Purification and characterization of a kanamycin nucleotidyltransferase from plasmid pUB110-carrying cells of Bacillus subtilis.

The nucleotidyltransferase encoded by plasmid pUB110 was purified to greater than 95% purity with a 33% yield. The enzyme is a monomeric protein with a molecular weight of 34,000. The optimum pH for activity is 5, and the optimum MgCl2 concentration for activity is 18 mM. The enzyme, which is synthesized constitutively, is stable for several weeks at 4 degrees C. This enzyme would appear to be a good model gene product for the development of a pUB110 deoxyribonucleic acid-dependent in vitro protein-synthesizing system from Bacillus subtilis.     

Highly Stable Plasmonic Nanostructures on a Nickel-Sputtered Glass and Polymeric Optical Fiber Sensors

Plasmonics - This study shows development of highly sensitive and stable localized surface plasmon resonance (LSPR)-active U-bent glass and polymeric optical fiber (GOF and POF) sensor probes by a...     

Nucleotide excision repair endonuclease genes in Drosophila melanogaster

Nucleotide excision repair (NER) is the primary pathway for the removal of ultraviolet light-induced damage and bulky adducts from DNA in eukaryotes. During NER, the helix is unwound around the damaged site, and incisions are made on the 5' and 3' sides, to release an oligonucleotide carrying the lesion. Repair synthesis can then proceed, using the intact strand as a template. The incisions flanking the lesion are catalyzed by different structure-specific endonucleases. The 5' incision is made by a heterodimer of XPF and ERCC1 (Rad1p-Rad10p in Saccharomyces cerevisiae), and the 3' incision is made by XPG (Rad2p in S. cerevisiae). We previously showed that the Drosophila XPF homologue is encoded by the meiotic recombination gene mei-9. We report here the identification of the genes encoding the XPG and ERCC1 homologues (XPG(Dm) and ERCC1(Dm)). XPG(Dm) is encoded by the mus201 gene; we found frameshift mutations predicted to produce truncated XPG(Dm) proteins in each of two mus201 alleles. These mutations cause defects in nucleotide excision repair and hypersensitivity to alkylating agents and ultraviolet light, but do not cause hypersensitivity to ionizing radiation and do not impair viability or fertility. ERCC1(Dm) interacts strongly in a yeast two-hybrid assay with MEI-9, indicative of the presumed requirement for these polypeptides to dimerize to form the functional endonuclease. The Drosophila Ercc1 gene maps to polytene region 51D1-2. The nucleotide excision repair gene mus210 maps nearby (51E-F) but is distinct from Ercc1.     

Improving consensus structure by eliminating averaging artifacts

Background  

Common structural biology methods (i.e., NMR and molecular dynamics) often produce ensembles of molecular structures. Consequently, averaging of 3D coordinates of molecular structures (proteins and RNA) is a frequent approach to obtain a consensus structure that is representative of the ensemble. However, when the structures are averaged, artifacts can result in unrealistic local geometries, including unphysical bond lengths and angles.     

Leaf Litter Fuels Methanogenesis Throughout Decomposition in a Forested Peatland

Decomposing leaf litter is a large supply of energy and nutrients for soil microorganisms. How long decaying leaves continue to fuel anaerobic microbial activity in wetland ecosystems is poorly understood. Here, we compare leaf litter from 15 tree species with different growth forms (angiosperms and gymnosperms, deciduous, and longer life span), using litterbags positioned for up to 4 years in a forested peatland in New York State. Periodically, we incubated partially decayed residue per species with fresh soil to assess its ability to fuel microbial methane (CH₄) production and concomitant anaerobic carbon dioxide (CO₂) production. Decay rates varied by leaf type: deciduous angiosperm > evergreen gymnosperm > deciduous gymnosperm. Decay rates were slower in leaf litter with a large concentration of lignin. Soil with residue of leaves decomposed for 338 days had greater rates of CH₄ production (5.8 µmol g^?1 dry mass d^?1) than less decomposed (<0.42 µmol g^?1 dry mass d^?1) or more decomposed (2.1 µmol g^?1 dry mass d^?1) leaf residue. Species-driven differences in their ability to fuel CH₄ production were evident throughout the study, whereas concomitant rates of CO₂ production were more similar among species and declined with degree of decomposition. Methane production rates exhibited a positive correlation with pectin and the rate of pectin decomposition. This link between leaf litter decay rates, biochemical components in leaves, and microorganisms producing greenhouse gases should improve predictions of CH₄ production in wetlands.     

A microfluidic competitive immuno-aggregation assay for high sensitivity cell secretome detection

We report a high-sensitivity cell secretome detection method using competitive immuno-aggregation and a micro-Coulter counter. A target cell secretome protein competes with anti-biotin-coated microparticles (MPs) to bind with a biotinylated antibody (Ab), causing decreased aggregation of the functionalized MPs and formation of a mixture of MPs and aggregates. In comparison, without the target cell secretome protein, more microparticles are functionalized, and more aggregates are formed. Thus, a decrease in the average volume of functionalized microparticles/aggregates indicates an increase in cell secretome concentration. This volume change is measured by the micro-Coulter counter, which is used to quantitatively estimate the cell secretome concentration. Vascular endothelial growth factor (VEGF), one of the key cell secretome proteins that regulate angiogenesis and vascular permeabilization, was used as the target protein to demonstrate the sensing principle. A standard calibration curve was generated by testing samples with various VEGF concentrations. A detection range from 0.01 ng/mL to 100.00 ng/mL was achieved. We further demonstrated the quantification of VEGF concentration in exogenous samples collected from the secretome of human mesenchymal stem cells (hMSCs) at different incubation times. The results from the assay agree well with the results of a parallel enzyme-linked immunoabsorbent assay (ELISA) test, indicating the specificity and reliability of the competitive immuno-aggregation assay. With its simple structure and easy sample preparation, this assay not only enables high sensitivity detection of VEGF but also can be readily extended to other types of cell secretome analysis as long as the specific Ab is known.