Invariant chain processing is independent of cathepsin variation between primary human B cells/dendritic cells and B-lymphoblastoid cells

As part of the endocytic antigen processing pathway, proteolytic cleavage of the invariant chain (Ii) is important for the generation of class II-associated invariant chain peptide (CLIP). CLIP remains associated with the major histocompatibility complex (MHC) class II molecule to prevent premature loading of antigenic peptides. Cysteine proteases, such as Cathepsin S (CatS), CatL, or CatV, play a pivotal role in the final stage of Ii degradation depending on the cell type studied. Less is known regarding the early stages of Ii processing. We therefore explored whether the serine protease CatG is involved in the initial step of Ii degradation in primary antigen presenting cells (APC), since the cathepsin distribution differs between primary APC and cell lines. While primary human B cells and dendritic cells (DC) do harbor CatG, this protease is absent in B-lymphoblastoid cells (BLC) or monocyte-derived DC generated in vitro. In addition, other proteases, such as CatC, CatL, and the asparagine endoprotease (AEP), are active in BLC and monocyte-derived DC. Here we demonstrate that CatG progressively degraded Ii in vitro resulting in several intermediates. However, pharmacological inhibition of CatG in primary B cells and DC did not alter Ii processing, indicating that CatG is dispensable in Ii degradation. Interestingly, stalling of cysteine proteases by inhibition in BLC vs. primary B cells and DC did not result in any differences in the generation of distinct Ii intermediates between the cells tested, suggesting that Ii processing is independent of the cathepsin variation within professional human APC.     

Klotho inhibits transforming growth factor-beta1 (TGF-beta1) signaling and suppresses renal fibrosis and cancer metastasis in mice

Fibrosis is a pathological process characterized by infiltration and proliferation of mesenchymal cells in interstitial space. A substantial portion of these cells is derived from residing non-epithelial and/or epithelial cells that have acquired the ability to migrate and proliferate. The mesenchymal transition is also observed in cancer cells to confer the ability to metastasize. Here, we show that renal fibrosis induced by unilateral ureteral obstruction and metastasis of human cancer xenografts are suppressed by administration of secreted Klotho protein to mice. Klotho is a single-pass transmembrane protein expressed in renal tubular epithelial cells. The extracellular domain of Klotho is secreted by ectodomain shedding. Secreted Klotho protein directly binds to the type-II TGF-β receptor and inhibits TGF-β1 binding to cell surface receptors, thereby inhibiting TGF-β1 signaling. Klotho suppresses TGF-β1-induced epithelial-to-mesenchymal transition (EMT) responses in cultured cells, including decreased epithelial marker expression, increased mesenchymal marker expression, and/or increased cell migration. In addition to TGF-β1 signaling, secreted Klotho has been shown to inhibit Wnt and IGF-1 signaling that can promote EMT. These results have raised the possibility that secreted Klotho may function as an endogenous anti-EMT factor by inhibiting multiple growth factor signaling pathways simultaneously.     

Exogenous gibberellic acid increases the fruit weight of ‘Comte de Paris’ pineapple by enlarging flesh cells without negative effects on fruit quality

In mainland China, the most popular pineapple cultivar is ‘Comte de Paris’. Gibberellic acids have been widely applied to enhance fruit growth in various species. To evaluate the effect of gibberellic acid (GA₃) on ‘Comte de Paris’ pineapple production and quality, pineapple fruits were sprayed with GA₃ at concentrations of 5, 20, 50, or 100 mg l⁻¹ at both 0 and 15 days after flowering (DAF). Fruits were sampled every 15 days from 0 to 60 DAF (maturation) for flow cytometric analysis and histological observation. The results showed that the treatments with the three highest concentrations of GA₃ significantly increased fruit weight, and the most effective concentration was 50 mg l⁻¹ GA₃, which increased the flesh weight by 20.3% compared to the control. Although treatment with GA₃ had little effect on the total soluble solids and fruit juice pH, it increased the vitamin C content. Although flow cytometric analysis showed that the 50 mg l⁻¹ GA₃ treatment had only a slight impact on the number of S phase cells, histological observations indicated that the increase of fruit volume and flesh weight under this GA₃ treatment was not due to the increase of cell number but a result of the increase of cell area in the fruit flesh.     

Construction of an effective screening system for detection of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> quorum sensing inhibitors and its application in bioautographic thin-layer chromatography

In Pseudomonas aeruginosa, quorum sensing (QS) regulates dozens of genes and proteins, many of which contribute to the virulence of this pathogen. QS inhibitory (QSI) compounds have been proposed as potential agents for treatment of bacterial infections. To search for Ps. aeruginosa QS inhibitors, we constructed an effective screening system, QSIS-lasI selector, based on the PlasI-sacB reporter, in which QS could be induced with 20 nM 3-oxo-N-[(3S)-tetrahydro-2-oxo-3-furanyl]-dodecanamide (3-oxo-C₁₂-HSL). During screening of the crude extracts from 65 marine fungi, an isolate of Penicillium atramentosum was found to have QSI activity. Thin-layer chromatography assay of the fungal extracts for bioautographic identification of QSIS-lasI indicated that this fungus produced several QSI compounds, including QS inhibitors other than penicillic acid or patulin.     

Fungistatic Intensity of Agricultural Soil Against Fungal Agents and Phylogenetic Analysis on the Actinobacteria Involved

A total of 287 agricultural soil samples collected from 26 provinces or autonomous regions of China were tested on their ability to suppress the conidial germination of nine biocontrol fungal agents. These soil samples showed great differences in the degree to inhibit the germination of conidia (22.8% < mean inhibition rate < 97.5%), but all exhibited fungistatic activities above the moderate levels (mean inhibition rate > 50%) to most of tested fungi. Ten soil samples that have stronger fungistatic intensity (germination inhibition rate > 68.3%) to the target fungi, Trichoderma viride and Paecilomyces lilacinus, were selected to evaluate their soil actinobacteria involved fungistasis in soil. Of the 1,000 isolates from those soil samples, 345 actinobacteria exhibited fungistatic activity to conidial germination of T. viride and P. lilacinus with germination inhibition rates higher than 10%. Sequences encoding 16S rRNA gene of the 345 actinobacteria were analyzed by ARDRA and resulted 44 different ARDRA types. Fifty-six isolates, at least one from each unique ARDRA type, were selected for 16S rDNA sequencing and phylogenetic analysis. Results indicated that the actinobacteria involved in the soil fungistasis had close phylogenetic relationship with the members of Sterptomycetaceae, Microbacteriaceae, Micrococcaceae, and Nocardiacea.     

Morphological and Molecular Differences in Two Strains of <Emphasis Type="Italic">Ustilago esculenta</Emphasis>

Ustilago esculenta is a fungal endophyte of Zizania latifolia that plays an important agricultural role in this vegetable crop. The purpose of this study was to characterize sporidial (T) and mycelial (M-T) strains of U. esculenta isolated from sporulating and non-sporulating galls on plants growing in Zhejiang province, China. Morphological comparisons of the T strain and M-T strain were made by optical and scanning electron microscope observation. Genetic differences were examined by sequencing the ITS region of the fungus and examining differential protein expression by two-dimensional gel electrophoresis and MALDI-TOF-MS/MS. The sporidial (T) and mycelial (M-T) strains differed in morphological characteristics of their in vitro single colony formations and in cell shape. Alignment of ITS sequences of the T strain and M-T strain revealed a single mutation between the T strain and M-T strain, but the sequences were the same within strains. A total of 146 proteins were only expressed in the M-T strain, and 242 proteins were only expressed in the T strain isolated from infected plants. A total of 222 proteins were up-regulated or down-regulated in the T strain when compared with the M-T strain. Of these, 18 proteins were identified and eight were associated with processes involving energy metabolism and the cytoskeleton. Two morphology-related proteins, MAP kinase kinase and actin, were differentially expressed. The differences noted in the T strain and M-T strain may lead to a better understanding of the life cycle and morphogenesis in U. esculenta.     

Uracils at nucleotide position 9-11 are required for the rapid turnover of miR-29 family

MicroRNAs are endogenous small RNA molecules that regulate gene expression. Although the biogenesis of microRNAs and their regulation have been thoroughly elucidated, the degradation of microRNAs has not been fully understood. Here by using the pulse-chase approach, we performed the direct measurement of microRNA lifespan. Five representative microRNAs demonstrated a general feature of relatively long lifespan. However, the decay dynamic varies considerably between these individual microRNAs. Mutation analysis of miR-29b sequence revealed that uracils at nucleotide position 9-11 are required for its rapid decay, in that both specific nucleotides and their position are critical. The effect of uracil-rich element on miR-29b decay dynamic occurs in duplex but not in single strand RNA. Moreover, analysis of published data on microRNA expression profile during development reveals that a substantial subset of microRNAs with the uracil-rich sequence tends to be down-regulated compared to those without the sequence. Among them, Northern blotting shows that miR-29c and fruit fly bantam possess a relatively rapid turnover rate. The effect of uracil-rich sequence on microRNA turnover depends on the sequence context. The present work indicates that microRNAs contain sequence information in the middle region besides the sequence element at both ends.