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.     

Roles of NO Signaling in Long-Term Memory Formation in Visual Learning in an Insect

Many insects exhibit excellent capability of visual learning, but the molecular and neural mechanisms are poorly understood. This is in contrast to accumulation of information on molecular and neural mechanisms of olfactory learning in insects. In olfactory learning in insects, it has been shown that cyclic AMP (cAMP) signaling critically participates in the formation of protein synthesis-dependent long-term memory (LTM) and, in some insects, nitric oxide (NO)-cyclic GMP (cGMP) signaling also plays roles in LTM formation. In this study, we examined the possible contribution of NO-cGMP signaling and cAMP signaling to LTM formation in visual pattern learning in crickets. Crickets that had been subjected to 8-trial conditioning to associate a visual pattern with water reward exhibited memory retention 1 day after conditioning, whereas those subjected to 4-trial conditioning exhibited 30-min memory retention but not 1-day retention. Injection of cycloheximide, a protein synthesis inhibitor, into the hemolymph prior to 8-trial conditioning blocked formation of 1-day memory, whereas it had no effect on 30-min memory formation, indicating that 1-day memory can be characterized as protein synthesis-dependent long-term memory (LTM). Injection of an inhibitor of the enzyme producing an NO or cAMP prior to 8-trial visual conditioning blocked LTM formation, whereas it had no effect on 30-min memory formation. Moreover, injection of an NO donor, cGMP analogue or cAMP analogue prior to 4-trial conditioning induced LTM. Induction of LTM by an NO donor was blocked by DDA, an inhibitor of adenylyl cyclase, an enzyme producing cAMP, but LTM induction by a cAMP analogue was not impaired by L-NAME, an inhibitor of NO synthase. The results indicate that cAMP signaling is downstream of NO signaling for visual LTM formation. We conclude that visual learning and olfactory learning share common biochemical cascades for LTM formation.     

Skin Autofluorescence Is Associated with the Progression of Chronic Kidney Disease: A Prospective Observational Study

Background

Advanced glycation end product (AGE) accumulation is thought to be a measure of cumulative metabolic stress that has been reported to independently predict cardiovascular disease in diabetes and renal failure. The aim of this study was to evaluate the association between AGE accumulation, measured as skin autofluorescence, and the progression of renal disease in pre-dialysis patients with chronic kidney disease (CKD).

Methods

Skin autofluorescence was measured noninvasively with an autofluorescence reader at baseline in 449 pre-dialysis patients with CKD. The primary end point was defined as a doubling of serum creatinine and/or need for dialysis.

Results

Thirty-three patients were lost to follow-up. Forty six patients reached the primary end point during the follow-up period (Median 39 months). Kaplan-Meier analysis showed a significantly higher risk of development of the primary end points in patients with skin autofluorescence levels above the optimal cut-off level of 2.31 arbitrary units, derived by receiver operator curve analysis. Cox regression analysis revealed that skin autofluorescence was an independent predictor of the primary end point, even after adjustment for age, gender, smoking history, diabetes, estimated glomerular filtration rate and proteinuria (adjusted hazard ratio 2.58, P = 0.004).

Conclusions

Tissue accumulation of AGEs, measured as skin autofluorescence, is a strong and independent predictor of progression of CKD. Skin autofluorescence may be useful for risk stratification in this group of patients; further studies should clarify whether AGE accumulation could be one of the therapeutic targets to improve the prognosis of CKD.