Although cocaine abuse and addiction continue to cause serious health and societal problems, an FDA-approved medication to treat cocaine addiction has yet to be developed. Employing a pharmacokinetic strategy, an anticocaine vaccine provides an attractive avenue to address these issues; however, current vaccines have shown varying degrees of efficacy, indicating that further formulation is necessary. As a means to improve vaccine efficacy, we examined the effects of varying anticocaine vaccine formulations by combining a Toll-like receptor 9 (TLR9) agonist with a TLR5 agonist in the presence of alum. The TLR9 agonist used was cytosine-guanine oligodeoxynucleotide 1826 (CpG 1826), while the TLR5 agonist was flagellin (FliC). Formulations with the TLR9 agonist elicited superior anticocaine antibody titers and blockade of hyperlocomotor effects compared to vaccines without CpG 1826. This improvement was seen regardless of whether the TLR5 agonist, FliC, or the nonadjuvanting Tetanus Toxoid (TT) was used as the carrier protein. Additional insights into the value of FliC as a carrier versus adjuvant was also investigated by generating two unique formats of the protein, wild-type and mutated flagellin (mFliC). While the mFliC conjugate retained its ability to stimulate mTLR5, it yielded reduced cocaine sequestration and functional blockade relative to FliC and TT. Overall, this work indicates that activation of TLR9 can improve the function of cocaine vaccines in the presence of TLR5 activation by FliC, with any potential additive effects limited by the inefficiency of FliC as a carrier protein as compared to TT.
Amino acid substitutions at two residues downstream from the active-site histidine of polyhydroxyalkanoate (PHA) synthases are effective for changing the composition and the molecular weight of PHA. In this study, saturation mutagenesis at the position Ala505 was applied to PHA synthase (PhaCAc) from Aeromonas caviae to investigate the effects on the composition and the molecular weight of PHA synthesized in Ralstonia eutropha. The copolymer composition and molecular weight of PHA were varied by association with amino acid substitutions. There was a strong relationship between copolymer composition and PHA synthase activity of the cells. This finding will serve as a rationale for producing tailor-made PHAs.
Pseudozyma antarctica is a non-pathogenic phyllosphere yeast known as an excellent producer of mannosylerythritol lipids (MELs), multi-functional extracellular glycolipids, from vegetable oils. To clarify the genetic characteristics of P. antarctica, we analyzed the 18 Mb genome of P. antarctica T-34. On the basis of KOG analysis, the number of genes (219 genes) categorized into lipid transport and metabolism classification in P. antarctica was one and a half times larger than that of yeast Saccharomyces cerevisiae (140 genes). The gene encoding an ATP/citrate lyase (ACL) related to acetyl-CoA synthesis conserved in oleaginous strains was found in P. antarctica genome: the single ACL gene possesses the four domains identical to that of the human gene, whereas the other oleaginous ascomycetous species have the two genes covering the four domains. P. antarctica genome exhibited a remarkable degree of synteny to U. maydis genome, however, the comparison of the gene expression profiles under the culture on the two carbon sources, glucose and soybean oil, by the DNA microarray method revealed that transcriptomes between the two species were significantly different. In P. antarctica, expression of the gene sets relating fatty acid metabolism were markedly up-regulated under the oily conditions compared with glucose. Additionally, MEL biosynthesis cluster of P. antarctica was highly expressed regardless of the carbon source as compared to U. maydis. These results strongly indicate that P. antarctica has an oleaginous nature which is relevant to its non-pathogenic and MEL-overproducing characteristics. The analysis and dataset contribute to stimulate the development of improved strains with customized properties for high yield production of functional bio-based materials.
In vitro and in situ enzymatic polymerization of polyhydroxyalkanoate (PHA) on two hydrophobic surfaces, a highly oriented pyrolytic graphite (HOPG) and an alkanethiol self-assembled monolayer (SAM), was studied by atomic force microscopy (AFM) and quartz crystal microbalance (QCM), using purified Ralstonia eutropha PHA synthase (PhaC(Re)) as a biocatalyst. (R)-Specific enoyl-CoA hydratase was used to prepare R-enantiomer monomers [(R)-3-hydroxyacyl-CoA] with an acyl chain length of 4-6 carbon atoms. PHA homopolymers with different side-chain lengths, poly[(R)-3-hydroxybutyrate] [P(3HB)] and poly[(R)-3-hydroxyvalerate] [P(3HV)] were successfully synthesized from such R-enantiomer monomers on HOPG substrates. After the reaction, the surface morphologies were analyzed by AFM, revealing a nanometer thick PHA film. The same biochemical polymerization process was observed on an alkanethiol (C18) SAM surface fabricated on a gold electrode using QCM. This analysis showed that a complex sequence of PhaC(Re) adsorption and PHA polymerization has occurred on the hydrophobic surface. On the basis of these observations, the possible mechanisms of the PhaC(Re)-catalyzed polymerization reaction on the surface of hydrophobic substrates are proposed.
Bioethanol has recently become an important resource for chemical industries. The chemical compositions of 17 different types of bioethanol were investigated with a focus on impurities that could affect catalytic performances in the downstream chemical processes. Lignocellulosic ethanol contained higher concentrations and a greater variety of organic impurities compared to sugar-or starch-derived bioethanol. Twenty-nine impurities were identified in lignocellulosic ethanol, whereas 16 impurities were in sugar-or starch-derived bioethanol. Lignocellulosic ethanol contained high concentrations of acetic acid, acetaldehyde, methanol, and furan-related compounds such as furfural. In contrast, with the exception of molasses-derived bioethanol obtained by crude distillation, the concentrations of these components were lower in sugar-or starch-derived bioethanol samples. Lignocellulosic ethanol contained dimethyl disulfide and thiazole, whereas the only organosulfur compounds found in sugar-or starch-derived bioethanol were dimethyl sulfide and dimethyl sulfoxide. These sulfur-containing impurities can cause catalyst deactivation in the bioethanol transformation processes. In lignocellulosic ethanol, more than 0.1 mg/mL of Si was detected.
This study was undertaken to examine the antimicrobial property of azuki beans (Vigna angularis). The water extracts of green, black and red colored azuki beans showed antibacterial effects against Staphylococcus aureus, Aeromonas hydrophila and Vibrio parahaemolyticus. In contrast, the extract of white azuki beans showed no inhibition towards any of the microorganisms examined. The extracts of colored azuki beans contained larger amounts of polyphenols including proanthocyanidins than the extracts of white azuki beans. The counts of S. aureus cells, inoculated in the medium containing the extracts of colored azuki beans, were significantly reduced in comparison with those of control and white azuki beans after 24 h (p < 0.05). These results suggest that polyphenols including proanthocyanidins in colored azuki beans may be responsible for their antibacterial activity.
Eggs play important roles as food resources and nutraceuticals, to alleviate malnutrition and to improve health status in the world. Since free amino acids contribute to the nutritional values and food tastes, we investigated a total of 81 eggs from five chicken breeds, which are Australorp, Nagoya (NGY), Rhode Island Red (RIR), Shamo (SHA), Ukokkei, and two F1 hybrids (NGYxRIR and SHAxRIR) to test impact on genetic differences in 10 egg traits, 20 yolk amino acid traits, and 18 albumen amino acid traits. One-way ANOVA revealed significant breed effects on 10 egg traits, 20 yolk amino acid traits, and 15 albumen amino acid traits. Moreover, a significant heterosis effect on yolk aspartic acid was identified. In addition, positive correlations were found broadly among traits within each trait category (egg traits, yolk amino acid traits, and albumen amino acid traits), whereas there were basically no or weak correlations among the trait categories. These results suggest that almost all traits can be dramatically modified by genetic factor, and there will be partially independent production systems of amino acids into yolk and albumen. Since there will be typical quantitative genetic architecture of egg contents, further genetic analyses will be needed.
We investigated the expression of (R)-specific enoyl coenzyme A hydratase (PhaJ) in Pseudomonas putida KT2440 accumulating polyhydroxyalkanoate (PHA) from sodium octanoate in order to identify biosynthesis pathways of PHAs from fatty acids in pseudomonads. From a database search through the P. putida KT2440 genome, an additional phaJ gene homologous to phaJ4(Pa) from Pseudomonas aeruginosa, termed phaJ4(Pp), was identified. The gene products of phaJ1(Pp), which was identified previously, and phaJ4(Pp) were confirmed to be functional in recombinant Escherichia coli on PHA synthesis from sodium dodecanoate. Cytosolic proteins from P. putida grown on sodium octanoate were subjected to anion exchange chromatography and one of the eluted fractions with hydratase activity included PhaJ4(Pp), as revealed by western blot analysis. These results strongly suggest that PhaJ4(Pp) forms a channeling route from β-oxidation to PHA biosynthesis in P. putida. Moreover, the substrate specificity of PhaJ1(Pp) was suggested to be different from that of PhaJ1(Pa) from P. aeruginosa although these two proteins share 67% amino acid sequence identity.
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