Biochemical properties of a native β-1,4-mannanase from Aspergillus aculeatus QH1 and partial characterization of its N-glycosylation

N-glycosylation plays critical roles in protein secretion, sorting, stability, activity modulation, and interactions to other molecules in the eukaryotic organisms. Fungal β-1,4-mannanases have been widely used in the agri-food industry and contribute to the pathogenesis on plants. However, the information on N-glycosylation of a specific fungal carbohydrate-active enzyme (CAZyme) is currently limited. Herein, a cDNA was cloned from Aspergillus aculeatus QH1, displaying a full length of 1302 bp with an open reading frame of 1134 bp encoding for a GH5 subfamily 7 β-1, 4-mannanase, namely AacMan5_7A. The enzyme was purified and exhibited an optimal activity at pH 4.6 and 60 °C, hydrolyzing glucomannan and galactomannan, but not yeast mannan. AacMan5_7A is an N-glycosylated protein decorated with a high-mannose type glycan. Further through UPLC-ESI-MS/MS analysis, one of the four predicted N-glycosylation sites at N255 position was experimentally verified. The present study expands the information of N-glycosylation in fungal CAZymes, providing scientific bases for enhancing the production of fungal enzymes and their applications in food, feed, and plant biomass conversions.     

Bone morphogenetic protein 9 overexpression reduces osteosarcoma cell migration and invasion

Transforming growth factor-β (TGF-β) is known to promote tumor migration and invasion. Bone morphogenetic proteins (BMPs) are members of the TGF-β family expressed in a variety of human carcinoma cell lines. The role of bone morphogenetic protein 9 (BMP9), the most powerful osteogenic factor, in osteosarcoma (OS) progression has not been fully clarified. The expression of BMP9 and its receptors in OS cell lines was analyzed by RT-PCR. We found that BMP9 and its receptors were expressed in OS cell lines. We further investigated the influence of BMP9 on the biological behaviors of OS cells. BMP9 overexpression in the OS cell lines 143B and MG63 inhibited in vitro cell migration and invasion. We further investigated the expression of a panel of cancer-related genes and found that BMP9 overexpression increased the phosphorylation of Smad1/5/8 proteins, increased the expression of ID1, and reduced the expression and activity of matrix metalloproteinase 9 (MMP9) in OS cells. BMP9 silencing induced the opposite effects. We also found that BMP9 may not affect the chemokine (C-X-C motif) ligand 12 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis to regulate the invasiveness and metastatic capacity of OS cells. Interestingly, CXCR4 was expressed in both 143B and MG63 cells, while CXCL12 was only detected in MG63 cells. Taken together, we hypothesize that BMP9 inhibits the migration and invasiveness of OS cells through a Smad-dependent pathway by downregulating the expression and activity of MMP9.     

Morphogenesis and Molecular Phylogeny of a New Freshwater Ciliate,Notohymena apoaustralis n. sp. (Ciliophora,Oxytrichidae)

The live morphology, infraciliature, and morphogenesis of a new oxytrichid ciliate, Notohymena apoaustralis n. sp. collected from a freshwater pond in Qingdao (Tsingtao), China, were studied in vivo and after protargol impregnation. Notohymena apoaustralis n. sp. is characterized as follows: undulating membranes in Notohymena‐pattern; cortical granules yellow‐green, grouped around the marginal cirri and dorsal bristles, and in short irregular rows elsewhere in the cell; single contractile vacuole positioned at anterior 1/3 of the body length; two macronuclear nodules and one micronucleus; about 39 adoral membranelles; 18 frontoventral transverse cirri in typical Oxytricha‐pattern; one right and one left marginal row, almost confluent posteriorly; dorsal ciliature in typical Oxytricha‐pattern; 8–10 caudal cirri arranged in three rows, one each at the posterior end of dorsal kineties 1, 2, and 4, indistinguishable from marginal cirri in life. The morphogenetic process in N. apoaustralis n. sp. is consistent with that of the type species, Notohymena rubescens Blatterer and Foissner, 1988. Phylogenetic analyses based on small subunit rDNA sequence data suggest a sister relationship between N. apoaustralis n. sp. and Paraurostyla weissei, which cluster in a clade with Rubrioxytricha ferruginea.     

The primary culture of mirror carp snout and caudal fin tissues and the isolation of Koi herpesvirus

The explosive Koi herpesvirus (KHV) epidemic has caused the deaths of a large number of carp and carp variants and has produced serious economic losses. The mirror carp (Cyprinus carpio var. specularis) exhibits strong environmental adaptability and its primary cells can be used to isolate KHV. This study utilized the tissue explant method to systematically investigate primary cell culture conditions for mirror carp snout and caudal fin tissues. We demonstrated that cells from these two tissue types had strong adaptability, and when cultured in Medium 199 (M199) containing 20% serum at 26 to 30°C, the cells from the snout and caudal fin tissues exhibited the fastest egress and proliferation. Inoculation of these two cell types with KHV-infected fish kidney tissues produced typical cytopathic effects; additionally, identification by electron microscopy, and PCR indicated that KHV could be isolated from both cell types.     

Sonochemical synthesis of Ag nanoclusters: electrogenerated chemiluminescence determination of dopamine

We report a facile one‐pot sonochemical approach to preparing highly water‐soluble Ag nanoclusters (NCs) using bovine serum albumin as a stabilizing agent and reducing agent in aqueous solution. Intensive electrogenerated chemiluminescence (ECL) was observed from the as‐prepared Ag (NCs) and successfully applied for the ECL detection of dopamine with high sensitivity and a wide detection range. A possible ECL mechanism is proposed for the preparation of Ag NCs. With this method, the dopamine concentration was determined in the range of 8.3 × 10^–9 to 8.3 × 10^–7 mol/L without the obvious interference of uric acid, ascorbic acid and some other neurotransmitters, such as serotonin, epinephrine and norepinephrine, and the detection limit was 9.2 × 10^–10 mol/L at a signal/noise ratio of 3. Copyright © 2013 John Wiley & Sons, Ltd.     

Activated Cyclin-Dependent Kinase 5 Promotes Microglial Phagocytosis of Fibrillar β-Amyloid by Up-regulating Lipoprotein Lipase Expression

Amyloid plaques are crucial for the pathogenesis of Alzheimer disease (AD). Phagocytosis of fibrillar β-amyloid (Aβ) by activated microglia is essential for Aβ clearance in Alzheimer disease. However, the mechanism underlying Aβ clearance in the microglia remains unclear. In this study, we performed stable isotope labeling of amino acids in cultured cells for quantitative proteomics analysis to determine the changes in protein expression in BV2 microglia treated with or without Aβ. Among 2742 proteins identified, six were significantly up-regulated and seven were down-regulated by Aβ treatment. Bioinformatic analysis revealed strong over-representation of membrane proteins, including lipoprotein lipase (LPL), among proteins regulated by the Aβ stimulus. We verified that LPL expression increased at both mRNA and protein levels in response to Aβ treatment in BV2 microglia and primary microglial cells. Silencing of LPL reduced microglial phagocytosis of Aβ, but did not affect degradation of internalized Aβ. Importantly, we found that enhanced cyclin-dependent kinase 5 (CDK5) activity by increasing p35-to-p25 conversion contributed to LPL up-regulation and promoted Aβ phagocytosis in microglia, whereas inhibition of CDK5 reduced LPL expression and Aβ internalization. Furthermore, Aβ plaques was increased with reducing p25 and LPL level in APP/PS1 mouse brains, suggesting that CDK5/p25 signaling plays a crucial role in microglial phagocytosis of Aβ. In summary, our findings reveal a potential role of the CDK5/p25-LPL signaling pathway in Aβ phagocytosis by microglia and provide a new insight into the molecular pathogenesis of Alzheimer disease.Alzheimer disease (AD)¹ is one of the most common neurodegenerative disorders, which is characterized by pathological hallmarks such as neuronal and synaptic loss, neurofibrillary tangles (NFTs), and senile plaques. The intracellular NFTs are mainly composed of hyper-phosphorylated microtubule-associated protein tau, whereas toxic fibrillar β-amyloid (fAβ) as the main component of senile plaques is generated by sequential proteolytic cleavage of trans-membrane β-amyloid precursor protein (APP) by β- and γ-secretases. fAβ can induce oxidative stress-mediated neuronal cell death and cause cognitive impairment in mouse brains (1). Many reports suggest that fAβ induces dysregulation of two pivotal kinases CDK5 (2, 3) and GSK-3 (4), which are crucial regulators of hyperphosphorylated tau and increased production of Aβ from APP, and thereby triggers the cascade of signal transduction events underlying neuronal cell death in AD pathogenesis.As the resident immune cells in the brain, microglia can be activated in response to fAβ and often accumulate around the amyloid deposits in the brains of AD patients. Activated microglia trigger the production of inflammatory factors, reactive oxygen species, and chemokines, which may cause neuronal cell death (5). Furthermore, increasing evidence supports that activated microglia exert a vital beneficial role in the clearance of Aβ by phagocytosis. Many receptors, including scavenger receptor A (SR-A) (6), scavenger receptor class B type I (SR-BI) (7), lipopolysaccharide receptor (CD14) (8), CD33 (9), B-class scavenger receptor CD36 (10), CD47 (11), β1 integrin (12), toll-like receptor 2 (TLR2) (13), and toll-like receptor 4 (TLR4) (14), have been implicated in microglial phagocytosis of fAβ via direct or indirect binding to Aβ. Microglial phagocytosis of fAβ is also regulated by proinflammatory cytokines (15) and chemokine receptor CX3CR1 (16). Farfara et al. reported that the γ-secretase component presenilin, which is responsible for APP cleavage and Aβ production in neurons, is important for microglial fAβ clearance, indicating a dual role for presenilin in neuronal cell death and microglial phagocytosis (17). In addition, accumulating evidence suggests a critical role of lipids and lipoproteins in microglial fAβ phagocytosis and clearance. Lee et al. reported that apolipoprotein E (ApoE) enhances fAβ trafficking and degradation, indicating a role of cholesterol in fAβ degradation (18). After internalization, fAβ is degraded through the lysosome pathway (19, 20). However, the mechanism underlying microglial internalization of fAβ remains unclear.Stable isotope labeling of amino acids in cell culture (SILAC) is an accurate and reproducible mass spectrometry-based quantitative proteomics approach for examining changes in protein expression or post-translational modifications at a large scale (21, 22). Here, we used the SILAC quantitative proteomics strategy to investigate changes in the protein levels in BV2 microglia treated with fAβ. We found that 6 proteins were up-regulated and 7 were down-regulated significantly by Aβ treatment. Interestingly, bioinformatic analysis revealed that most of these up- or down-regulated proteins, including lipoprotein lipase (LPL), were mainly distributed in the cell membrane. We verified that LPL was up-regulated at both gene and protein levels in BV2 and primary microglia in response to fAβ, thereby indicating its role in the microglial phagocytosis of Aβ. Importantly, we further demonstrated that CDK5, which is a critical serine/threonine kinase in the pathogenesis of AD, regulated the expression of LPL and played a critical role in Aβ phagocytosis of microglia. Moreover, we found that increase in the p35-to-p25 conversion contributed to the enhanced CDK5 activity under Aβ stimulus and played a vital role in regulation of LPL expression and microglial Aβ phagocytosis. Our results suggest a role of the CDK5/p25-LPL signaling pathway in Aβ phagocytosis of microglia and provide valuable information to understand the molecular mechanism underlying microglial fAβ phagocytosis.     

Inhibition of hydrogen sulfide synthesis provides protection for severe acute pancreatitis rats via apoptosis pathway

We aimed to investigate the relationship between the synthesis of hydrogen sulfide (H₂S) and the pancreatic acinar cell apoptosis in severe acute pancreatitis (SAP) rats, as well as analyse the potential apoptotic pathway involved in this process. Sixty rats had been equally divided into four groups: sham, SAP, SAP + sodium hydrosulfide (NaHS) and SAP + DL-propargylglycine (PAG). 24 h after SAP induction, all surviving animals of each group were sacrificed to collect blood and tissue samples for the following measurements: the level of serum H₂S as well as the levels of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), H₂S synthesizing activity, CSE mRNA and protein expression, maleic dialdehyde (MDA) and myeloperoxidase (MPO) activity, the expression of Bax, Bcl-2, caspase-3, -8 and -9, the release of cytochrome c and the activation of nuclear factor-kappa B (NF-κB), ERK1/2, JNK1/2 and p38 in pancreas. Furthermore, in situ detection of cell apoptosis was examined and the severity of pancreatic damage was analyzed by pathological grading and scoring. Results Significant differences in every index except IL-10 had been found between the SAP, NaHS and PAG groups (P < 0.05). Treatment with PAG obviously induced the pancreatic acinar cell apoptosis as well as improved all the pathological changes and inflammatory parameters. In contrast, administration of NaHS significantly attenuated apoptosis in the pancreas and aggravated the severity of pancreatic damage. Moreover, the expressions of caspase-3, -8, -9 and the release of cytochrome c were all increased in the apoptotic cells, and the activity of NF-κB as well as the phosphorylation of ERK1/2, JNK1/2 and p38 decreased accompanying with the reduction of the serum H₂S level. H₂S plays a pivotal role in the regulation of pancreatic acinar cell apoptosis in SAP rats. The present results showed that inhibition of H₂S synthesis provided protection for SAP rats via inducing acinar cell apoptosis. This process acted through both extrinsic and intrinsic apoptotic pathways, and may be regulated by reducing the activity of NF-κB.     

Development of InDel markers linked to Fusarium wilt resistance in cabbage

Cabbage Fusarium wilt (CFW) is a destructive disease causing great losses to cabbage (Brassica oleracea L. var. capitata L.) production worldwide. At present, there are few reports concerning molecular marker research on cabbage resistance to CFW. In this study, 160 double haploid (DH) lines were obtained from the F1 population of a 99–77 (highly resistant to CFW) × 99–91 (highly susceptible to CFW) cross. Insertion–deletion (InDel) markers were designed according to the reference genome sequence of cabbage and the whole-genome re-sequencing data of the two parents. A genetic map of chromosome C06 including seven InDel markers was constructed based on the DH population. Thus, FOC (resistance gene to Fusarium oxysporum f. sp. conglutinans) was located on chromosome C06 and two InDel markers out of the seven, M10 and A1, flanked the gene at 1.2 and 0.6 cM, respectively. Marker A1 revealed a significant consistency with the phenotype assay in the F2 population as well as in 40 inbred lines (96 and 82 %, respectively). This study lays the foundation for fine mapping and cloning of the FOC gene and for marker-assisted selection in cabbage resistance breeding.     

Isolation and Quantification of MicroRNAs from Urinary Exosomes/Microvesicles for Biomarker Discovery

Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs in patients with various kidney diseases was validated with real-time PCR. The result shows that miRNAs extracted from the exosomal fraction were resistant to RNase digestion and with high quality confirmed by agarose electrophoresis. 16ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×10⁵ copies/ul for miR-200c. Exosomes was stable at 4℃ 24h for shipping before stored at -80℃ and was stable in urine when stored at -80°C for 12months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The detection of miRNA by quantitative PCR showed high reproducibility (>94% for intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for CKD patients), broad dynamic range (8-log wide) and good linearity for quantification (R²>0.99). miR-29c and miR-200c showed different expression in different types of kidney disease. In summary, the presence of urinary exosomal miRNA was confirmed for patients with a diversity of chronic kidney disease. The conditions of urine collection, storage and miRNA detection determined in this study may be useful for future biomarker discovery efforts.