Signaling, Actin Dynamics and Endocytosis

１ＲｅｇｕｌａｔｉｏｎｏｆａｃｔｉｎｏｒｇａｎｉｚａｔｉｏｎｂｙｓｉｇｎａｌｉｎｇＴｈｅａｃｔｉｎｃｙｔｏｓｋｅｌｅｔｏｎｒｅｏｒｇａｎｉｚｅｓｉｔｓｓｔｒｕｃｔｕｒｅｔｈｒｏｕｇｈｒａｐｉｄｌｙｐｏｌｙｍｅｒｉｚａｔｉｏｎ，ｄｅｐｏｌｙｍｅｒｉｚａｔｉｏｎ，ｂｒａｎｃｈｅｄｎｕｃｌｅａｔｉｏｎ，ａｎｄｆｏｒｍａｔｉｏｎｏｆｂｕｎｄｌｅｓｆｏｒｄｉｆｆｅｒｅｎｔｃｅｌｌｕｌａｒａｃｔｉｖｉｔｉｅｓ．Ｒｈｏ -ｆａｍｉｌｙＧＴＰａｓｅｓｐｌａｙｅｓｓｅｎｔｉａｌｒｏｌｅｉｎｃｏｏｒｄｉｎａｔｉｎｇａ…     

The Endocytosis of Cellulose Synthase in Arabidopsis Is Dependent on μ2, a Clathrin-Mediated Endocytosis Adaptin

Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells. Plants appear to possess all of the molecular components necessary to carry out CME; however, functional characterization of the components is still in its infancy. A yeast two-hybrid screen identified μ2 as a putative interaction partner of CELLULOSE SYNTHASE6 (CESA6). Arabidopsis (Arabidopsis thaliana) μ2 is homologous to the medium subunit 2 of the mammalian ADAPTOR PROTEIN COMPLEX2 (AP2). In mammals, the AP2 complex acts as the central hub of CME by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis. We confirmed that μ2 interacts with multiple CESA proteins through the μ-homology domain of μ2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent with its role in mediating the endocytosis of cargos at the plasma membrane, μ2-YELLOW FLUORESCENT PROTEIN localized to transient foci at the plasma membrane, and loss of μ2 resulted in defects in bulk endocytosis. Furthermore, loss of μ2 led to increased accumulation of YELLOW FLUORESCENT PROTEIN-CESA6 particles at the plasma membrane. Our results suggest that CESA represents a new class of CME cargo proteins and that plant cells might regulate cellulose synthesis by controlling the abundance of active CESA complexes at the plasma membrane through CME.Cellulose microfibrils, as the major load-bearing polymers in plant cell walls, are the predominant component that enforces asymmetric cell expansion (Green, 1962). In higher plants, cellulose is synthesized by multimeric rosettes, which are also referred to as cellulose synthase complexes (CSCs; Kimura et al., 1999). Genetic and coimmunoprecipitation studies have indicated that CELLULOSE SYNTHASE1 (CESA1), CESA3, and CESA6-like (CESA6, CESA2, CESA5, and CESA9) isoforms are constituents of CSCs during primary cell wall synthesis (Persson et al., 2005; Desprez et al., 2007; Persson et al., 2007; Wang et al., 2008), whereas CESA4, CESA7, and CESA8 are implicated in the cellulose synthesis of secondary cell walls (Taylor et al., 1999, 2003; Brown et al., 2005). Knowledge about cellulose synthesis has recently been enhanced by the development of a system whereby the dynamics of CESA can be imaged in living cells (Paredez et al., 2006; Desprez et al., 2007). In agreement with earlier transmission electron microscopy studies in which rosettes were visualized in Golgi cisternae, vesicles, and at the plasma membrane (Haigler and Brown, 1986), fluorescent protein tagging of CESA has identified CESA localization at the plasma membrane, in Golgi bodies, and in small intracellular compartments (Paredez et al., 2006; Desprez et al., 2007; Crowell et al., 2009; Gutierrez et al., 2009; Gu et al., 2010; Lei et al., 2012; Li et al., 2012b).Assuming that cellulose synthesis occurs solely at the plasma membrane, the trafficking of CSCs to and from the plasma membrane may act as a significant regulatory mechanism. Although the mechanistic details of CESA trafficking are lacking, live cell imaging has shown that CESA localizes to various subcellular compartments. A subset of CESAs colocalize with markers of the trans-Golgi network (TGN)/early endosome (EE), an organelle that is part of both the secretory and endocytic pathways in Arabidopsis (Arabidopsis thaliana; Dettmer et al., 2006; Lam et al., 2007; Crowell et al., 2009, 2010; Viotti et al., 2010). CESAs also localize to microtubule-associated cellulose synthase compartments (MASCs) and small CESA-containing compartments (SmaCCs). The exact function of SmaCCs/MASCs is unknown, but it has been proposed that SmaCCs/MASCs might result from the internalization of CSCs or might act in the delivery of CSCs to the plasma membrane (Crowell et al., 2009, 2010; Gutierrez et al., 2009).Clathrin-mediated endocytosis (CME) has been shown to be a major endocytic pathway in Arabidopsis (Holstein, 2002; Samaj et al., 2005; Dhonukshe et al., 2007; Kleine-Vehn and Friml, 2008; Chen et al., 2011; Beck et al., 2012; Wang et al., 2013), although there is also evidence of clathrin-independent endocytosis mechanisms (Bandmann and Homann, 2012). The function of many CME proteins has been extensively characterized in mammals (McMahon and Boucrot, 2011), and homologs of many CME components are encoded by the Arabidopsis genome, including multiple copies of clathrin H chain and clathrin light chain (CLC), all four subunits of the heterotetrameric ADAPTOR PROTEIN COMPLEX2 (AP2) complex, dynamin-related proteins, and accessory proteins such as AP180 (Holstein, 2002; Chen et al., 2011); however, many CME components have yet to be characterized in plants.It has been suggested that CME might also function in controlling cell wall metabolism. For example, dividing and growing cells internalize cross-linked cell wall pectins, which might allow for cell wall remodeling (Baluska et al., 2002, 2005; Samaj et al., 2004). Moreover, the importance of endocytosis for cell wall morphogenesis is apparent from the functional characterization of proteins involved in CME. A dynamin-related protein, DRP1A, plays a significant role in endocytosis and colocalizes with CLC (Collings et al., 2008; Konopka and Bednarek, 2008). Defective endocytosis in RADIAL SWELLING9 (rsw9) plants, which contain a mutation in DRP1A, results in cellulose deficiency and defects in cell elongation (Collings et al., 2008). A mutation in rice, brittle culm3 (bc3), was mapped to the dynamin-related gene OsDRP2A, which has been proposed to function in CME. The brittle-culm phenotype in this mutant was attributed to cellulose deficiency (Xiong et al., 2010). Although the abundance of OsCESA4 was also altered in bc3, it remains unclear whether the cellulose deficiency of either bc3 or rsw9 results directly from perturbations in CESA trafficking.To identify proteins involved in the regulation of cellulose biosynthesis, a yeast two-hybrid (Y2H) screen was performed in which the central domain of CESA6 (CESA6CD) was used as bait to screen an Arabidopsis complementary DNA library for potential interaction partners of CESA6 (Gu et al., 2010; Gu and Somerville, 2010). The Y2H screen identified μ2 as a putative interaction partner of CESA6CD. The mammalian homolog of μ2 is the medium subunit of the tetrameric AP2, which acts as the core of the CME machinery by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis (Jackson et al., 2010; McMahon and Boucrot, 2011; Cocucci et al., 2012). In this study, we provide evidence that μ2 plays a role in CME in Arabidopsis, that CESAs are a new set of CME cargo proteins, and that plant cells might regulate cellulose synthesis by controlling the abundance of CSCs at the plasma membrane through CME. To our knowledge, this study is the first to show the affect of an AP2 complex component on endocytosis in plants and the first to visualize an AP2 complex component in living plant cells. Furthermore, our data suggest that the role of AP2 in plants may differ from what has been shown in animals.     

Endocytosis: why not wait to deubiquitinate?

Deubiquitination by the Fat facets protein - a regulator of photoreceptor differentiation during Drosophila eye development - has been found to activate endocytosis, while ubiquitination inhibits endocytosis. Surprisingly, this is the opposite effect that ubiquitination has on endocytosis of many plasma membrane proteins.     

A Hyaluronan Receptor for Endocytosis (HARE) Link Domain N-Glycan Is Required for Extracellular Signal-regulated Kinase (ERK) and Nuclear Factor-κB (NF-κB) Signaling in Response to the Uptake of Hyaluronan but Not Heparin,Dermatan Sulfate,or Acetylated Low Density Lipoprotein (LDL)

The human hyaluronan (HA) receptor for endocytosis (HARE; the 190-kDa C terminus of Stab2) is a major clearance receptor for multiple circulating ligands including HA, heparin (Hep), acetylated LDL (AcLDL), dermatan sulfate (DS), apoptotic debris, and chondroitin sulfate types A, C, D, and E. We previously found that HARE contains an N-glycan in the HA binding Link domain (at Asn²²⁸⁰), and cells expressing membrane-bound HARE(N2280A) bind and endocytose HA normally (Harris, E. N., Parry, S., Sutton-Smith, M., Pandey, M. S., Panico, M., Morris, H. R., Haslam, S. M., Dell, A., and Weigel, P. H. (2010) Glycobiology 20, 991–1001). Also, NF-κB-mediated signaling is activated by HARE-mediated endocytosis of HA, Hep, AcLDL, or DS but not by chondroitin sulfates (Pandey, M. S., and Weigel, P. H. (2014) J. Biol. Chem. 289, 1756–1767). Here we investigated the role of Link N-glycans in ligand uptake and NF-κB and ERK1/2 signaling. HA·HARE-mediated ERK1/2 activation was HA size- dependent, as found for NF-κB activation. HARE(N2280A) cells internalized HA, Hep, AcLDL, and DS normally. No ERK1/2 activation occurred during HA endocytosis by HARE(N2280A) cells, but activation did occur with Hep. Dual-luciferase recorder assays showed that NF-κB-mediated gene expression occurred normally in HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but did not occur with HA. Activation of NF-κB by endogenous degradation of IκB-α was observed for HARE(N2280A) cells endocytosing Hep, AcLDL, or DS but not HA. We conclude that a Link domain complex N-glycan is required specifically for HARE·HA-mediated activation of ERK1/2 and NF-κB-mediated gene expression and that this initial activation mechanism is different from and independent of the initial mechanisms for HARE-mediated signaling in response to Hep, AcLDL, or DS uptake.     

Pancreatic α-Amylase Controls Glucose Assimilation by Duodenal Retrieval through N-Glycan-specific Binding,Endocytosis, and Degradation

α-Amylase, a major pancreatic protein and starch hydrolase, is essential for energy acquisition. Mammalian pancreatic α-amylase binds specifically to glycoprotein N-glycans in the brush-border membrane to activate starch digestion, whereas it significantly inhibits glucose uptake by Na⁺/glucose cotransporter 1 (SGLT1) at high concentrations (Asanuma-Date, K., Hirano, Y., Le, N., Sano, K., Kawasaki, N., Hashii, N., Hiruta, Y., Nakayama, K., Umemura, M., Ishikawa, K., Sakagami, H., and Ogawa, H. (2012) Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane. J. Biol. Chem. 287, 23104–23118). However, how the inhibition is stopped was unknown. Here, we show a new mechanism for the regulation of intestinal glucose absorption. Immunohistochemistry revealed that α-amylase in the duodena of non-fasted, but not fasted, pigs was internalized from the pancreatic fluid and immunostained. We demonstrated that after N-glycan binding, pancreatic α-amylase underwent internalization into lysosomes in a process that was inhibited by α-mannoside. The internalized α-amylase was degraded, showing low enzymatic activity and molecular weight at the basolateral membrane. In a human intestinal Caco-2 cell line, Alexa Fluor 488-labeled pancreatic α-amylase bound to the cytomembrane was transported to lysosomes through the endocytic pathway and then disappeared, suggesting degradation. Our findings indicate that N-glycan recognition by α-amylase protects enterocytes against a sudden increase in glucose concentration and restores glucose uptake by gradual internalization, which homeostatically controls the postprandial blood glucose level. The internalization of α-amylase may also enhance the supply of amino acids required for the high turnover of small intestine epithelial cells. This study provides novel and significant insights into the control of blood sugar during the absorption stage in the intestine.     

Activity-Dependent Bulk Synaptic Vesicle Endocytosis—A Fast,High Capacity Membrane Retrieval Mechanism

Central nerve terminals are placed under considerable stress during intense stimulation due to large numbers of synaptic vesicles (SVs) fusing with the plasma membrane. Classical clathrin-dependent SV endocytosis cannot correct for the large increase in nerve terminal surface area in the short term, due to its slow kinetics and low capacity. During such intense stimulation, an additional SV retrieval pathway is recruited called bulk endocytosis. Recent studies have shown that bulk endocytosis fulfils all of the physiological requirements to remedy the acute changes in nerve terminal surface area to allow the nerve terminal to continue to function. This review will summarise the recent developments in the field that characterise the physiology of bulk endocytosis which show that it is a fast, activity-dependent and high capacity mechanism that is essential for the function of central nerve terminals.     

丙酸睾酮对鸡的法氏囊表面上皮内吞作用(Endocytosis)的影响

在莱杭雏鸡和仔鸡的肛唇上进行胶质碳处理,证明法氏囊附着滤泡上皮具有内吞作用。被内吞的碳粒24小时以后,主要存在于淋巴滤泡髓部的星状细胞及胞间隙中。在法氏囊发育的不同时期,用丙酸睾酮处理,均可损害附着滤泡上皮细胞。它们的亚微结构发生了变化,同时正常的内吞作用也受到明显地抑制。特别是用高剂量的丙酸睾酮处理以后,附着滤泡上皮细胞的内吞作用受到完全抑制。此外也揭示了丙酸睾酮处理以后,法氏囊淋巴滤泡表面的形态变化与法氏囊自然退化的情形大致相似。进一步证明法氏囊的自然退化与体内性激素浓度升高有关。     

Inhibition of Swarming of Proteus by Sodium Tetradecyl Sulfate, β-Phenethyl Alcohol,and p-Nitrophenylglycerine

The effects of sodium tetradecyl sulfate (STS), β-phenethyl alcohol (PEA), and p-nitrophenylglycerol (PNPG) on motility, swarming, flagellation, and growth of Proteus were examined. Growth-inhibitory concentrations (GIC) and swarming-inhibitory concentrations (SIC) were determined. A characterization of the swarming-inhibitory efficacy of these compounds was based on their GIC/SIC ratio and their concentration inhibition curves. Using the homologous series of sodium alkyl sulfates as a standard reference, we showed that PNPG was more effective than STS, which was the most effective of the homologous series. PEA was less effective than sodium decyl sulfate but more effective than sodium octyl sulfate. Motility tests in liquid medium and electron microscope investigations indicated that the modes of action of the three compounds, all of which effectively inhibit the swarming of Proteus, are different. Whereas STS and PEA inhibit swarming by inhibition of motility, PNPG seems to act on the swarming mechanism sensu strictori, without impairment of motility. STS immobilizes by inhibition of flagellum formation or by some lytic action on the flagella already synthesized. PEA acts by impairing flagellar function, but leaves the flagella morphologically intact.     

Endocytosis and Autophagy: Exploitation or Cooperation?

Autophagy is a lysosome-mediated degradative system that is a highly conserved pathway present in all eukaryotes. In all cells, double-membrane autophagosomes form and engulf cytoplasmic components, delivering them to the lysosome for degradation. Autophagy is essential for cell health and can be activated to function as a recycling pathway in the absence of nutrients or as a quality-control pathway to eliminate damaged organelles or even to eliminate invading pathogens. Autophagy was first identified as a pathway in mammalian cells using morphological techniques, but the Atg (autophagy-related) genes required for autophagy were identified in yeast genetic screens. Despite tremendous advances in elucidating the function of individual Atg proteins, our knowledge of how autophagosomes form and subsequently interact with the endosomal pathway has lagged behind. Recent progress toward understanding where and how both the endocytotic and autophagic pathways overlap is reviewed here.Autophagy is a lysosome-mediated pathway for the degradation of cytosolic proteins and organelles, which is essential for cell homeostasis, development, and for the prevention of several human diseases and infection (Choi et al. 2013). Importantly, autophagy cannot occur without an active lysosome. However, it is becoming increasingly recognized that the endosomal pathway plays a greater role than just providing the degradative enzymes found in the lysosome. Recent data suggest that in mammalian cells multiple contributions from several stages of the endocytic pathway are essential for efficient autophagy. Here we outline the autophagic pathway and then address the recent data on how different endosomal compartments contribute to autophagy, and the molecular machinery required for the interaction of the endosome and lysosome during the formation, and consumption of the autophagosome. Given the model emerging that the amino-acid-sensitive autophagic pathway originates from the endoplasmic reticulum (ER), several questions arise, including how do recognition and productive interaction occur between an ER-derived membrane and endosomes? How are these interactions mediated, and which are essential for efficient autophagy?     

Endocytosis of KATP Channels Drives Glucose-Stimulated Excitation of Pancreatic β Cells

1. Download high-res image (129KB)
2. Download full-size image

     

Unconventional Myosins,Actin Dynamics and Endocytosis: A Ménage à Trois?

Ever since the discovery of class I myosins, the first nonmuscle myosins, about 30 years ago, the history of unconventional myosins has been linked to the organization and working of actin filaments. It slowly emerged from studies of class I myosins in lower eukaryotes that they are involved in mechanisms of endocytosis. Most interestingly, a flurry of recent findings assign a more active role to class I myosins in regulating the spatial and temporal organization of actin filament nucleation and elongation. The results highlight the multiple links between class I myosins and the major actin nucleator, the Arp2/3 complex, and its newly described activators. Two additional types of unconventional myosins, myosinIX, and Dictyostelium discoideum MyoM, have recently been tied to the signaling pathways controlling actin cytoskeleton remodeling. The present review surveys the links between these three classes of molecular motors and the complex cellular processes of endocytosis and actin dynamics, and concentrates on a working model accounting for the function of class I myosins via recruitment of the machinery responsible for actin nucleation and elongation .     

β 2 -Glycoprotein I (Apolipoprotein H) Modulates Uptake and Endocytosis Associated Chemiluminescence in Rat Kupffer Cells

&#103 ₂-Glycoprotein I (&#103 ₂GPI) is known to influence macrophage uptake of particles with phosphatidylserine containing surfaces, as apoptotic thymocytes and unilamellar vesicles in vitro. Nevertheless, effects upon macrophage activation induced by this interaction are still unknown. &#103 ₂GPI influence upon the reactive species production by Kupffer cells was evaluted in order to investigate whether &#103 ₂GPI modulates the macrophage response to negatively charged surfaces. Chemiluminescence of isolated non-parenchymal rat liver cells was measured after phagocytosis of opsonized zymosan or phorbolmyristate acetate (PMA) stimulation, in the presence and absence of large unilamellar vesicles (LUVs) containing 25mol% phosphatidylserine (PS) or 50mol% cardiolipin (CL) and complementary molar ratio of phosphatidylcholine (PC). &#103 ₂GPI decreased by 50% the chemiluminescence response induced by opsonized zymosan, with a 66% reduction of the initial light emission rate. PMA stimulated Kupffer cell chemiluminescence was insensitive to human or rat &#103 ₂GPI. Albumin (500 &#119 g/ml) showed no effect upon chemiluminescence. &#103 ₂GPI increased PS/PC LUV uptake and degradation by Kupffer cells in a concentration-dependent manner, without leakage of the internal contents of the LUVs, as shown by fluorescence intensity enhancement. LUVs opsonized with antiphospholipid antibodies (aPL) from syphilitic patients increased light emission by Kupffer cells. Addition of &#103 ₂GPI to the assay reduced chemiluminescence due to opsonization with purified IgG antibodies from systemic lupus erythematosus (SLE or syphilis (Sy) patient sera. A marked net increase in chemiluminescence is observed in the presence of Sy aPL antibodies, whereas a decrease was found when SLE aPL were added to the assay, in the presence or absence of &#103 ₂GPI. At a concentration of 125 &#119 g/ml, &#103 2 GPI significantly reduced Kupffer cell Candida albicans phagocytosis index and killing score by 50 and 10%, respectively. The present data strongly suggest that particle uptake in the presence of &#103 ₂GPI is coupled to an inhibition of reactive species production by liver macrophages during the respiratory burst, supporting the role of &#103 ₂GPI as a mediator of senescent cell removal.     

Adenovirus Endocytosis via αv Integrins Requires Phosphoinositide-3-OH Kinase

Integrins mediate cell adhesion and motility on the extracellular matrix, yet they also promote viral attachment and/or entry. Evidence is presented that adenovirus internalization by α_v integrins requires activation of phosphoinositide-3-OH kinase (PI3K), whereas α_v integrin-mediated cell motility depends on the ERK1/ERK2 mitogen-activated protein kinase pathway. Interaction of adenovirus with α_v integrins induced activation of PI3K. Pharmacologic or genetic disruption of endogenous PI3K activity blocked adenovirus internalization and virus-mediated gene delivery yet had no effect on integrin-mediated cell adhesion or motility. Therefore, integrin ligation engages distinct signaling pathways that promote viral endocytosis or cell movement.Adenovirus entry into host cells depends on α_v integrin binding to the penton base viral coat protein (2, 20, 48). A highly mobile protrusion on the adenovirus penton base contains the arginine-glycine-aspartic acid (RGD) sequence which mediates α_v integrin binding (42). Integrins are more noted for their ability to mediate cell surface recognition of the extracellular matrix, thereby facilitating adhesion, migration (24), and cell growth and differentiation (28). These interactions have been associated with cell differentiation and tissue development, angiogenesis, wound repair, cancer, and inflammation (22).A number of cell signaling molecules that are associated with integrin-mediated cellular processes, including adhesion, survival, and motility, have recently been identified (18, 32, 34). For example, the signaling molecule pp125^FAK focal adhesion kinase (FAK) (35) is localized to clustered integrins following ligation by extracellular matrix proteins. Engagement (clustering) of integrins by its ligands increases tyrosine phosphorylation and activation of FAK (29). Potential downstream substrates of FAK are the ERK1/ERK2 mitogen-activated protein (MAP) kinases (8, 40) and phosphoinositide-3-OH kinase (PI3K) (7, 17).Recent studies have demonstrated that ligation of α_v and β1 integrins by the extracellular matrix leads to engagement of the ERK1/ERK2 MAP kinase pathway (24). Integrin-mediated regulation of the ERK1/ERK2 MAP kinase pathway results in the activation of myosin light chain kinase and subsequently to phosphorylation of myosin light chains. These molecular events culminate in enhanced cell motility. Cell motility, but not cell adhesion or spreading, can be blocked by ERK antisense oligonucleotides or by the compound PD98059, a specific inhibitor of MEK MAP kinase (24), indicating that the ERK1/ERK2 MAP kinase pathway plays a specific role in cell movement.PI3K (44) is another downstream effector of FAK. PI3K is a member of a family of lipid kinases comprised of a p85 regulatory subunit and a p110 catalytic subunit. The p85 subunit of PI3K binds directly to phosphorylated FAK (6). The products of PI3K activation, phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate (PIP₃), are increased in the plasma membrane of activated but not quiescent cells and have been proposed to act as second messengers for a number of cell functions (5), including cell cycle progression (9) and cytoskeletal changes underlying the cell plasma membrane (47). PI3K activation also modulates intracellular protein trafficking (41), although a direct role of PI3K in receptor-mediated endocytosis has not been established.While integrins play an important role in adenovirus entry and in cell migration, the precise mechanisms by which these receptors promote these distinct biological functions are not known. In the studies reported here, we demonstrate that a specific signaling event is involved in the cell entry of a human viral pathogen. Evidence is provided that PI3K is activated upon adenovirus interaction with α_v integrins and that this event is required for adenovirus internalization. Surprisingly, activation of ERK1/ERK2 following integrin ligation was necessary for cell migration but not for internalization of adenovirus.     

The demonstration of a novel sulfur cycle-based wastewater treatment process: Sulfate reduction, autotrophic denitrification, and nitrification integrated (SANI®) biological nitrogen removal process

Saline water supply has been successfully practiced for toilet flushing in Hong Kong since 1950s, which saves 22% of freshwater in Hong Kong. In order to extend the benefits of saline water supply into saline sewage management, we have recently developed a novel biological organics and nitrogen removal process: the Sulfate reduction, Autotrophic denitrification, and Nitrification Integrated (SANI®) process. The key features of this novel process include elimination of oxygen demand in organic matter removal and production of minimal sludge. Following the success of a 500‐day lab‐scale trial, this study reports a pilot scale evaluation of this novel process treating 10 m³/day of 6‐mm screened saline sewage in Hong Kong. The SANI® pilot plant consisted of a sulfate reduction up‐flow sludge bed (SRUSB) reactor, an anoxic bioreactor for autotrophic denitrification and an aerobic bioreactor for nitrification. The plant was operated at a steady state for 225 days, during which the average removal efficiencies of both chemical oxygen demand (COD) and total suspended solids (TSS) at 87% and no excess sludge was purposefully withdrawn. Furthermore, a tracer test revealed 5% short circuit flow and a 34.6% dead zone in the SRUSB, indicating a good possibility to further optimize the treatment capacity of the process for full‐scale application. Compared with conventional biological nitrogen removal processes, the SANI® process reduces 90% of waste sludge, which saves 35% of the energy and reduces 36% of fossil CO₂ emission. The SANI® process not only eliminates the major odor sources originating from primary treatment and subsequent sludge treatment and disposal during secondary saline sewage treatment, but also promotes saline water supply as an economic and sustainable solution for water scarcity and sewage treatment in water‐scarce coastal areas. Biotechnol. Bioeng. 2012; 109: 2778–2789. © 2012 Wiley Periodicals, Inc.     

Matrex Cellufine Sulfate在重组HBsAg纯化中的应用

建立纯化重组CHO细胞HBsAg的新工艺。将含有HBsAg的重组CHO细胞培养收获液,采用Butyl S Sepharose疏水作用柱层析、Matrex Cellufine Sulfate亲和柱层析、Sepharose 4FF凝胶过滤柱层析进行纯化,得到HBsAg纯品,该HBsAg经检定合格。HBsAg回收率为72％。     

Indoxyl Sulfate诱导腹膜间皮细胞转分化的实验研究

目的硫酸吲哚酚(IS)刺激永生化人腹膜间皮细胞株(HMrSV5)后,研究细胞是否发生上皮细胞-间充质转分化(EMT),同时探索转化生长因子β1(TGF-β1)可能在其中的作用。方法体外培养HMrSV5,传代后,随机将人腹膜间皮细胞株分成以3组:对照组:加入4.25﹪腹膜透析液与培养液共培养;IS处理组:分别用含250μmol/LIS、500μmol/LIS、1 000μmol/LIS浓度的培养液与4.25﹪腹膜透析液共培养;TGF-β1受体抑制剂组:用含2μmol/L LY364947+IS的培养液与4.25﹪腹膜透析液共培养。分别在培养0、4、12、24、48和72 h时间下,采用细胞免疫荧光法观察细胞形态变化,Western Blot法研究细胞α平滑肌肌动蛋白(α-SMA)的表达规律,48 h时用ELISA法测定纤维连接蛋白(FN),层粘连蛋白(LN5)两种基质蛋白的分泌情况。结果 (1)免疫荧光法显示:与正常细胞比较,IS处理组细胞由典型的铺路样鹅卵石状,逐渐变细变长,呈梭形改变;而TGF-β1受体抑制剂组细胞形态变化不明显,不如IS处理组;(2)Western Blot法检测结果显示:与对照组比较,IS处理组α-SMA的表达水平明显增加,48 h时1 000μmol/LIS表达最明显(39.929±2.610 vs 0.996±0.001;P0.01),差异有统计学意义;与IS处理组比较,TGF-β1受体抑制剂组的表达则明显减少,其中48 h时抑制明显(7.418±3.075 vs 39.929±2.610,P0.01),差异有统计学意义;(3)ELISA法结果显示:与对照组比较,IS处理组基质蛋白FN(1.368±0.040 vs 1.094±0.036,P0.01)、LN5(1.031±0.085vs 0.860±0.063,P0.01)的表达均上调,差异有统计学意义;与IS处理组比较,TGF-β1受体抑制剂组FN(0.925±0.15 vs 1.368±0.040,P0.01)、LN5(0.795±0.426 vs 1.031±0.085,P0.01)分泌均减少,差异有统计学意义。结论 IS可诱导腹膜间皮细胞发生EMT,TGF-β1的参与可能起重要作用,这为治疗腹膜纤维化可能提供新的靶点。