Study for the structures of the HA complexes produced by Clostridium botulinum type A mutant strain

Clostridium botulinum produces seven neurotoxin (NTX) serotypes, classified from as A to G. In culture, NTX forms protein complexes by association with non-toxic components, such as nontoxic-nonhemagglutinin (NTNH) and hemagglutinins (HA1, HA2, HA3). C. botulinum serotype A produces three types of toxin complexes, M-toxin (NTX and NTNH), L-toxin (M-toxin and HAs) and LL-toxin (dimer of L-toxin). In this study, I found three HA complexes in the culture of a nontoxigenic mutant serotype A lacking ntx and ntnh expression. The HA complexes displayed similar banding patterns on SDS-PAGE, but the staining intensities of the HA1 and HA2 bands were different. In addition, their native-PAGE banding profiles exhibited different behaviors. The large-molecular-size HA complex showed the highest activity, similar to that of an L-toxin. Based on the combined results of the PAGE and gel-filtration profiles, the differences in molecular size among the three HA complexes were thought to be caused by different numbers of HA1 and HA2 molecules. This paper reports for the first time the purification and characterization of a native HA complex of serotype A, and suggests that the HA can form complex structures without NTX and NTNH. This may help in understanding the toxin complex assembly pathway

Clostridium botulinum Type E Toxins Bind to Caco-2 Cells by a Different Mechanism from That of Type A Toxins

Cultured Clostridium botulinum strains produce progenitor toxins designated as 12S, 16S, and 19S toxins. The 12S toxin consists of a neurotoxin (NTX, 7S) and a non-toxic non-hemagglutinin (NTNH). The 16S and 19S toxins are formed by conjugation of the 12S toxin with hemagglutinin (HA), and the 19S toxin is a dimer of the 16S toxin. Type A cultures produce all 3 of these progenitor toxins, while type E produces only the 12S toxin. The 7S toxin is cleaved into heavy (H) and light (L) chains by a protease(s) in some strains, and the H chain has 2 domains, the N-terminus (Hn) and C-terminus (Hc). It has been reported that type A toxins bind to the intestinal cells or cultured cells via either HA or Hc. In this study, we investigated the binding of type A and E toxins to Caco-2 cells using Western blot analysis. Both the type E 7S and 12S toxins bound to the cells, with the 7S toxin binding more strongly, whereas, in the type A strain, only the 16S/19S toxins showed obvious binding. Pre-incubation of the type E 7S toxin with IgG against recombinant type E Hc significantly inhibited the 7S toxin binding, indicating that Hc might be a main binding domain of the type E toxin

Separation and purification of the bovine milk fat globule membrane protein and its effect on improvement of C2C12 mouse skeletal muscle cell proliferation

A novel method to improve the proliferation activity of C2C12 cells by the bovine milk fat globule membrane (MFGM) protein was established in this study. The MFGM protein was extracted and isolated into 4 fractions using an electric cream separator, and purified by a cellulose DEAE-52 column. Fraction 2 accounted for 57.8% of the total MFGM protein, and was used in the following study. The MTT assay showed that it induced cell proliferation activity, increased the cell survival rate and the cell number using flow cytometry and fluorescence microscopy analysis. There were only subtle changes in the morphology as observed using confocal scanning laser microscopy, but the number of mitochondria was significantly increased as observed using transmission electron microscopy analysis. Furthermore, the mRNA expression of MyoD, cyclin D1, p70S6K and mTOR was up-regulated as determined utilizing the quantitative real-time PCR assay, and the activation of Akt and mTOR phosphorylation was up regulated as determined using the Western blot assay. The main protein in fraction 2, assayed by 1-D gel electrophoresis and MALDI TOF-TOF, was identified as milk fat globule-EGF factor 8, the content was 65.6% of the total protein in fraction 2. The results elucidate a new molecular mechanism of the MFGM protein fraction 2: the activation of the Akt signal pathway in promoting cell proliferation

MFG-E8 induced differences in proteomic profiles in mouse C2C12 cells and its effect on PI3K/Akt and ERK signal pathways

Milk fat globule-EGF factor 8 (MFG-E8) is one of the major proteins in milk fat globule membrane. In this study, mouse-derived C2C12 myoblast cells were served as an experimentally tractable model system for investigating the molecular basis of skeletal muscle cell specification and development. To examine the biochemical adaptations associated with myocytes formation comprehensively, a liquid chromatography coupled with tandem mass spectrometry label-free semi-quantitative  approach was used to analyse the myogenic C2C12 proliferation program. Over 1987  proteins were identified in C2C12 cells. The MFG-E8 (200 mg/mL) and MFG-E8 (500 26 mg/mL) with significant differences were compared based on the relative abundance. The result profiles of regulation of MFG-E8 to the expression of proteins in C2C12 cells revealed that differential waves of expression of proteins linked to intracellular signaling, transcription, cytoarchitecture, adhesion, metabolism, and muscle contraction across during the C2C12 cell proliferation process. Based on the analysis of  KEGG and STRING database, further to verification the expression of PI3K and ERK phosphorylation levels by Western blot. This study found that the data of proteomic was complementary to recent MFG-E8 studies of protein expression patterns in developing myotubes and provided a holistic framework for understanding how diverse biochemical processes are coordinated at the cellular level during skeletal muscle development

Milk fat globule membrane protein promotes C2C12 cell proliferation through the PI3K/Akt signaling pathway

Milk fat globule membrane (MFGM) protein is known to have several health benefits, including an anti-sarcopenia effect; however, its mechanism is unclear. The aim of this study was to investigate the potential mechanism of action of the MFGM protein. The MFGM protein was extracted and separated into 4 fractions, and Fraction 2 (57 % of total MFGM) demonstrated the greatest effect on C2C12 cell proliferation. Milk fat globule-EGF factor 8 (MFG-E8) accounted for 82.35 % of the MFGM protein. The effects of whole Fraction 2 (100 μg/mL, 200 μg/mL and 300 μg/mL) on cell proliferation and morphology were measured. Using qRT-PCR or a Western blot assay, several regulatory factors, e.g., PI3K P85α, p-pI3K p85α (Tyr 508), Akt, p-Akt (Ser 473), mTOR and p-mTOR (Ser 2448), were measured in cells incubated with 200 μg/mL of Fraction 2 with or without wortmannin. The results demonstrated that Fraction 2 induced C2C12 cell proliferation in a dose-dependent manner, upregulated the mRNA expression of mTOR and p70S6K, and activated PI3K, Akt, mTOR and P70S6K phosphorylation; however, Fraction 2 inhibited FOXO3a and 4E-BP. The results demonstrate that the MFGM protein, predominantly MFG-E8, promotes cell proliferation through the PI3K/Akt/mTOR signaling pathway. This study elucidated the molecular mechanism of the MFGM protein, primarily MFG-E8, in promoting C2C12 cell proliferation via the PI3K/Akt/mTOR/P70S6K signal pathway

Transient analysis of arm locking controller

Arm locking is one of the key technologies to suppress the laser phase noise in spaced-based gravitational waves observatories. Since arm locking was proposed, phase margin criterion was always used as the fundamental design strategy for the controller development. In this paper, we find that this empirical method from engineering actually cannot guarantee the arm locking stability. Therefore, most of the advanced arm locking controllers reported so far may have instable problems. After comprehensive analysis of the single arm locking's transient responses, strict analytical stability criterions are summarized for the first time. These criterions are then generalized to dual arm locking, modified-dual arm locking and common arm locking, and special considerations for the design of arm locking controllers in different architectures are also discussed. It is found that PI controllers can easily meet our stability criterions in most of the arm locking systems. Using a simple high gain PI controller, it is possible to suppress the laser phase noise by 5 orders of magnitude within the science band. Our stability criterions can also be used in other feedback systems, where several modules with different delays are connected in parallel.Comment: 24 pages, 24 figure

Electromagnetic interaction of arbitrary radial-dependent anisotropic spheres and improved invisibility for nonlinear-transformation-based cloaks

An analytical method of electromagnetic wave interactions with a general radially anisotropic cloak is established. It is able to deal with arbitrary parameters ($\epsilon_r(r)$, $\mu_r(r)$, $\epsilon_t(r)$ and $\mu_t(r)$) of a radially anisotropic inhomogeneous shell. The general cloaking condition is proposed from the wave relations for the first time. We derive the parameters of a novel class of spherical nonlinear cloaks and examine its invisibility performance by the proposed method at various nonlinear situations. Spherical metamaterial cloaks with improved invisibility performance is achieved with optimal nonlinearity in transformation and core-shell ratio.Comment: 19 pages, 9 figure