Purification and characterization of a propanol-tolerant neutral protease from Bacillus sp. ZG20

Abstract

A propanol-tolerant neutral protease was purified and characterized from Bacillus sp. ZG20 in this study. This protease was purified to homogeneity with a specific activity of 26,655?U/mg. The recovery rate and purification fold of the protease were 13.7% and 31.5, respectively. The SDS-PAGE results showed that the molecular weight of the protease was about 29?kDa. The optimal temperature and pH of the protease were 45?°C and 7.0, respectively. The protease exhibited a good thermal- and pH stability, and was tolerant to 50% propanol. Mg²⁺, Zn²⁺, K⁺, Na⁺ and Tween-80 could improve its activity. The calculated K_m and V_max values of the protease towards α-casein were 12.74?mg/mL and 28.57?µg/(min mL), respectively. This study lays a good foundation for the future use of the neutral protease from Bacillus sp. ZG20.     

Enhanced intestinal epithelial barrier health status on European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides

The study assesses the effects of dietary mannan oligosaccharides (MOS) in European sea bass (Dicentrarchus labrax) posterior intestinal lipid class composition and its possible relation to the potential prostaglandins production and Gut Associated Lymphoid Tissue (GALT) stimulation.Fish were fed 4 g kg^?1 MOS (Bio-Mos^® Aquagrade, Alltech, Inc., USA) for eight weeks. Fish fed MOS presented higher (P ≤ 0.05) weight gain, total length, and specific and relative growth rates than fish fed the control diet. Stimulated posterior gut of fish fed MOS showed higher (P ≤ 0.05) prostaglandins production than fish fed the control diet. Lipid class analyses of posterior gut revealed a reduction (P ≤ 0.05) in the neutral lipid fraction in fish fed MOS compared to fish fed the control diet, particularly due to a reduction (P ≤ 0.05) in triacylglycerols content. The polar lipid fraction increased (P ≤ 0.05) in fish fed MOS compared to fish fed the control diet, mainly due to an increase (P ≤ 0.05) in phosphatidylethanolamine and phosphatidylcoline contents.Light microscopy of posterior gut revealed increased number or goblet cells as well as higher level of infiltrated eosinophilic granulocytes for fish fed MOS. Transmission electron microscopy qualitative observations revealed a better preserved cytoarchitecture of the intestinal epithelial barrier in the posterior gut of fish fed MOS. Posterior gut of fish fed MOS presented more densely packed non-damaged enterocytes, better preserved tight junctions structure, healthier and more organized microvilli, and a higher presence of infiltrated lymphocytes and granulocytes compared fish fed the control diet.The present study indicates that dietary MOS enhances European sea bass posterior gut epithelial defense by increasing membrane polar lipids content in relation to a stimulation of the eicosanoid cascade and GALT, promoting posterior gut health status.     

GalNAcβ1,3-linked paragloboside carries the epitope of a sperm maturation-related glycoprotein that is recognized by the monoclonal antibody MC121

The functional maturation of spermatozoa during epididymal transit in mammals accompanies the changes in their plasma membrane due to the binding or removal of proteins or interactions with the proteases, glycosidases and glycosyltransferases present in the epididymis. In order to study the surface changes in spermatozoa during their maturation in the epididymis, we previously established several monoclonal antibodies against the 54 kDa sialoglycoprotein of mouse cauda epididymal spermatozoa, which gradually increased the expression of antigenic determinants during epididymal transit. One of these monoclonal antibodies, MC121, reacted with mouse sperm glycoproteins on a polyvinylidene fluoride membrane after desialylation of the glycoproteins, and the treatment of the desialylated sperm glycoproteins with β-N-acetylhexosaminidase greatly decreased the expression of the antigenic determinants. In addition to reacting with mouse cauda epididymal spermatozoa, MC121 reacted with human red blood cells (hRBCs). MC121 induced agglutination of sialidase-treated hRBCs and stained hRBCs fixed with formalin vapor much more heavily than it stained hRBCs fixed with methanol. The thin layer chromatography (TLC) immunostaining of the sialidase-treated lipids of hRBCs with MC121 suggested that the epitope-bearing molecule is a glycosphingolipids (GSL), and that MC121 reacts with a pentaose-GSL. Analysis of sialidase-treated GSLs by TLC-Blot-Matrix Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI TOF MS) revealed that the GSL bound by MC121 was [HexNAc][HexNAc + Hex][Hex][Hex]-Cer. The lipid band stained with mAb TH2, which is specific for a GSL, GalNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1-ceramide. These results indicated that the epitope to which MC121 binds is present in a neolacto-series GSL, IV³GalNAcβ-nLc₄Cer² sequence.     

NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

Accumulating evidence indicates the involvement of N-methyl-d-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.     

CYP18A1, a key enzyme of Drosophila steroid hormone inactivation, is essential for metamorphosis

Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.     

ßFTZ-F1 and Matrix metalloproteinase 2 are required for fat-body remodeling in Drosophila

During metamorphosis, holometabolous insects eliminate obsolete larval tissues via programmed cell death. In contrast, tissues required for further development are retained and often remodeled to meet the needs of the adult fly. The larval fat body is involved in fueling metamorphosis, and thus it escapes cell death and is instead remodeled during prepupal development. The molecular mechanisms by which the fat body escapes programmed cell death have not yet been described, but it has been established that fat-body remodeling requires 20-hydroxyecdysone (20E) signaling. We have determined that 20E signaling is required within the fat body for the cell-shape changes and cell detachment that are characteristic of fat-body remodeling. We demonstrate that the nuclear hormone receptor ßFTZ-F1 is a key modulator of 20E hormonal induction of fat body remodeling and Matrix metalloproteinase 2 (MMP2) expression in the fat body. We show that induction of MMP2 expression in the fat body requires 20E signaling, and that MMP2 is necessary and sufficient to induce fat-body remodeling.     

Development of anti-CD20 therapy for multiple sclerosis

The therapeutic utility of the targeting of B lymphocytes is currently being evaluated in a range of autoimmune diseases that include multiple sclerosis (MS). For MS, even though intrathecal immunoglobulin production is a hallmark of multiple sclerosis (MS), T cells have long been considered as the main effectors of pathogenesis. Recognition of the roles of autoreactive B cells has changed this conventional view of the disease and also provided a rationale for studies of anti-CD20 therapy in MS. Recent trials suggest that this approach may provide clinical benefits in some MS patients that equal or surpass currently approved approaches, yet not all patients may benefit. In this review we provide an overview on recent progress on these trials.     

Substrate specificity of diacylglycerol kinase-epsilon and the phosphatidylinositol cycle

We show that diacylglycerol kinase-ε (DGKε) has less preference for the acyl chain at the sn-1 position of diacylglycerol (DAG) than the one at the sn-2 position. Although DGKε discriminates between 1-stearoyl-2-arachidonoyl-DAG and 1-palmitoyl-2-arachidonoyl-DAG, it has similar substrate preference for 1-stearoyl-2-arachidonoyl-DAG and 1,2-diarachidonoyl-DAG. We suggest that in addition to binding to the enzyme, the acyl chain at the sn-1 position may contribute to the depth of insertion of the DAG into the membrane. Thus, the DAG intermediate of the PI-cycle, 1-stearoyl-2-arachidonoyl-DAG, is not the only DAG that is a good substrate for DGKε, the DGK isoform involved in PI-cycling.     

The mechanistic aspects of iron(III) porphyrin catalyzed oxidation reactions in mixed solvents

In the iron(III) porphyrin catalyzed oxidation reactions, the formation of various reactive intermediates have been observed to depend upon the nature of the catalyst, the oxidant and the solvent used for the study. The various iron(III) porphyrin catalysts such as F₂₀TPPFeCl, F₁₆TPPFeCl, F₁₂TPPFeCl and F₈TPPFeCl have been used in the present study to understand the effect of solvent system in the activation of the catalysts. As the terminal oxidant t-BuOOH has been used. It has been observed that acetonitrile contaminated with water activates all the catalysts. It has been noted that ∼9% of water in acetonitrile is the best solvent system for the activation of all the catalysts. The results obtained have been applied to successfully oxidize cyclohexene and cyclohexane by these oxidizing systems. It has also been observed that CH₃OH mixed with CH₂Cl₂ play a very important role in the activation of catalyst in hydroperoxide oxidizing system. The 33 ± 3% ratio of CH₃OH in CH₂Cl₂ acts as the most suitable solvent system to convert organopalladium compound 1a-c to 2a-c.     

Design and construction of improved new vectors for Zymomonas mobilis recombinants

Zymomonas mobilis is a very important gram-negative bacterium having a potential application to simultaneous co-production of biofuel and other high value-added products through biorefinery process technology development. Up to now, pLOI193 has been used as the plasmid of choice for Z. mobilis strains. However, its application has been limited due to its relatively low transformation efficiency, a large plasmid size (13.4 kb), and limited choice of cloning sites for gene manipulations. Some of these limitations can be overcome by the newly designed and constructed plasmid pHW20a, which provides significantly higher transformation efficiency (about two orders of magnitude greater), better stability (for at least 120 generation times), and an ease of gene manipulations. The pHW20a contains three complete cis-acting genes (repA, repB, and repC) encoding the Rep proteins for primosome formation. It has the origin of replication (oriV) to ensure replication in gram-negative bacteria, two mob genes that enhances transformation efficiency, a screening marker (lacZα), expanded multiple cloning sites (MCS) that enables easy gene manipulation, and the tetracycline resistance gene (tc(r) ). The utility of screening marker, lacZα with MCS, was confirmed by the blue-white screening test. Several examples of applications of gene expression in Z. mobilis ZM4 have been demonstrated in this article by using several new pHW20a-derived plasmids and expressing the homologous genes (gfo and ppc) and the heterologous genes (bglA, mdh, and fdh1). The results show that pHW20a is a very useful new vector for construction of new Z. mobilis recombinant strains that will enable simultaneous co-production of biofuel and high value added products.