autoecious rust fungus, is reported to occur on several Meliosma species widely distributed in Asia. Despite the apparent broad host range, a recent molecular phylogenetic study indicated that two rust populations on Meliosma myriantha and Meliosma tenuis respectively in Japan were biologically distinct. To clarify the biological and taxonomic relationships of these populations, cross inoculations and comparative morphological examinations were carried out. Cross inoculations using basidiospores and aeciospores confirmed the macrocyclic, autoecious nature of the life cycle in both rust populations and showed that the two populations were distinct in their host specificity. Furthermore, they were found to be distinct in the structure of the aecial peridium surface, the size and wall thickness of uredinial paraphyses, and the urediniospore size and shape. Consequently, the fungal population on M. tenuis is taxonomically separated from P. meliosmae originally proposed for the fungus on M. myriantha. A new name, Phakopsora orientalis, is proposed for the fungus on M. tenuis.     

Heterotrimeric G protein β subunit GPB1 and MAP kinase MPK1 regulate hyphal growth and female fertility in Fusarium sacchari

Heterotrimeric GTP-binding proteins (G proteins) and mitogen-activated protein kinase (MAPK) cascades involve vegetative hyphal growth, development of infection-related structure, colonization in host plant and female fertility in phytopathogenic ascomycete fungi. In this study, a heterotrimeric G protein β subunit (Gβ), GPB1, and MAPK, MPK1, were characterized from Fusarium sacchari (= Gibberella sacchari; mating population B of the G. fujikuroi-species complex). GPB1 and MPK1 showed high homology to known Gβ and Fus3/Kss1 MAP kinases of other filamentous ascomycetes, respectively. Disruption (Δ) of gpb1 suppressed hyphal branching and accelerated aerial hyphae formation in F. sacchari. Oppositely, disruption of mpk1 caused delayed aerial hyphae formation. These indicated that GPB1 regulates vegetative hyphal growth negatively, and MPK1 does positively in F. sacchari. Both Δgpb1 and Δmpk1 showed female sterility. Level of intracellular cAMP in Δgpb1 was lower than wild type. Exogenous cyclic AMP (cAMP) partially restored enhanced aerial hyphae formation. These suggested that abnormal hyphal growth was caused by depletion of intracellular cAMP in Δgpb1. cAMP has been reported to suppress development of perithecia in crossing between wild type strains. Thus, precise regulation of intracellular cAMP level via Gβ/MAPK is essential for normal hyphal growth and fertility.     

Infectivity of Cryptosporidium andersoni Kawatabi type relative to the small number of oocysts in immunodeficient and immunocompetent neonatal and adult mice

Cryptosporidium andersoni is a protozoan parasite found in many countries that invades the stomachs of primarily adult cattle. Unlike the isolates of C. andersoni in cattle from other countries, C. andersoni isolates from Japanese cattle can infect mice and were identified as a novel type and later defined as C. andersoni Kawatabi type. The biological characteristics of C. andersoni Kawatabi type have not yet been well documented. In the present study, we assess the infectivity of this type isolate in mice with different immune competence status and age. We found that inoculation of more than 1 × 10⁴ oocysts is needed to establish infection in mature mice irrespective of immune status. All of the infected immunocompetent mice recovered after a patent period of approximately 20 days. In immunodeficient mice, the pre-patent period was prolonged compared with that of 1 × 10⁶ oocysts, but the pattern and the maximum shedding measured by the number of oocysts per day were almost identical. In neonatal immunocompetent and immunodeficient mice, inoculation with 1 × 10⁴ to 10⁵ oocysts was also needed to establish infection. Our results indicate that there is a threshold of oocysts needed to establish patent infection in the acidic conditions of the stomach.     

Apoptosis inhibitor of macrophage (AIM) expression in alveolar macrophages in COPD

Background

Marked accumulation of alveolar macrophages (AM) conferred by apoptosis resistance has been implicated in pathogenesis of chronic obstructive pulmonary disease (COPD). Apoptosis inhibitor of macrophage (AIM), has been shown to be produced by mature tissue macrophages and AIM demonstrates anti-apoptotic property against multiple apoptosis-inducing stimuli. Accordingly, we attempt to determine if AIM is expressed in AM and whether AIM is involved in the regulation of apoptosis in the setting of cigarette smoke extract (CSE) exposure.

Methods

Immunohistochemical evaluations of AIM were performed. Immunostaining was assessed by counting total and positively staining AM numbers in each case (n = 5 in control, n = 5 in non-COPD smoker, n = 5 in COPD). AM were isolated from bronchoalveolar lavage fluid (BALF). The changes of AIM expression levels in response to CSE exposure in AM were evaluated. Knock-down of anti-apoptotic Bcl-xL was mediated by siRNA transfection. U937 monocyte-macrophage cell line was used to explore the anti-apoptotic properties of AIM.

Results

The numbers of AM and AIM-positive AM were significantly increased in COPD lungs. AIM expression was demonstrated at both mRNA and protein levels in isolated AM, which was enhanced in response to CSE exposure. AIM significantly increased Bcl-xL expression levels in AM and Bcl-xL was involved in a part of anti-apoptotic mechanisms of AIM in U937 cells in the setting of CSE exposure.

Conclusions

These results suggest that AIM expression in association with cigarette smoking may be involved in accumulation of AM in COPD.     

Ex vivo characterization of γδ T-cell repertoire in patients after adoptive transfer of Vγ9Vδ2 T cells expressing the interleukin-2 receptor β-chain and the common γ-chain

Background aimsAdoptive immunotherapy is emerging as a potent anti-tumor treatment modality; Vγ9Vδ2 T cells may represent appropriate agents for such cancer immunotherapy. To improve the currently limited success of Vγ9Vδ2 T-cell–based immunotherapy, we examined the in vivo dynamics of these adoptively-transferred cells and hypothesized that interleukin (IL)-15 is the potential factor for Vγ9δ2 T cell in vivo survival.MethodsWe conducted a clinical trial of adoptive Vγ9Vδ2 T-cell transfer therapy in six colorectal cancer patients who received pulmonary metastasectomy. Patients' peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and IL-2 (1000 IU/mL) for 14 d. Harvested cells, mostly Vγ9Vδ2 T cells, were given intravenously weekly without additional IL-2 eight times in total. The frequency, phenotype and common γ-chain cytokine receptor expression of Vγ9Vδ2 T cells in peripheral blood was monitored by flow cytometry at each time point during treatment and 4 and 12 weeks after the last administration.ResultsAdoptively transferred Vγ9Vδ2 T cells expanded well without exogenous IL-2 administration or lymphodepleting preconditioning. They maintained effector functions in terms of interferon-γ secretion and prompt release of cytotoxic granules in response to PMA/ionomycin or isopentenyl pyrophosphate–positive cells. Because they are IL-2Rα^?IL-7Rα^?IL-15Rα^?IL-2Rβ⁺γ_c⁺, it is likely that IL-2 or IL-15 is required for their maintenance.ConclusionsThe persistence of large numbers of functionally active adoptively transferred Vγ9Vδ2 T cells in the absence of exogenous IL-2 implies that an endogenous factor, such as IL-15 transpresentation, is adequate to support these cells in vivo.