Preparation and properties of an immobilized cellulase on the reversibly soluble matrix Eudragit L-100

Abstract

To prepare a smart biocatalyst, cellulase was immobilized on the reversibly soluble matrix Eudragit L-100 by non-covalent and covalent methods. Covalent immobilization using carbodiimide coupling exhibited superior enzyme loading and reusability compared with non-covalent immobilization, and the covalent loading was increased by almost 20% through the addition of N-hydroxysuccinimide. The temperature optimum of the cellulase was not improved apparently by immobilization but the pH optimum increased from 4.75 to 5.25. Immobilized cellulase was more active than free cellulase above pH 5.0. Immobilized cellulase was more stable than free cellulase during storage at 4°C, room temperature and 50°C. K_m values of immobilized and free cellulase were 85.55 and 73.84 g L^?1, respectively. About 50% productivity was retained after five cycles for hydrolysis of steam-exploded straw.     

Probing antioxidant activity of 2′-hydroxychalcones: Crystal and molecular structures, in vitro antiproliferative studies and in vivo effects on glucose regulation

In order to better understand the antioxidant behavior of a series of polyphenolic 2′-hydroxychalcones, we describe the results of several chemical and biological studies, in vitro and in vivo. Single crystal X-ray methods elucidated their molecular structures and important intermolecular interactions such as H-bonding and molecular stacking in the crystal structures that contribute to our knowledge in explaining antioxidant activity. The results of experiments using the 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) UV–vis spectroscopic method indicate that a hydroxyl group in position 5′ induces the highest antioxidant activity. Consequently, 2,2′,5′-trihydroxychalcone was selected for further study in vitro towards ROS scavenging in L-6 myoblasts and THP-1 human monocytes, where it shows an excellent antioxidant activity in a concentration range lower than that reported by most studies of related molecules. In addition, this chalcone shows a very selective activity: it inhibits the proliferation of leukemic cells, but it does not affect the normal L-6 myoblasts and human fibroblasts. In studying 2,2′,5′-trihydroxychalcone's effect on weight gain and serum glucose and insulin levels in Zucker fatty (fa⁻/fa⁻) rats we found that supplementing the diet with a 10 mg/kg dose of this chalcone (3 times weekly) blunted the increase in glucose that co-occurs with weight gain over the 6-week treatment period. It is concluded that 2,2′,5′-trihydroxychalcone has the potential to serve as a protective agent for some debilitating diseases.     

Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody

Reactive oxygen species generated by NADPH oxidase 5 (Nox5) have been implicated in physiological and pathophysiological signaling pathways, including cancer development and progression. However, because immunological tools are lacking, knowledge of the role of Nox5 in tumor biology has been limited; the expression of Nox5 protein across tumors and normal tissues is essentially unknown. Here, we report the characterization and use of a mouse monoclonal antibody against a recombinant Nox5 protein (bp 600–746) for expression profiling of Nox5 in human tumors by tissue microarray analysis. Using our novel antibody, we also report the detection of endogenous Nox5 protein in human UACC-257 melanoma cells. Immunofluorescence, confocal microscopy, and immunohistochemical techniques were employed to demonstrate Nox5 localization throughout UACC-257 cells, with perinuclear enhancement. Tissue microarray analysis revealed, for the first time, substantial Nox5 overexpression in several human cancers, including those of prostate, breast, colon, lung, brain, and ovary, as well as in malignant melanoma and non-Hodgkin lymphoma; expression in most nonmalignant tissues was negative to weak. This validated mouse monoclonal antibody will promote further exploration of the functional significance of Nox5 in human pathophysiology, including tumor cell growth and proliferation.     

Up-regulation of protein tyrosine nitration in methamphetamine-induced neurotoxicity through DDAH/ADMA/NOS pathway

Protein tyrosine nitration is an important post-translational modification mediated by nitric oxide (NO) associated oxidative stress, occurring in a variety of neurodegenerative diseases. In our previous study, an elevated level of dimethylarginine dimethylaminohydrolase 1 (DDAH1) protein was observed in different brain regions of acute methamphetamine (METH) treated rats, indicating the possibility of an enhanced expression of protein nitration that is mediated by excess NO through the DDAH1/ADMA (Asymmetric Dimethylated l-arginine)/NOS (Nitric Oxide Synthase) pathway. In the present study, proteomic methods, including stable isotope labeling with amino acids in cell culture (SILAC) and two dimensional electrophoresis, were used to determine the relationship between protein nitration and METH induced neurotoxicity in acute METH treated rats and PC12 cells. We found that acute METH administration evokes a positive activation of DDAH1/ADMA/NOS pathway and results in an over-production of NO in different brain regions of rat and PC12 cells, whereas the whole signaling could be repressed by DDAH1 inhibitor N^ω-(2-methoxyethyl)-arginine (l-257). In addition, enhanced expressions of 3 nitroproteins were identified in rat striatum and increased levels of 27 nitroproteins were observed in PC12 cells. These nitrated proteins are key factors for Cdk5 activation, cytoskeletal structure, ribosomes function, etc. l-257 also displayed significant protective effects against METH-induced protein nitration, apoptosis and cell death. The overall results illustrate that protein nitration plays a significant role in the acute METH induced neurotoxicity via the activation of DDAH1/ADMA/NOS pathway.     

Regulatory effects of the L-lysine metabolites,L-2-aminoadipic acid and L-pipecolic acid,on protein turnover in C2C12 myotubes

We previously showed that L-lysine (Lys) and a metabolite of Lys, L-saccharopine, suppressed autophagic proteolysis in C2C12 myotubes. However, the effects of other metabolites of Lys on protein turnover were unknown. We here investigated the effect of the Lys metabolites, L-2-aminoadipic acid (2-AA) and L-pipecolic acid (Pip), on protein turnover in C2C12 myotubes. 2-AA suppressed myofibrillar protein degradation evaluated by the 3-methylhistidine and autophagy activity evaluated by light chain 3-II at lower concentration (100 μM) than did Lys. On the other hand, Pip stimulated the mammalian target of rapamycin signaling activity. Additionally, 100 μM Pip significantly increased the rates of protein synthesis whereas 100 μM Lys had no effect. These results indicate that in C2C12 myotubes, 2-AA could suppress autophagy and Pip could stimulate the rates of protein synthesis, and these metabolites may contribute to exert effect of Lys on protein turnover.     

Responses of Glutathione and Glutathione S-Transferase to Cadmium and Mercury Exposure in Pedunculate Oak (Quercus robur) Leaf Discs

Changes in chlorophyll, non-protein thiol and glutathione (GSH) levels, and the activity of glutathione S-transferase (GST) were investigated in cadmium(ll) and mercury(ll) cchloride treated leaf discs of mature pedunculate oak trees (Quercus robur). Both heavy metals caused decreases in chlorophyll content, but mercury was more toxic than cadmium. Cadmium treatments (30–250μiM) resulted in increasing non-protein thiol levels after 3d, but GSH contents decreased. Mercury (1–20μM) led to a concentration-dependent decline in both non-protein thiol and GSH levels. GST activities were not modified significantly by cadmium, but mercury treatments caused a dose- and time-dependent enzyme induction. Both the phytotoxic- and GST-inducing effect of mercury could be prevented by the cysteine precursor L-2-oxo-4-thiazolidinecarboxylic acid.     

Photosan脂质立方液晶纳米光敏剂的制备及光动力杀伤效应研究

目的:制备Photosan脂质立方液晶纳米光敏剂,通过体外实验探讨其介导的光动力疗法(photodynamic therapy,PDT)特异性杀伤肿瘤细胞效果。方法:以单油酸甘油酯(glyceryl monooleate,GMO)和泊洛沙姆407(poloxamer 407,P407)为液晶材料,以光敏剂Photosan为模型药物,制备Photosan立方液晶纳米粒,通过Malvern粒径仪和扫描电子显微镜等考察其理化性质;通过MTS法考察Photosan立方液晶纳米粒对正常肝细胞株和肝癌细胞株的暗毒性及光动力杀伤效果。结果:成功制备Photosan脂质立方液晶纳米粒,该纳米粒对人肝L-02细胞和人肝癌Hep G2细胞均没有暗毒性,其介导的PDT对人肝L-02细胞增殖有一定抑制作用[细胞抑制率为(32.9±1.19)%],而对肝癌Hep G2细胞增殖具有更显著的抑制作用[细胞抑制率为(77.9±2.06)%];Photosan立方液晶介导的光动力作用对人正常肝细胞的增殖抑制作用低于Photosan,但对肝癌细胞的增殖抑制作用高于Photosan。结论:Photosan脂质立方液晶纳米粒对人正常肝细胞安全性较好,对肝癌细胞的光动力杀伤效应明显优于Photosan,为光动力治疗癌症提供了创新性方法。     

Peripheral administration of oxytocin increases social affiliation in the naked mole-rat (Heterocephalus glaber)

The neuropeptide oxytocin regulates a wide variety of social behaviors across diverse species. However, the types of behaviors that are influenced by this hormone are constrained by the species in question and the social organization that a particular species exhibits. Therefore, the present experiments investigated behaviors regulated by oxytocin in a eusocial mammalian species by using the naked mole-rat (Heterocephalus glaber). In Experiment 1, adult non-breeding mole-rats were given intraperitoneal injections of either oxytocin (1 mg/kg or 10 mg/kg) or saline on alternate days. Animals were then returned to their colony and behavior was recorded for minutes 15–30 post-injection. Both doses of oxytocin increased huddling behavior during this time period. In Experiment 2, animals received intraperitoneal injections of either oxytocin (1 mg/kg), an oxytocin-receptor antagonist (0.1 mg/kg), a cocktail of oxytocin and the antagonist, or saline across 4 testing days in a counterbalanced design. Animals were placed in either a 2-chamber arena with a familiar conspecific or in a small chamber with 1 week old pups from their home colony and behaviors were recorded for minutes 15–30 post-injection. Oxytocin increased investigation of, and time spent in close proximity to, a familiar conspecific; these effects were blocked by the oxytocin antagonist. No effects were seen on pup-directed behavior. These data suggest that oxytocin is capable of modulating affiliative-like behavior in this eusocial species.     

Chylomicron formation and glucagon-like peptide 1 receptor are involved in activation of the nutritional anti-inflammatory pathway

Enteral administration of lipid-enriched nutrition effectively attenuates inflammation via a cholecystokinin (CCK)-mediated vagovagal anti-inflammatory reflex. Cholecystokinin release and subsequent activation of the vagus are dependent on chylomicron formation and associated with release of additional gut peptides. The current study investigates the intestinal processes underlying activation of the CCK-mediated vagal anti-inflammatory pathway by lipid-enriched nutrition. Rats and mice were subjected to hemorrhagic shock (HS) or endotoxemia, respectively. Prior to the experimental procedures, animals were fasted or fed lipid-enriched nutrition. Pluronic L-81 (L-81) was added to the feeding to investigate involvement of chylomicron formation in activation of mesenteric afferent fibers and the immune-modulating potential of lipid-enriched nutrition. Ob/Ob mice and selective receptor antagonists were used to study the role of leptin, glucagon-like peptide 1 and peptide YY in activation of the nutritional reflex. Electrophysiological analysis of mesenteric afferents in mice revealed that lipid-enriched nutrition-mediated neural activation was abrogated by L-81 (P<.05). L-81 blunted the beneficial effects of lipid-enriched nutrition on systemic inflammation and intestinal integrity in both species (all parameters, P<.01). Ob/Ob mice required a higher dose of nutrition compared with wild-type mice to attenuate plasma levels of TNF-α and ileum-lipid binding protein, a marker for enterocyte damage (both P<.01), suggesting a higher stimulation threshold in leptin-deficient mice. Administration of a glucagon-like peptide 1-receptor antagonist, but not leptin or peptide YY antagonists, suppressed the effects of lipid-enriched nutrition. These data indicate that chylomicron formation is essential and activation of the glucagon-like peptide 1-receptor is involved in activation of the nutritional anti-inflammatory pathway by lipid-enriched nutrition.