Purification and characterization of a methanol dehydrogenase derived fromMethylomicrobium sp. HG-1 cultivated using a compulsory circulation diffusion system

Methanotrophs are microorganisms that possess the unique ability to utilize methane as their sole source of carbon and energy. A novel culture system, known as the compulsory circulation diffusion system, was developed for rapid growth of methanotrophic bacteria. Methanol dehydrogenase (MDH, EC 1.1.99.8) fromMethylomicrobium sp. HG-1, which belongs to the type 1 group of methanotrophic bacteria, can catalyze the oxidation of methanol directly into formaldehyde. This enzyme was purified 8-fold to electrophoretic homogeneity by means of a 4 step procedure and was found in the soluble fraction. The relative molecular weight of the native enzyme was estimated by gel filtration to be 120 kDa. The enzyme consisted of two identical dimers which, in turn, consisted of large and small subunits in anα ₂ β ₂ conformation. The isoelectric point was 5.4. The enzymatic activity of purified MDH was optimum at pH 9.0 and 60°C, and remained stable at that temperature for 20 min. MDH was able to oxidize primary alcohols from methanol to octanol and formaldehyde.     

One-step enzymatic synthesis of blue pigments from geniposide for fabric dyeing

In this study, we describe a one-step chemoenzymatic reaction for the production of natural blue pigments, in which the geniposide from Gardenia extracts is transformed by glycosidases to genipin. Genipin is then allowed to react with amino acids, thereby generating a natural blue pigment. The β-glycosidases, most notably isolase (a variant of β-glucanase), recombinant β-glucosidase, Cellulase T, and amylases, were shown to hydrolyze geniposide to produce the desired pigments, whereas the α-glycosidases did not. Among the 20 tested amino acids, glycine and tyrosine were associated with the highest dye production yields. The optimal molar ratio of geniposide to glycine, two reactants relevant to pigment production, was unity. The natural blue pigments produced in this study were used to dye cotton, silk, and wool. The color yields of the pigments were determined to be significantly higher than those of other natural dyes. Furthermore, the color fastness properties of these dyes were fairly good, even in the absence of mordant.     

Fabrication of disposable protein chip for simultaneous sample detection

In this study, we have described a method for the fabrication of a protein chip on silicon substrate using hydrophobic thin film and microfluidic channels, for the simultaneous detection of multiple targets in samples. The use of hydrophobic thin film provides for a physical, chemical, and biological barrier for protein patterning. The microfluidic channels create four protein patterned strips on the silicon surfaces with a high signal-to-noise ratio. The feasibility of the protein chips was determined in order to discriminate between each protein interaction in a mixture sample that included biotin, ovalbumin, hepatitis B antigen. In the fabrication of the multiplexed assay system, the utilization of the hydrophobic thin film and the microfluidic networks constitutes a more convenient method for the development of biosensors or biochips. This technique may be applicable to the simultaneous evaluation of multiple protein-protein interactions.     

Gene targets for fungal and mycotoxin control

It was initially shown that gallic acid, from hydrolysable tannins in the pelliele of walnut kernels, dramatically inhibits biosynthesis of aflatoxin byAspergillus flavus. The mechanism of this inhibition was found to take place upstream from the gene cluster, including the regulatory gene,aflR, involved in aflatoxin biosynthesis. Additional research using other antioxidant phenolics showed similar antiaflatoxigenic activity to gallic acid. Treatment ofA. flavus withtert-butyl hydroperoxide resulted in an almost doubling of aflatoxin biosynthesis compared to untreated samples. Thus, antioxidative response systems are potentially useful molecular targets for control ofA. flavus. A high throughput screening system was developed using yeast,Saccharomyces cerevisiae, as a model fungus. This screening provided an avenue to quickly identify fungal genes that were vulnerable to treatment by phenolic compounds. The assay also provided a means to quickly assess effects of combinations of phenolics and certain fungicides affecting mitochondrial respiration. For example, theS. cerevisiae sod2† mutant was highly sensitive to treatment by certain phenolics and strobilurins/antimycin A, fungicides which inhibit complex III of the mitochondrial respiratory chain. Verification of stress to this system in the target fungus,A. flavus, was shown through complementation analysis, wherein the mitochondrial superoxide dismutase (Mn-SOD) gene (sodA) ofA. flavus in the ortholog mutant,sod2†, ofS. cerevisiae, relieved phenolic-induced stress. Mitochondrial antioxidative stress systems play an important role in fungal response to antifungals. Combined treatment of fungi with phenolics and inhibitors of mitochondrial respiration can effectively suppress growth ofA. flavus in a synergistic fashion.     

The presence of natural human antibodies reactive against pharmacologically active pectic polysaccharides from herbal medicines

Direct ELISA was performed using normal human sera and human colostrum, to analyse the presence of antibodies which react with pharmacologically active pectic polysaccharides isolated from plants used in traditional Japanese herbal (Kampo) medicine. All sera and colostrum were shown to contain IgM, IgG, IgA and secretory IgA class antibodies which react with the active pectic polysaccharides to different degrees. The reacting IgG antibody in normal human serum recognized the ramified regions (rhamnogalacturonan core with carbohydrate side-chains) of the pharmacologically active pectic polysaccharides as the active sites for complement-activating activity. Correlation analysis indicated that a significant and positive correlation was observed between reactivity with the reacting antibody of IgG class and the degree of complement-activating activity of the active polysaccharides.The reacting IgG class antibody, which was purified from normal human serum by affinity chromatography on bupleuran 2IIc (a pharmacologically active pectic polysaccharide from the roots of Bupleurum falcatum)-immobilized Sepharose, showed cross-reactivity not only with some other pharmacologically active pectic polysaccharides from other medicinal herbs but also with autoantigens such as single-strand DNA, myosin and tublin from mammals.     

Efficient q-gram filters for finding all epsilon-matches over a given length.

Fast and exact comparison of large genomic sequences remains a challenging task in biosequence analysis. We consider the problem of finding all epsilon-matches between two sequences, i.e., all local alignments over a given length with an error rate of at most epsilon. We study this problem theoretically, giving an efficient q-gram filter for solving it. Two applications of the filter are also discussed, in particular genomic sequence assembly and BLAST-like sequence comparison. Our results show that the method is 25 times faster than BLAST, while not being heuristic.     

MuPIT interactive: webserver for mapping variant positions to annotated, interactive 3D structures

Mutation position imaging toolbox (MuPIT) interactive is a browser-based application for single-nucleotide variants (SNVs), which automatically maps the genomic coordinates of SNVs onto the coordinates of available three-dimensional (3D) protein structures. The application is designed for interactive browser-based visualization of the putative functional relevance of SNVs by biologists who are not necessarily experts either in bioinformatics or protein structure. Users may submit batches of several thousand SNVs and review all protein structures that cover the SNVs, including available functional annotations such as binding sites, mutagenesis experiments, and common polymorphisms. Multiple SNVs may be mapped onto each structure, enabling 3D visualization of SNV clusters and their relationship to functionally annotated positions. We illustrate the utility of MuPIT interactive in rationalizing the impact of selected polymorphisms in the PharmGKB database, somatic mutations identified in the Cancer Genome Atlas study of invasive breast carcinomas, and rare variants identified in the exome sequencing project. MuPIT interactive is freely available for non-profit use at http://mupit.icm.jhu.edu.     

The Reverse Roles of Transient Receptor Potential Canonical Channel-3 and -6 in Neuronal Death Following Pilocarpine-Induced Status Epilepticus

Transient receptor potential canonical channel (TRPC) is a nonselective cation channel permeable to Ca²⁺, which is expressed in many cell types, including neurons. However, the alterations in TRPC receptor expressions in response to status epilepticus (SE) have not been explored. Therefore, the present study was designated to elucidate the roles of TRPC3 and TRPC6 in neuronal death following SE. In non-SE animals, TRPC3 and TRPC6 immunoreactivity was abundantly detected in the dendrites of pyramidal cells and the cell bodies of dentate granule cells. Following SE, TRPC3 expression was significantly elevated in CA1-, CA3 pyramidal cells, and dentate granule cells, while TRPC6 expression was reduced in these regions. Pyrazole-3 (a TRPC3 inhibitor) effectively prevented up-regulation of neuronal TRPC3 expression induced by SE. Hyperforin (a TRPC6 activator) effectively prevented down-regulation of neuronal TRPC6 expression induced by SE. In addition, both Pyr3 and hyperforin effectively protected neuronal damages from SE. Therefore, the present study yields novel information regarding the role of TRPC3 and 6 in epileptogenic insults and suggests that TRPC 3 and 6 may be involved in neurodegeneration following SE.