Characterization of the cellulase complex from Cellulomonas grown on bagasse pith

Summary The effect of physico-chemical parameters on the cellulolytic activity of Cellulomonas sp. IIbc grown on sugarcane bagasse pith was investigated, and the optimum ranges for enzyme activity were established. The cellulases were more stable when incubated at the optimum growth temperature (32°C) than under optimum activity conditions (45°C for -glucosidases and 50°C for CMC- and FP-cellulases). The -glucosidases were the thermostability-limiting enzymes of the complex. Two types of endoglucanases could be recognized according to their adsorption properties on bagasse: one weakly-bound and one tightly-bound type, the latter constituting approximately 73% of the extracellular endoglucanases at exponential growth phase. Four forms active on filter paper and three active on CMC were obtained by HPLC separation of the extracellular fraction of the culture at stationary phase.Abbreviations CMC carboxymethylcellulose - FP filter paper     

Bioguided Fractionation and Isolation of an Antiplasmodial Saponin from the Roots of Nauclea xanthoxylon (A.Chev.) Aubrév. (Rubiaceae)

The root extract of Nauclea xanthoxylon (A.Chev.) Aubrév. displayed significant 50 % inhibition concentration (IC₅₀s) of 0.57 and 1.26 μg/mL against chloroquine resistant and sensitive Plasmodium falciparum (Pf) Dd2 and 3D7 strains, respectively. Bio-guided fractionation led to an ethyl acetate fraction with IC₅₀s of 2.68 and 1.85 μg/mL and subsequently, to the new quinovic acid saponin named xanthoxyloside ( 1 ) with IC₅₀s of 0.33 and 1.30 μM, respectively against the tested strains. Further compounds obtained from ethyl acetate and hexane fractions were the known clethric acid ( 2 ), ursolic acid ( 3 ), quafrinoic acid ( 4 ), quinovic acid ( 5 ), quinovic acid 3-O-β-D-fucopyranoside ( 6 ), oleanolic acid ( 7 ), oleanolic acid 3-acetate ( 8 ), friedelin ( 9 ), β-sitosterol ( 10a ), stigmasterol ( 10b ) and stigmasterol 3-O-β-D-glucopyranoside ( 11 ). Their structures were characterised with the aid of comprehensive spectroscopic methods (1 and 2D NMR, Mass). Bio-assays were performed using nucleic acid gel stain (SYBR green I)-based fluorescence assay with chloroquine as reference. Extracts and compounds exhibited good selectivity indices (SIs) of >10. Significant antiplasmodial activities measured for the crude extract, the ethyl acetate fraction and xanthoxyloside ( 1 ) from that fraction can justify the use of the root of N. xanthoxylon in ethnomedicine to treat malaria.     

Polar uncoating of tobacco mosaic virus (TMV) with dimethylsulfoxide (DMSO) and subsequent reassembly of partially stripped TMV

Summary Increasing concentrations of dimethylsulfoxide (DMSO) strip tobacco mosaic virus (TMV) stepwise from the 3 end. The RNA tail increases in length up to 2,000 nucleotides (nu) reaching a region of very strong protein-RNA affinity. Thereafter, uncoating occurs from the other end and produces a second RNA tail 500 nu long. Further stripping of TMV proceeds from both ends, the long tail increasing in length up to 4,000 nu and the short one increasing more moderately and remaining below 2,000 nu. The region of strongest protein-RNA affinity is located between 4,000 and 5,000 nu away from the 3 end.Using the same conditions as for in vitro TMV reassembly, it is possible to recoat the RNA tails with viral protein preferentially in the 5 direction.The advantages of DMSO in studies of TMV protein-RNA interactions are discussed.     

Packing analysis of carbohydrates and polysaccharides. V. Crystal structures of two polymorphs of pachyman triacetate

The crystal structures of two polymorphic forms of pachyman triacetate, the fully acetylated derivative of a naturally occuring β-(1 → 3)-D -glucan, were determined by a combination of stereochemical and x-ray diffraction analysis. The two polymorphs could be obtained depending on the temperature and the degree of stretching of film specimens of the substance: polymorph I resulted from stretching 25–50% at 125°C and polymorph II resulted from further stretching to 300% at 215°C. Both polymorphs had previously been shown to have sixfold helical chain conformations, but of unequal pitch. Subsequent detailed structure refinement performed with bond lengths, bond angles, conformational angles, and helix-packing parameters as refinement variables, and the simultaneous minimization of packing and conformational energy and the crystallographic R-factor as refinement criteria, resulted in a complete determination of the two crystal structures. Pachyman triacetate I was found to be a right-handed helix packing with antiparallel polarity and space group P2₁2₁2₁ symmetry (unit-cell parameters a = 11.0, b = 19.0, c (fiber repeat) = 22.38 Å). The acetate groups were nearly planar and the O(2) and O(4) acetates were oriented in such a fashion that the carbonyl double-bond nearly eclipsed the corresponding C—H bond of the ring. The O(6) was in the tg position and its acetate was oriented in such a fashion that the bond sequence C(6)—O(6)—C(6C)—C(6M) was nearly trans-planar, with the carbonyl double-bond bisecting the tetrahedral angle formed by C(6) and its two hydrogens. The final R = 0.221. Pachyman triacetate II was similarly found to be a right-handed helix, but packing as a 50:50 mixture of parallel and antiparallel polarities (unit-cell parameters a = 11.49, b = 20.13, c (fiber repeat) = 18.6 Å). The acetate positions in pachyman triacetate II were substantially the same as in pachyman triacetate I. The final R for the 50:50 mixture was 0.234. Probable reasons for the change in packing polarities are discussed, as are the difficulties encountered in the structure refinement of acetate derivatives.     

Regression of Fibrosis and Reversal of Cirrhosis in Rats by Galectin Inhibitors in Thioacetamide-Induced Liver Disease

Galectin-3 protein is critical to the development of liver fibrosis because galectin-3 null mice have attenuated fibrosis after liver injury. Therefore, we examined the ability of novel complex carbohydrate galectin inhibitors to treat toxin-induced fibrosis and cirrhosis. Fibrosis was induced in rats by intraperitoneal injections with thioacetamide (TAA) and groups were treated with vehicle, GR-MD-02 (galactoarabino-rhamnogalaturonan) or GM-CT-01 (galactomannan). In initial experiments, 4 weeks of treatment with GR-MD-02 following completion of 8 weeks of TAA significantly reduced collagen content by almost 50% based on Sirius red staining. Rats were then exposed to more intense and longer TAA treatment, which included either GR-MD-02 or GM-CT-01 during weeks 8 through 11. TAA rats treated with vehicle developed extensive fibrosis and pathological stage 6 Ishak fibrosis, or cirrhosis. Treatment with either GR-MD-02 (90 mg/kg ip) or GM-CT-01 (180 mg/kg ip) given once weekly during weeks 8–11 led to marked reduction in fibrosis with reduction in portal and septal galectin-3 positive macrophages and reduction in portal pressure. Vehicle-treated animals had cirrhosis whereas in the treated animals the fibrosis stage was significantly reduced, with evidence of resolved or resolving cirrhosis and reduced portal inflammation and ballooning. In this model of toxin-induced liver fibrosis, treatment with two galectin protein inhibitors with different chemical compositions significantly reduced fibrosis, reversed cirrhosis, reduced galectin-3 expressing portal and septal macrophages, and reduced portal pressure. These findings suggest a potential role of these drugs in human liver fibrosis and cirrhosis.     

Phytochemical composition of Cymbopogon citratus and Eucalyptus citriodora essential oils and their anti-inflammatory and analgesic properties on Wistar rats

Cymbopogon citratus and Eucalyptus citriodora are widely used herbs/plants as a source of ethnomedicines in tropical regions of the world. In this work, we studied the anti-inflammatory and gastroprotective effects of C. citratus and E. citriodora essential oils on formol-induced edema, and acetic acid induced abdominal cramps in Wistar rats. To fully understand the chemically induced anti-inflammatory properties of these plants, we first analyzed the chemical composition of the essential oils. A total of 16 chemical constituents accounting for 93.69 % of the oil, were identified in C. citratus among which, Geranial (27.04 %), neral (19.93 %) and myrcene (27.04 %) were the major constituents. For E. citriodora, 19 compounds representing 97.2 % of the extracted oil were identified. The dominant compound of E. citriodora essential oil was citronellal (83.50 %). In vivo analysis and histological assay showed that the two essential oils displayed significant dose dependent edema inhibition effect over time. They displayed strong analgesic and antipyretic properties similar to that induced by 50 mg/kg of acetylsalicylate of lysine. However, the E. citriodora essential oil was more effective than that of C. citratus. We identified significant numbers of aldehyde molecules in both essential oils mediating antioxidant activity that may contribute to the anti-inflammatory effects observed on the rats. Altogether, this work demonstrates the anti-inflammatory property of C. citratus and E. citriodora suggesting their potential role as adjuvant therapeutic alternatives in dealing with inflammatory-related diseases.     

Analysis of Allele-Specific Expression in Mouse Liver by RNA-Seq: A Comparison With Cis-eQTL Identified Using Genetic Linkage

We report an analysis of allele-specific expression (ASE) and parent-of-origin expression in adult mouse liver using next generation sequencing (RNA-Seq) of reciprocal crosses of heterozygous F₁ mice from the parental strains C57BL/6J and DBA/2J. We found a 60% overlap between genes exhibiting ASE and putative cis-acting expression quantitative trait loci (cis-eQTL) identified in an intercross between the same strains. We discuss the various biological and technical factors that contribute to the differences. We also identify genes exhibiting parental imprinting and complex expression patterns. Our study demonstrates the importance of biological replicates to limit the number of false positives with RNA-Seq data.     

Linkers of Cell Polarity and Cell Cycle Regulation in the Fission Yeast Protein Interaction Network

The study of gene and protein interaction networks has improved our understanding of the multiple, systemic levels of regulation found in eukaryotic and prokaryotic organisms. Here we carry out a large-scale analysis of the protein-protein interaction (PPI) network of fission yeast (Schizosaccharomyces pombe) and establish a method to identify ‘linker’ proteins that bridge diverse cellular processes - integrating Gene Ontology and PPI data with network theory measures. We test the method on a highly characterized subset of the genome consisting of proteins controlling the cell cycle, cell polarity and cytokinesis and identify proteins likely to play a key role in controlling the temporal changes in the localization of the polarity machinery. Experimental inspection of one such factor, the polarity-regulating RNB protein Sts5, confirms the prediction that it has a cell cycle dependent regulation. Detailed bibliographic inspection of other predicted ‘linkers’ also confirms the predictive power of the method. As the method is robust to network perturbations and can successfully predict linker proteins, it provides a powerful tool to study the interplay between different cellular processes.