Composition and Role of Extracellular Polymers in Methanogenic Granules

Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an anaerobic granular consortium grown on a fatty acid mixture. The extracellular polysaccharides (EPS) were extracted from Methanobacterium formicicum and Methanosarcina mazeii and from the methanogenic granules to examine their role in granular development. The EPS made up approximately 20 to 14% of the extracellular polymer extracted from the granules, Methanobacterium formicicum, and Methanosarcina mazeii. The EPS produced by Methanobacterium formicicum was composed mainly of rhamnose, mannose, galactose, glucose, and amino sugars, while that produced by Methanosarcina mazeii contained ribose, galactose, glucose, and glucosamine. The same sugars were also present in the EPS produced by the granules. These results indicate that the two methanogens, especially Methanobacterium formicicum, contributed significantly to the production of the extracellular polymer of the anaerobic granules. Growth temperature, substrates (formate and H(inf2)-CO(inf2)), and the key nutrients (nitrogen and phosphate concentrations) affected polymer production by Methanobacterium formicicum.     

Plasmid-encoded degradation of p-nitrophenol and 4-nitrocatechol by Arthrobacter protophormiae

Arthrobacter protophormiae strain RKJ100 is capable of utilizing p-nitrophenol (PNP) as well as 4-nitrocatechol (NC) as the sole source of carbon, nitrogen and energy. The degradation of PNP and NC by this microorganism takes place through an oxidative route, as stoichiometry of nitrite molecules was observed when the strain was grown on PNP or NC as sole carbon and energy sources. The degradative pathways of PNP and NC were elucidated on the basis of enzyme assays and chemical characterization of the intermediates by TLC, GC, (1)H NMR, GC-MS, UV spectroscopy, and HPLC analyses. Our studies clearly indicate that the degradation of PNP proceeds with the formation of p-benzoquinone (BQ) and hydroquinone (HQ) and is further degraded via the beta-ketoadipate pathway. Degradation of NC involved initial oxidation to generate 1,2,4-benzenetriol (BT) and 2-hydroxy-1,4-benzoquinone; the latter intermediate is then reductively dehydroxylated, forming BQ and HQ, and is further cleaved via beta-ketoadipate to TCA intermediates. It is likely, therefore, that the same set of genes encode the further metabolism of HQ in PNP and NC degradation. A plasmid of approximately 65 kb was found to be responsible for harboring genes for PNP and NC degradation in this strain. This was based on the fact that PNP(-) NC(-) derivatives were devoid of the plasmid and had simultaneously lost their capability to grow at the expense of these nitroaromatic compounds.     

Stereoselective enzymatic hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol diacetate ester in a biphasic system

Summary A key chiral intermediate lactol(3)[3aS (3a,4,7,7a)]-hexahydro-4,7-epoxy-isobenzofuran-1 (3H)-one was prepared for the total synthesis of a new thromboxane antagonist. The stereoselective hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol, diacetate ester (1) to the corresponding chiral monoacetate ester (2) was carried out with lipases, among which Amano P-30 lipase from Pseudomonas sp. was most effective since it gave the desired enantiomer of monoacetate ester. A yield of 75 mol% and optical purity of >99% was obtained when the reaction was conducted in a biphasic system with 10% toluene at 5 g/l of the substrate. Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (five cycles) without loss of enzyme activity, productivity or optical purity. The reaction process was scaled-up to 80 1 (400 g substrate) and monoacetate (2) was isolated in 80 mol% yield with 99.3% optical purity as determined by chiral HPLC and nuclear magnetic resonance (NMR) analysis. A gas chromatography of 99.5% and specific rotation, []_D of -7.6° was obtained. The chiral monoacetate ester (2) was oxidized to its corresponding aldehyde and subsequently hydrolyzed to give lactol (3).     

Bayesian-based selection of metabolic objective functions

MOTIVATION: A critical component of in silico analysis of underdetermined metabolic systems is the identification of the appropriate objective function. A common assumption is that the objective of the cell is to maximize growth. This objective function has been shown to be consistent in a few limited experimental cases, but may not be universally appropriate. Here a method is presented to quantitatively determine the most probable objective function. RESULTS: The genome-scale metabolism of Escherichia coli growing on succinate was used as a case-study for analysis. Five different objective functions, including maximization of growth rate, were chosen based on biological plausibility. A combination of flux balance analysis and linear programming was used to simulate cellular metabolism, which was then compared to independent experimental data using a Bayesian objective function discrimination technique. After comparing rates of oxygen uptake and acetate production, minimization of the production rate of redox potential was determined to be the most probable objective function. Given the appropriate reaction network and experimental data, the discrimination technique can be applied to any bacterium to test a variety of different possible objective functions. SUPPLEMENTARY INFORMATION: Additional files, code and a program for carrying out model discrimination are available at http://www.engr.uconn.edu/~srivasta/modisc.html.     

Evaluation of cyclooxygenase 2 derived endogenous prostacyclin in mouse preimplantation embryo development in vitro

Cyclooxygenase (COX) plays an important role in prostaglandin (PG) synthesis and has two isoforms, COX1 and COX2. PGI synthase (PGIS) catalyzes the isomeization of PGH(2) to prostacyclin (PGI(2)). It is reported that COX2 derived PGI2(2) plays a critical role in blastocyst implantation and decidualization and PGI2 mediates its function via PPARdelta receptor. It is also known that cyclooxygenase derived prostaglandins play an important role in mouse blastocyst hatching in vitro. In this study we hypothesized that COX2 derived PGI2 plays an important role in preimplantation embryonic development by increasing the cell number. To examine this hypothesis, 8-cell stage mouse embryos were cultured in the presence of selective inhibitors of COX1 (SC560), COX2 (NS398) and PGIS (U51605) respectively. COX2 and PGIS inhibitor significantly reduced the blastocyst development and presence of PGI2 analogue along with these inhibitors restored the blastocyst development by increasing the total number of embryonic cells. Our immunohistochemical analysis showed that COX1 is expressed at 2-cell, 8-cell, compaction and blastocyst stage whereas COX2 expression starts from eight cell stage embryos. PGIS and PPARdelta expression starts at 2-cell stage of development. Our results suggest that PGI(2) may affect blastomeres number via the so called hypothesis of PPARdelta nuclear receptor in autocrine manner.     

Myogenesis defect due to Toca-1 knockdown can be suppressed by expression of N-WASP

Skeletal muscle formation is a multistep process involving proliferation, differentiation, alignment and fusion of myoblasts to form myotubes which fuse with additional myoblast to form myofibers. Toca-1 (Transducer of Cdc42-dependent actin assembly), is an adaptor protein which activates N-WASP in conjunction with Cdc42 to facilitate membrane invagination, endocytosis and actin cytoskeleton remodeling. Expression of Toca-1 in mouse primary myoblasts and C2C12 myoblasts was up-regulated on day 1 of differentiation and subsequently down-regulated during differentiation. Knocking down Toca-1 expression in C2C12 cells (Toca-1^KD cells) resulted in a significant decrease in myotube formation and expression of shRNA-resistant Toca-1 in Toca-1^KD cells rescued the myogenic defect, suggesting that the knockdown was specific and Toca-1 is essential for myotube formation. Toca-1^KD cells exhibited elongated spindle-like morphology, expressed myogenic markers (MyoD and MyHC) and localized N-Cadherin at cell periphery similar to control cells suggesting that Toca-1 is not essential for morphological changes or expression of proteins critical for differentiation. Toca-1^KD cells displayed prominent actin fibers suggesting a defect in actin cytoskeleton turnover necessary for cell–cell fusion. Toca-1^KD cells migrated faster than control cells and had a reduced number of vinculin patches similar to N-WASP^KO MEF cells. Transfection of N-WASP-expressing plasmid into Toca-1^KD cells restored myotube formation of Toca-1^KD cells. Thus, our results suggest that Toca-1^KD cells have defects in formation of myotubes probably due to reduced activity of actin cytoskeleton regulators such as N-WASP. This is the first study to identify and characterize the role of Toca-1 in myogenesis.     

A Novel Folate-Targeted Nanoliposomal System of Doxorubicin for Cancer Targeting

Targeted drug delivery systems for cancer improves anti-tumor efficacy and reduces systemic toxicity by restricting availability of cytotoxic drugs within tumors. Targeting moieties, such as natural ligands (folic acid, transferrin, and biotin) which are overexpressed on tumors, have been used to enhance liposome-encapsulated drug accumulation within tumors and resulted in better control. In this report, we explored the scope of targeting ligand folic acid, which is incorporated in liposome systems using folic acid-modified cholesterol (CPF), enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in increased cytotoxicity within tumors. Folate-tagged poloxamer-coated liposomes (FDL) were found to have significantly higher cellular uptake than conventional poloxamer-coated liposomes (DL), as confirmed by fluorometric analysis in B16F10 melanoma cells. Biodistribution study of the radiolabeled liposomal system indicated the significantly higher tumor uptake of FDL as compared to DL. Anti-tumor activity of FDL against murine B16F10 melanoma tumor-bearing mice revealed that FDL inhibited tumor growth more efficiently than the DL. Taken together, the results demonstrated the significant potential of the folate-conjugated nanoliposomal system for drug delivery to tumors.     

Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM

Over the last three decades, Cryo-TEM has developed into a powerful technique for high-resolution imaging of biological macromolecules in their native vitrified state. However, the method for vitrifying specimens onto EM grids is essentially unchanged - application of ～3 μL sample to a grid, followed by blotting and rapid plunge freezing into liquid ethane. Several trials are often required to obtain suitable thin (few hundred nanometers or less) vitrified layers amenable for cryo-TEM imaging, which results in waste of precious sample and resources. While commercially available instruments provide some level of automation to control the vitrification process in an effort to increase quality and reproducibility, obtaining satisfactory vitrified specimens remains a bottleneck in the Cryo-TEM pipeline. We describe here a completely novel method for EM specimen preparation based on small volume (picoliter to nanoliter) dispensing using inkjet technology. A first prototype system (Spotiton v0.5) demonstrates feasibility of this new approach for specimen vitrification. A piezo-electric inkjet dispenser is integrated with optical real-time cameras (100 Hz frame rate) to analyze picoliter to nanoliter droplet profiles in-flight and spreading dynamics on the grid, and thus provides a method to optimize timing of the process. Using TEM imaging and biochemical assays we demonstrate that the piezo-electric inkjet mechanism does not disrupt the structural or functional integrity of macromolecules. These preliminary studies provide insight into the factors and components that will need further development to enable a robust and repeatable technique for specimen vitrification using this novel approach.