Reorganization of the Endosomal System in Salmonella-Infected Cells: The Ultrastructure of Salmonella-Induced Tubular Compartments

During the intracellular life of Salmonella enterica, a unique membrane-bound compartment termed Salmonella-containing vacuole, or SCV, is formed. By means of translocated effector proteins, intracellular Salmonella also induce the formation of extensive, highly dynamic membrane tubules termed Salmonella-induced filaments or SIF. Here we report the first detailed ultrastructural analyses of the SCV and SIF by electron microscopy (EM), EM tomography and live cell correlative light and electron microscopy (CLEM). We found that a subset of SIF is composed of double membranes that enclose portions of host cell cytosol and cytoskeletal filaments within its inner lumen. Despite some morphological similarities, we found that the formation of SIF double membranes is independent from autophagy and requires the function of the effector proteins SseF and SseG. The lumen of SIF network is accessible to various types of endocytosed material and our CLEM analysis of double membrane SIF demonstrated that fluid phase markers accumulate only between the inner and outer membrane of these structures, a space continual with endosomal lumen. Our work reveals how manipulation of the endosomal membrane system by an intracellular pathogen results in a unique tubular membrane compartmentalization of the host cell, generating a shielded niche permissive for intracellular proliferation of Salmonella.     

Application of a Novel Alkali-Tolerant Thermostable DyP-Type Peroxidase from Saccharomonospora viridis DSM 43017 in Biobleaching of Eucalyptus Kraft Pulp

Saccharomonospora viridis is a thermophilic actinomycete that may have biotechnological applications because of its dye decolorizing activity, though the enzymatic oxidative system responsible for this activity remains elusive. Bioinformatic analysis revealed a DyP-type peroxidase gene in the genome of S. viridis DSM 43017 with sequence similarity to peroxidase from dye-decolorizing microbes. This gene, svidyp, consists of 1,215 bp encoding a polypeptide of 404 amino acids. The gene encoding SviDyP was cloned, heterologously expressed in Escherichia coli, and then purified. The recombinant protein could efficiently decolorize several triarylmethane dyes, anthraquinonic and azo dyes under neutral to alkaline conditions. The optimum pH and temperature for SviDyP was pH 7.0 and 70°C, respectively. Compared with other DyP-type peroxidases, SviDyP was more active at high temperatures, retaining>63% of its maximum activity at 50–80°C. It also showed broad pH adaptability (>35% activity at pH 4.0–9.0) and alkali-tolerance (>80% activity after incubation at pH 5–10 for 1 h at 37°C), and was highly thermostable (>60% activity after incubation at 70°C for 2 h at pH 7.0). SviDyP had an accelerated action during the biobleaching of eucalyptus kraft pulp, resulting in a 21.8% reduction in kappa number and an increase of 2.98% (ISO) in brightness. These favorable properties make SviDyP peroxidase a promising enzyme for use in the pulp and paper industries.     

14-3-3 and aggresome formation: Implications in neurodegenerative diseases

Protein misfolding and aggregation underlie the pathogenesis of many neurodegenerative diseases. In addition to chaperone-mediated refolding and proteasomal degradation, the aggresome-macroautophagy pathway has emerged as another defense mechanism for sequestration and clearance of toxic protein aggregates in cells. Previously, the 14-3-3 proteins were shown to be indispensable for the formation of aggresomes induced by mutant huntingtin proteins. In a recent study, we have determined that 14-3-3 functions as a molecular adaptor to recruit chaperone-associated misfolded proteins to dynein motors for transport to aggresomes. This molecular complex involves a dimeric binding of 14-3-3 to both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3). As 14-3-3 has been implicated in various neurodegenerative diseases, our findings may provide mechanistic insights into its role in managing misfolded protein stress during the process of neurodegeneration.     

Flavonoids from tartary buckwheat induce G2/M cell cycle arrest and apoptosis in human hepatoma HepG2 cells

The cytotoxicity and antioxidant activity on human hepa toma cell line HepG2 of three flavonoids homogenous com pounds from tartary buckwheat seeds and bran, namely quercetin, isoquercetin, and rutin, were investigated. The total antioxidant competency detection results indicated that the antioxidant capacity of quercetin was the strongest in a biological response system. A [3-（4,5-dimethylthiazol-2-yl）-2,5diphenyltetrazolium bromide] assay showed that quercetin exhibited the strongest cytotoxic effects against the HepG2 cell line. Flow cytometric analysis indicated that quercetin significantly increased the production of reactive oxygen species, and led to the G2/M phase arrest accom panied by an increase of apoptotic cell death after 48 h of incubation. Quercetininduced cell apoptosis was shown to involve p53 and p21 upregulation, Cyclin D1, Cdk2, and Cdk7 downregulation. These results suggested that the in duction of G2/M arrest, apoptosis, and cell death by quer cetin may associate with increased expression of p53 and p21, decrease of Cyclin D1, Cdk2, and Cdk7 levels, and generation of reactive oxygen species in cells. This study will help to better understand and fully utilize medicinal resources of plant flavonoids.     

Targeted NGF siRNA Delivery Attenuates Sympathetic Nerve Sprouting and Deteriorates Cardiac Dysfunction in Rats with Myocardial Infarction

Nerve growth factor (NGF) is involved in nerve sprouting, hyper-innervation, angiogenesis, anti-apoptosis, and preservation of cardiac function after myocardial infarction (MI). Positively modulating NGF expression may represent a novel pharmacological strategy to improve post-infarction prognosis. In this study, lentivirus encoding NGF short interfering RNA (siRNA) was prepared, and MI was modeled in the rat using left anterior descending coronary artery ligation. Rats were randomly grouped to receive intramyocardial injection of lentiviral solution containing NGF-siRNA (n = 19, MI-SiNGF group), lentiviral solution containing empty vector (n = 18, MI-GFP group) or 0.9% NaCl solution (n = 18, MI-control group), or to receive thoracotomy and pericardiotomy (n = 17, sham-operated group). At 1, 2, 4, and 8 wk after transduction, rats in the MI-control group had higher levels of NGF mRNA and protein than those in the sham-operated group, rats in the MI-GFP group showed similar levels as the MI-control group, and rats in the MI-SiNGF group had lower levels compared to the MI-GFP group, indicating that MI model was successfully established and NGF siRNA effectively inhibited the expression of NGF. At 8 wk, echocardiographic and hemodynamic studies revealed a more severe cardiac dysfunction in the MI-siRNA group compared to the MI-GFP group. Moreover, rats in the MI-siRNA group had lower mRNA and protein expression levels of tyrosine hydroxylase (TH) and growth-associated protein 43-positive nerve fibers (GAP-43) at both the infarcted border and within the non-infarcted left ventricles (LV). NGF silencing also reduced the vascular endothelial growth factor (VEGF) expression and decreased the arteriolar and capillary densities at the infarcted border compared to the MI-GFP group. Histological analysis indicated a large infarcted size in the MI-SiNGF group. These findings suggested that endogenous NGF silencing attenuated sympathetic nerve sprouting and angiogenesis, enlarged the infarct size, aggravated cardiac dysfunction, and potentially contributed to an unfavorable prognosis after MI.     

Enhanced isopropanol and <Emphasis Type="Italic">n</Emphasis>-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.     

Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids

Cannabinoids and related drugs generate profound behavioral effects (such as analgesic effects) through activating CNR1 (cannabinoid receptor 1 [brain]). However, repeated cannabinoid administration triggers lysosomal degradation of the receptor and rapid development of drug tolerance, limiting the medical use of marijuana in chronic diseases. The pathogenic mechanisms of cannabinoid tolerance are not fully understood, and little is known about its prevention. Here we show that a protein involved in macroautophagy/autophagy (a conserved lysosomal degradation pathway), BECN2 (beclin 2), mediates cannabinoid tolerance by preventing CNR1 recycling and resensitization after prolonged agonist exposure, and deletion of Becn2 rescues CNR1 activity in mouse brain and conveys resistance to analgesic tolerance to chronic cannabinoids. To target BECN2 therapeutically, we established a competitive recruitment model of BECN2 and identified novel synthetic, natural or physiological stimuli of autophagy that sequester BECN2 from its binding with GPRASP1, a receptor protein for CNR1 degradation. Co-administration of these autophagy inducers effectively restores the level and signaling of brain CNR1 and protects mice from developing tolerance to repeated cannabinoid usage. Overall, our findings demonstrate the functional link among autophagy, receptor signaling and animal behavior regulated by psychoactive drugs, and develop a new strategy to prevent tolerance and improve medical efficacy of cannabinoids by modulating the BECN2 interactome and autophagy activity.     

A sensitive and specific ELISA detects methionine sulfoxide-containing apolipoprotein A-I in HDL

Oxidized HDL has been proposed to play a key role in atherogenesis. A wide range of reactive intermediates oxidizes methionine residues to methionine sulfoxide (MetO) in apolipoprotein A-I (apoA-I), the major HDL protein. These reactive species include those produced by myeloperoxidase, an enzyme implicated in atherogenesis. The aim of the present study was to develop a sensitive and specific ELISA for detecting MetO residues in HDL. We therefore immunized mice with HPLC-purified human apoA-I containing MetO(86) and MetO(112) (termed apoA-I(+32)) to generate a monoclonal antibody termed MOA-I. An ELISA using MOA-I detected lipid-free apoA-I(+32), apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Detection was concentration dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radical or metal ions, or LDL and methionine-containing proteins other than apoA-I modified by 2e-oxidants. Because the ELISA we have developed specifically detects apoA-I containing MetO in HDL and plasma, it should provide a useful tool for investigating the relationship between oxidized HDL and coronary artery disease.     

Modulation of aggressive behaviour by fighting experience: mechanisms and contest outcomes

Experience in aggressive contests often affects behaviour during, and the outcome of, later contests. This review discusses evidence for, variations in, and consequences of such effects. Generally, prior winning experiences increase, and prior losing experiences decrease, the probability of winning in later contests, reflecting modifications of expected fighting ability. We examine differences in the methodologies used to study experience effects, and the relative importance and persistence of winning and losing experiences within and across taxa. We review the voluminous, but somewhat disconnected, literature on the neuroendocrine mechanisms that mediate experience effects. Most studies focus on only one of a number of possible mechanisms without providing a comprehensive view of how these mechanisms are integrated into overt behaviour. More carefully controlled work on the mechanisms underlying experience effects is needed before firm conclusions can be drawn.Behavioural changes during contests that relate to prior experience fall into two general categories. Losing experiences decrease willingness to engage in a contest while winning experiences increase willingness to escalate a contest. As expected from the sequential assessment model of contest behaviour, experiences become less important to outcomes of contests that escalate to physical fighting.A limited number of studies indicate that integration of multiple experiences can influence current contest behaviour. Details of multiple experience integration for any species are virtually unknown. We propose a simple additive model for this integration of multiple experiences into an individual's expected fighting ability. The model accounts for different magnitudes of experience effects and the possible decline in experience effects over time.Predicting contest outcomes based on prior experiences requires an algorithm that translates experience differences into contest outcomes. We propose two general types of model, one based solely on individual differences in integrated multiple experiences and the other based on the probability contests reach the escalated phase. The difference models include four algorithms reflecting possible decision rules that convert the perceived fighting abilities of two rivals into their probabilities of winning. The second type of algorithm focuses on how experience influences the probability that a subsequent contest will escalate and the fact that escalated contests may not be influenced by prior experience. Neither type of algorithm has been systematically investigated.Finally, we review models for the formation of dominance hierarchies that assume that prior experience influences contest outcome. Numerous models have reached varied conclusions depending on which factors examined in this review are included. We know relatively little about the importance of and variation in experience effects in nature and how they influence the dynamics of aggressive interactions in social groups and random assemblages of individuals. Researchers should be very active in this area in the next decade. The role of experience must be integrated with other influences on contest outcome, such as prior residency, to arrive at a more complete picture of variations in contest outcomes. We expect that this integrated view will be important in understanding other types of interactions between individuals, such as mating and predator-prey interactions, that also are affected significantly by prior experiences.