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.     

The More the Merrier: Recent Hybridization and Polyploidy in Cardamine

This article describes the use of cytogenomic and molecular approaches to explore the origin and evolution of Cardamine schulzii, a textbook example of a recent allopolyploid, in its ∼110-year history of human-induced hybridization and allopolyploidy in the Swiss Alps. Triploids are typically viewed as bridges between diploids and tetraploids but rarely as parental genomes of high-level hybrids and polyploids. The genome of the triploid semifertile hybrid Cardamine × insueta (2n = 24, RRA) was shown to combine the parental genomes of two diploid (2n = 2x = 16) species, Cardamine amara (AA) and Cardamine rivularis (RR). These parental genomes have remained structurally stable within the triploid genome over the >100 years since its origin. Furthermore, we provide compelling evidence that the alleged recent polyploid C. schulzii is not an autohexaploid derivative of C. × insueta. Instead, at least two hybridization events involving C. × insueta and the hypotetraploid Cardamine pratensis (PPPP, 2n = 4x−2 = 30) have resulted in the origin of the trigenomic hypopentaploid (2n = 5x−2 = 38, PPRRA) and hypohexaploid (2n = 6x−2 = 46, PPPPRA). These data show that the semifertile triploid hybrid can promote a merger of three different genomes and demonstrate how important it is to reexamine the routinely repeated textbook examples using modern techniques.     

Naemacyclus culmigenus,a newly reported potential pathogen to Miscanthus sinensis,new to Japan

Since the summer of 2010, a discomycete with erumpent apothecia associated with a leaf blight of Miscanthus leaves, were often collected. The morphological characteristics of the fungus suggested it was a member of the Helotiales rather than the Rhytismatales and this was supported by a phylogenetic analysis. Based on a morphological comparison with the type specimen of Naemacyclus culmigenus, currently known from Poaceae (Andropogon and Panicum), it was identified as N. culmigenus, new to Japan. The molecular phylogenetic analysis showed that the generic delimitation of Naemacyclus and related species requires clarification, as does their higher classification within the Leotiomycetes.     

Increase of Circulating CD11b+Gr1+ cells and Recruitment into the Synovium in Osteoarthritic Mice with Hyperlipidemia

Although recent studies suggest that hyperlipidemia is a risk factor for osteoarthritis (OA), the link between OA and hyperlipidemia is not fully understood. As the number of activated, circulating myeloid cells is increased during hyperlipidemia, we speculate that myeloid cells contribute to the pathology of OA. Here, we characterized myeloid cells in STR/Ort mice, a murine osteoarthritis model, under hyperlipidemic conditions. Ratios of myeloid cells in bone marrow, the spleen, and peripheral blood were determined by flow cytometry. To examine the influence of the hematopoietic environment, including abnormal stem cells, on the hematopoietic profile of STR/Ort mice, bone marrow transplantations were performed. The relationship between hyperlipidemia and abnormal hematopoiesis was examined by evaluating biochemical parameters and spleen weight of F₂ animals (STR/Ort x C57BL/6J). In STR/Ort mice, the ratio of CD11b⁺Gr1⁺ cells in spleens and peripheral blood was increased, and CD11b⁺Gr1⁺ cells were also present in synovial tissue. Splenomegaly was observed and correlated with the ratio of CD11b⁺Gr1⁺ cells. When bone marrow from GFP-expressing mice was transplanted into STR/Ort mice, no difference in the percentage of CD11b⁺Gr1⁺ cells was observed between transplanted and age-matched STR/Ort mice. Analysis of biochemical parameters in F₂ mice showed that spleen weight correlated with serum total cholesterol. These results suggest that the increase in circulating and splenic CD11b⁺Gr1⁺ cells in STR/Ort mice originates from hypercholesterolemia. Further investigation of the function of CD11b⁺Gr1⁺ cells in synovial tissue may reveal the pathology of OA in STR/Ort mice.     

Legacies of Prehistoric Agricultural Practices Within Plant and Soil Properties Across an Arid Ecosystem

Closely integrated research between archaeologists and ecologists provides a long-term view of human land use that is rare in the ecological literature, allowing for investigation of activities that lead to enduring environmental outcomes. This extended temporal perspective is particularly important in aridlands where succession occurs slowly and ecosystem processes are mediated by abiotic, geomorphic factors. Numerous studies show that impacts from ancient human actions can persist, but few have explored the types of practices or mechanisms that lead to either transient or long-term environmental change. We compared plant and soil properties and processes from a range of landscape patch types in the Sonoran Desert of the US Southwest that supported different, well-documented prehistoric farming practices from AD 750–1300. Our results show that the types of ancient human activities that leave long-term ecological legacies in aridlands are those that fundamentally alter “slow variables” such as soil properties that regulate the timing and supply of water. Prehistoric Hohokam floodwater-irrigation practices, but not dryland farming techniques, substantially altered soil texture, which was strongly associated with desert plant community and functional composition. However, prehistoric agriculture did not consistently alter long-term nutrient availability and thus had no impact on “fast variables” such as production of seasonal annual plants that are restricted to periods of ample rainfall. In this arid ecosystem, the inverse texture model explained patterns in plant functional composition at large scales, but is less predictive of production of short-lived desert annuals that experience a more mesic precipitation regime.     

Ras guanyl nucleotide releasing protein 2 affects cell viability and cell–matrix adhesion in ECV304 endothelial cells

Ras guanyl nucleotide releasing proteins (RasGRPs) are guanine nucleotide exchange factors that activate Ras and Rap. We recently reported that xrasgrp2, which is a homolog of the human rasgrp2, plays a role in vasculogenesis and/or angiogenesis during early development of Xenopus embryos. However, the function of RasGRP2 in human vascular endothelium remains unknown. Therefore we aimed to analyze the function of human RasGRP2 in vascular endothelial cells. RasGRP2 overexpression did not increase Ras activation. However, it slightly increased Ras expression and increased proliferation in ECV304 cells. Furthermore, RasGRP2 overexpression increased Rap1 activation and cell–matrix adhesion in ECV304 cells. These data demonstrate that RasGRP2 increases cell viability and cell–matrix adhesion through increased Ras expression and Rap1 activation, respectively, in endothelial cells.     

Matrix-Embedded Osteocytes Regulate Mobilization of Hematopoietic Stem/Progenitor Cells

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Urinary Fetuin-A Is a Novel Marker for Diabetic Nephropathy in Type 2 Diabetes Identified by Lectin Microarray

We analyzed the urine samples of patients with type 2 diabetes at various stages of diabetic nephropathy by lectin microarray to identify a biomarker to predict the progression of diabetic nephropathy. Japanese patients with type 2 diabetes at various stages of nephropathy were enrolled and we performed lectin microarray analyses (n = 17) and measured urinary excretion of fetuin-A (n = 85). The increased signals of urine samples were observed in Siaα2-6Gal/GalNAc-binding lectins (SNA, SSA, TJA-I) during the progression of diabetic nephropathy. We next isolated sialylated glycoproteins by using SSA-lectin affinity chromatography and identified fetuin-A by liquid chromatography–tandem mass spectrometer. Urinary excretion of fetuin-A significantly increased during the progression of albuminuria (A1, 0.40±0.43; A2, 0.60±0.53; A3 1.57±1.13 ng/gCr; p = 7.29×10⁻⁸) and of GFR stages (G1, 0.39±0.39; G2, 0.49±0.45; G3, 1.25±1.18; G4, 1.34±0.80 ng/gCr; p = 3.89×10⁻⁴). Multivariate logistic regression analysis was employed to assess fetuin-A as a risk for diabetic nephropathy with microalbuminuria or GFR<60 mL/min. Fetuin-A is demonstrated as a risk factor for both microalbuminuria and reduction of GFR in diabetic nephropathy with the odds ratio of 4.721 (1.881–11.844) and 3.739 (1.785–7.841), respectively. Collectively, the glycan profiling analysis is useful method to identify the urine biomarkers and fetuin-A is a candidate to predict the progression of diabetic nephropathy.     

Basal and Ischemia-Induced Transcardiac Troponin Release into the Coronary Circulation in Patients with Suspected Coronary Artery Disease

Background

Cardiac troponin is a specific biomarker for cardiomyocyte necrosis in acute coronary syndromes. Troponin release from the coronary circulation remains to be determined because of the lower sensitivity of the conventional assay. We sought to determine basal and angina-induced troponin release using a highly sensitive troponin assay.

Methods and Results

The cardiac troponin T levels in serum sampled from the peripheral vein (PV), the aortic root (AO), and the coronary sinus (CS) were measured in 105 consecutive stable patients with coronary risk factor(s) and suspected coronary artery disease (CAD) and in 33 patients without CAD who underwent an acetylcholine provocation test. At baseline, there was a significant increase in the troponin levels from AO [9.0 (6.4, 13.1) pg/mL for median (25^th, 75^th percentiles)] to CS [10.3 (7.3, 15.5) pg/mL, p<0.001] in 96 (91.4%) patients and the difference was 1.1 (0.4, 2.1) pg/mL, which reflected basal transcardiac troponin release (TTR). TTR was positively correlated with PV levels (r = 0.22, p = 0.03). Male sex, left ventricular hypertrophy determined by echocardiography, T-wave inversion, and CAD correlated with elevated TTR defined as above: median, 1.1 pg/mL. A significant increase in TTR was noted in 17 patients with coronary spasms [0.6 (0.2, 1.2) pg/mL, p<0.01] but not in 16 patients without spasms [0.0 (−0.5, 0.9) pg/mL, p = 0.73] after the acetylcholine provocation.

Conclusion

Basal TTR in the coronary circulation was observed in most of the patients with suspected CAD and risk factor(s). This sensitive assay detected myocardial ischemia-induced increases in TTR caused by coronary spasms.     

Measuring Airway Surface Liquid Depth in Ex Vivo Mouse Airways by X-Ray Imaging for the Assessment of Cystic Fibrosis Airway Therapies

In the airways of those with cystic fibrosis (CF), the leading pathophysiological hypothesis is that an ion channel defect results in a relative decrease in airway surface liquid (ASL) volume, producing thick and sticky mucus that facilitates the establishment and progression of early fatal lung disease. This hypothesis predicts that any successful CF airway treatment for this fundamental channel defect should increase the ASL volume, but up until now there has been no method of measuring this volume that would be compatible with in vivo monitoring. In order to accurately monitor the volume of the ASL, we have developed a new x-ray phase contrast imaging method that utilizes a highly attenuating reference grid. In this study we used this imaging method to examine the effect of a current clinical CF treatment, aerosolized hypertonic saline, on ASL depth in ex vivo normal mouse tracheas, as the first step towards non-invasive in vivo ASL imaging. The ex vivo tracheas were treated with hypertonic saline, isotonic saline or no treatment using a nebuliser integrated within a small animal ventilator circuit. Those tracheas exposed to hypertonic saline showed a transient increase in the ASL depth, which continued for nine minutes post-treatment, before returning to baseline by twelve minutes. These findings are consistent with existing measurements on epithelial cell cultures, and therefore suggest promise for the future development of in vivo testing of treatments. Our grid-based imaging technique measures the ASL depth with micron resolution, and can directly observe the effect of treatments expected to increase ASL depth, prior to any changes in overall lung health. The ability to non-invasively observe micron changes in the airway surface, particularly if achieved in an in vivo setting, may have potential in pre-clinical research designed to bring new treatments for CF and other airway diseases to clinical trials.