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.     

Mitochondrial reactive oxygen species originating from Romo1 exert an important role in normal cell cycle progression by regulating p27Kip1 expression

Reactive oxygen species (ROS) steady-state levels are required for entry into the S phase of the cell cycle in normal cells, as well as in tumour cells. However, the contribution of mitochondrial ROS to normal cell proliferation has not been well investigated thus far. A previous report showed that Romo1 was responsible for the high ROS levels in tumour cells. Here, we show that endogenous ROS generated by Romo1 are indispensable for cell cycle transition from G1 to S phase in normal WI-38 human lung fibroblasts. The ROS level in these cells was down-regulated by Romo1 knockdown, resulting in cell cycle arrest in the G1 phase. This arrest was associated with an increase in the level of p27^Kip1. These results demonstrate that mitochondrial ROS generated by Romo1 expression is required for normal cell proliferation and it is suggested that Romo1 plays an important role in redox signalling during normal cell proliferation.     

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.     

Novel cytotoxic exhibition mode of antimicrobial peptide anoplin in MEL cells,the cell line of murine Friend leukemia virus‐induced leukemic cells

Anoplin is a recently discovered antimicrobial peptide (AMP) isolated from the venom sac of the spider wasp Anoplius samariensis, and it is one of the shortest α‐helical AMP found naturally to date consisting of only ten amino acids. Previous results showed that anoplin exhibits potent antimicrobial activity but little hemolytic activity. In this study, we synthesized anoplin, studied its cytotoxicity in Friend virus‐induced leukemia cells [murine erythroleukemia (MEL) cells], and proposed its possible mechanism. Our results showed that anoplin could inhibit the proliferation of MEL cells in a dose‐dependent and time‐dependent manner via disrupting the integrity of cell membrane, which indicated that anoplin exerts its cytotoxicity efficacy. In addition, the cell cycle distribution of MEL cells was arrested in the G₀/G₁ phase significantly. However, anoplin could not induce obvious apoptosis in MEL cells, as well as anoplin could not induce visible changes on morphology and quantity in the bone marrow cells isolated from normal mice. All of these results indicate that anoplin, as generally believed, is a selective AMP, a value characteristic in the design of safe therapeutic agents. The cytotoxicity of anoplin on MEL cells was mainly attributable to the plasma membrane perturbation and also to the intracellular events such as the arrest of cell cycle. Although this is an initial study that explored the activity of anoplin in vitro rather than in vivo, with the increasing resistance of conventional chemotherapy, there is no doubt that anoplin has desirable feature to be developed as a novel and selective anticancer agent. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Inhibition of protein tyrosine phosphatase 1B by lupeol and lupenone isolated from Sorbus commixta

Protein tyrosine phosphatase 1B (PTP1B) appears to be an attractive target for the development of new drugs for type 2 diabetes and obesity. In our preliminary test, a MeOH extract of the stem barks of Sorbus commixta Hedl. (Rosaceae) showed strong PTP1B inhibitory activity. Bioassay?guided fractionation of the MeOH extract resulted in the isolation of two lupane?type triterpenes, lupenone (1) and lupeol (2). Compounds 1 and 2 inhibited PTP1B with IC₅₀ values of 13.7 ± 2.1 and 5.6 ± 0.9 μM, respectively. Kinetic studies revealed that both the compounds 1 and 2 are non?competitive inhibitors of PTP1B that decrease V_max values with no effect on K_m values.     

WormFarm: a quantitative control and measurement device toward automated Caenorhabditis elegans aging analysis

Caenorhabditis elegans is a leading model organism for studying the basic mechanisms of aging. Progress has been limited, however, by the lack of an automated system for quantitative analysis of longevity and mean lifespan. To address this barrier, we developed ‘WormFarm’, an integrated microfluidic device for culturing nematodes. Cohorts of 30–50 animals are maintained throughout their lifespan in each of eight separate chambers on a single WormFarm polydimethylsiloxane chip. Design features allow for automated removal of progeny and efficient control of environmental conditions. In addition, we have developed computational algorithms for automated analysis of video footage to quantitate survival and other phenotypes, such as body size and motility. As proof‐of‐principle, we show here that WormFarm successfully recapitulates survival data obtained from a standard plate‐based assay for both RNAi‐mediated and dietary‐induced changes in lifespan. Further, using a fluorescent reporter in conjunction with WormFarm, we report an age‐associated decrease in fluorescent intensity of GFP in transgenic worms expressing GFP tagged with a mitochondrial import signal under the control of the myo‐3 promoter. This marker may therefore serve as a useful biomarker of biological age and aging rate.     

Synthesis and interaction of bovine serum albumin with p‐hydroxybenzoic acid derivatives

Three novel p‐hydroxybenzoic acid derivatives (HSOP, HSOX, HSCP) were synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfamonomethoxine sodium, sulfamethoxazole and sulfachloropyridazine sodium) and characterized by elemental analysis, HNMR and MS. Interactions between derivatives and bovine serum albumin (BSA) were studied by fluorescence quenching spectra, UV–vis absorption spectra and time‐resolved fluorescence spectra. Based on fluorescence quenching calculation and Förster's non‐radioactive energy transfer theory, the values of the binding constants, basic thermodynamic parameters and binding distances were obtained. Experimental results indicated that the three derivatives had a strong ability to quench fluorescence from BSA and that the binding reactions of the derivatives with BSA were a static quenching process. Thermodynamic parameters showed that binding reactions were spontaneous and exothermic and hydrogen bond and van der Waals force were predominant intermolecular forces between the derivatives and BSA. Synchronous fluorescence spectra suggested that HSOX and HSCP had little effect on the microenvironment and conformation of BSA in the binding reactions but the microenvironments around tyrosine residues were disturbed and polarity around tyrosine residues increased in the presence of HSOP. Copyright © 2012 John Wiley & Sons, Ltd.     

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.