REAL-TIME OBSERVATION OF CELLULOSE BIODEGRADATION BY ATOMIC FORCE MICROSCOPY

Cellulose, the major structural component of plant cell walls, is a homopolymer of β-1,4-linked glucose residues. As cellulose is the most abundant biopolymer on Earth comprising approx. 50% of the bioshpere, it has attracted renewed interest as a potential source of energy through its biodegradation and fermentation to biofuels. The biodegradation of cellulose involves the concerted action of three types of enzymes, cellulases (EC 3.2.1.4, endo-β-1,4-glucanases), cellobiohydrolases (EC 3.2.1.91; cellulose 1,4-β-cellobiosidase), and β-glucosidases (EC 3.2.1.21; β-d-glucoside glucohydrolase). The former two classes of enzymes function to hydrolyze insoluble cellulose into soluble oligosaccharides which then serve as substrates for β-glucosidases to release free glucose. In many cases, these enzymes are multi-modular, being comprised of distinct catalytic and carbohydrate-binding modules (CBMs). The CBMS appear to aid in both the adsorption of the enzymes to the insoluble cellulose substrate and the destabilization of the hydrogen-bonding network within the crystalline substrate. An understanding of this latter process is extremely important because it has been demonstrated that binding of the enzymes to the insoluble cellulose represents the rate-limiting step in its hydrolysis. To this end, we have developed a protocol for the direct and real-time observation of cellulose biodegradation by atomic force microscopy (AFM). Working electrodes for AFM experiments consisted of a 200 nm thick gold film vapor deposited onto a glass slide pre-treated with a deposition of a 2 nm thick layer of chromium. After annealing in a muffle furnace at 700°C for 60 s, the slides were treated with thioglucose to provide a highly-ordered monolayer of hydrophilic glucose for the stable adsorption of cellulose. Thin films of bacterial microcrystalline cellulose on these electrodes were prepared using the Langmuir-Blodgett technique. Optimized conditions were established to involve a dispersion of a 2 mg/ml suspension of cellulose in methanol/chloroform (1:5) on aqueous phosphate buffer using a compression of 5 mN/m. With this protocol, drying of the cellulose film thereby precluding any associated structural alterations. AFM images were captured using a Pico SPM Microscope with AFMS 182 scanner and Pico-scan 5.2 software system using silicon nitride tips which had a nominal spring constant of 0.06 N m-1 for contact mode, and magnetically coated silicon tips for MAC mode. Under these conditions, the diameters of the microfibrils in a 50 nm fiber were observed to be 3 - 4 nm, which is smaller than the 7.5 nm previously reported by others. Homogeneous samples of the cellulase CenA from the bacterium Cellulomonas fimi were introduced into the liquid cell through capillary ports for the in situ imaging of cellulose disruption and hydrolysis. This activity was monitored over the course of 19 hours and initial evidence of degradation of the fibers was observed within three minutes of enzyme addition. In addition, details of the process of fiber fraying could be readily discerned. Genetic engineering was used to provide a mutant form of CenA involving a replacement of its catalytic aspartate nucleophile with alanine. Studies with this catalytically inactive enzyme derivative permit the analysis of cellulose fibril destabilization prior to hydrolysis

Oral administration of Lactococcus lactis expressing synthetic genes of myelin antigens in decreasing experimental autoimmune encephalomyelitis in rats

ABSTRACT Background: Multiple sclerosis is a human autoimmunological disease that causes neurodegeneration. One of the potential ways to stop its development is induction of oral tolerance, whose effect lies in decreasing immune response to the fed antigen. It was shown on animal models that administration of specific epitopes of the three main myelin proteins, myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP) and proteolipid protein (PLP) results in induction of oral tolerance and suppression of disease symptoms. Application of bacterial cells to produce and deliver antigens to gut mucosa seems to be an attractive method for oral tolerance induction in treatment of diseases with autoimmune background. Material/Methods: Synthetic genes of MOG35-55, MBP85-97 and PLP139-151 myelin epitopes were generated and cloned in Lactococcus lactis under a CcpA-regulated promoter. The tolerogenic effect of bacterial preparations was tested on experimental autoimmune encephalomyelitis, the animal model of MS. EAE was induced in rats by intradermal injection of guinea pig homogenate into hind paws. Results: Rats were administered preparations containing whole-cell lysates of L. lactis producing myelin antigens using different feeding schemes. Our study demonstrates that 20-fold, but not 4-fold, intragastric administration of autoantigen-expressing L. lactis cells under specific conditions reduces the clinical symptoms of EAE in rats. Conclusions: The present study evaluates the use of myelin antigens produced in L. lactis in inhibiting the on-set of experimental autoimmune encephalomyelitis in rats. Obtained results indicate that application of such recombinant cells can be an attractive method of oral tolerance induction

Effect of recombinant Lactococcus lactis producing myelin peptides on neuroimmunological changes in rats with experimental allergic encephalomyelitis

Multiple sclerosis (MS) is a human autoimmune neurodegenerative disease with an unknown etiology. Despite various therapies, there is no effective cure for MS. Since the mechanism of the disease is based on autoreactive T-cell responses directed against myelin antigens, oral tolerance is a promising approach for the MS treatment. Here, the experiments were performed to assess the impact of oral administration of recombinant Lactococcus lactis producing encephalogenic fragments of three myelin proteins: myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein, on neuroimmunological changes in rats with experimental allergic encephalomyelitis (EAE) – an animal model of MS. Lactococcus lactis whole-cell lysates were administered intragastrically at two doses (103 and 106 colony forming units) in a twenty-fold feeding regimen to Lewis rats with EAE. Spinal cord slices were subjected to histopathological analysis and morphometric evaluation, and serum levels of cytokines (IL-1b, IL-10, TNF-α and IFN-γ) were measured. Results showed that administration of the L. lactis preparations at the tested doses to rats with EAE, diminished the histopathological changes observed in EAE rats and reduced the levels of serum IL-1b, IL-10 and TNF-α, previously increased by evoking EAE. This suggests that oral delivery of L. lactis producing myelin peptide fragments could be an alternative strategy to induce oral tolerance for the treatment of MS

In situ PM-IRRAS Studies of a Floating Bilayer Lipid Membrane at Au(111) Electrode Surface

应用电化学原位偏振红外反射光谱法研究了构建于金（111）电极表面的浮动磷脂双层膜。金电极表面先自组装一层巯基葡萄糖单层来增加表面的亲水性，浮动磷脂双层膜通过LB-LS技术构建在巯基葡萄糖单层上.双层膜由双肉豆蔻磷脂酰胆碱（DMPC），胆固醇和神经节苷脂GM1构成.GM1分子中的糖链可以物理吸附在巯基葡萄糖表面，在双层膜和基底间形成一个富含水的隔层.红外光谱表明浮动双层膜中的DMPC分子比传统的支撑双层膜中的DMPC分子有更强的水合作用，证实了双层膜和基底间水层的存在.该浮动双层膜更接近于实际的生物膜体系，并且在金电极表面有宽的电位区间，非常适合于进一步的离子通道蛋白质研究.In situ Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) was used to study the structure of a DMPC + cholesterol + GM1 floating bilayer lipid membrane (fBLM) at a Au(111) surface. 1-thio-beta-D-glucose (beta-Tg) was self-assembled onto the Au electrode to increase the overall hydrophilicity of the surface. The fBLM was deposited on the beta-Tg self-assembled monolayer (SAM) using a combination of Langmuir-Blodgett/Langmuir-Schaefer (LB/LS) techniques. The carbohydrate headgroups of the GM1 molecules were physically adsorbed to the beta-Tg SAM forming a water rich cushion between the fBLM and the modified gold substrate. The PM-IRRAS spectra indicate that the DMPC molecules within the fBLM are more hydrated than previous studies involving supported bilayer lipid membranes (sBLM) where the membrane is directly adsorbed onto the surface. The tilt angle of the DMPC acyl chains in the fBLM is smaller than that of the sBLM composed of similar components. The results from this work confirmed that the fBLM is stable over a wide range of electrode potentials and that a water rich region is present between the bilayer and gold electrode surface. The addition of this water region more closely mimics the natural environment of a biological membrane making the fBLM a desirable candidate for future in situ studies involving transmembrane proteins.This work was supported by Discovery grant from the Natural Sciences and Engineering Research Council of Canada. J.L. acknowledges support from the Canada Research Chairs (CRC) program.This work was supported by Discovery grant from the Natural Sciences and Engineering Research Council of Canada. J.L. acknowledges support from the Canada Research Chairs (CRC) program.作者联系地址：圭尔夫大学化学系，加拿大 安大略省 N1G 2W1, 圭尔夫Author's Address: Department of Chemistry, University of Guelph, Guelph, Ontario N1G 2W1, Canada通讯作者E-mail：[email protected]