N-Formylcytisine: A new alkaloid from Thermopsis chinensis

A new base have been isolated from Thermopsis chinensis along with N-methylcytisine, cytisine, anagyrine and lupanine. The new alkaloid have been shown to be N-formylcytisine.     

Efficient isolation of human parainfluenza viruses 1 and 3 using MNT‐1, a human malignant melanoma cell line system that exhibits an apparent cytopathic effect

Isolation of human parainfluenza virus (HPIV) serotypes 1 and 3 from clinical specimens is not very efficient because of the lack of a cell culture system capable of inducing CPE. In this study, the utility of a melanoma cell line, MNT‐1, that allows HPIV growth and displays CPE was demonstrated. In particularly, the efficiency of isolating HPIV1 and HPIV3 using MNT‐1 was greater than for cell lines conventionally used for HPIV isolation. Our demonstrated efficacy of HPIV1 and HPIV3 isolation with apparent CPE using the MNT‐1 cell culture system has the potential to improve virus isolation from clinical specimens.     

Knockdown of Carotenoid Cleavage Dioxygenase 4 (CCD4) via Virus-Induced Gene Silencing Confers Yellow Coloration in Peach Fruit: Evaluation of Gene Function Related to Fruit Traits

Plant Molecular Biology Reporter - Transgenic approach is an excellent way for the clarification of gene function, but it is generally difficult to create transgenic plants for most of the fruit...     

Yeast mutant with efficient secretion identified by a novel secretory reporter, Cluc

Yeast is an important host for the production of pharmaceutical or industrial proteins by virtue of its genetic information and easy handling. A number of heterologous proteins have been produced and purified from yeast cell cultures as secreted forms. Here, we describe a novel screening system of Saccharomyces cerevisiae and its application to improve the secretion efficiency of yeast. In our system, a natural secretory luciferase from Cypridina noctiluca is used as a reporter enzyme. The accumulation of enzymatically active luciferase in culture medium makes it possible to screen many samples simultaneously in a simple and sensitive assay. Using this system, we have discovered that the deletion mutant of MON2, which encoded a scaffold protein for vesicle formation located at the late Golgi, secreted luciferase highly efficiently to the extracellular space. Thus, we conclude that this new reporter assay is useful for the improvement and screening of yeast secretory strains.     

RCAI-17, 22, 24-26, 29, 31, 34-36, 38-40, and 88, the analogs of KRN7000 with a sulfonamide linkage: their synthesis and bioactivity for mouse natural killer T cells to produce Th2-biased cytokines

RCAI-17, 22, 24-26, 29, 31, 34-36, 38-40, and 88, the analogs of KRN7000 with a sulfonamide linkage instead of an amide bond, were synthesized to examine their bioactivity for mouse natural killer (NK) T cells. RCAI-17, 22, 24-26, 29, 31, 34-36, and 88 are the aromatic sulfonamide analogs, while RCAI-39 and 40 are the aliphatic ones. RCAI-38 is a C-galactoside analog of RCAI-26, which is the p-toluenesulfonamide analog of KRN7000. According to their bioassay, these sulfonamide analogs were shown to be the stimulants of mouse NKT cells to induce the production of Th2-biased cytokines in vitro, while RCAI-38 did not induce any cytokine production.     

Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenic tissue

A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on hygromycin and kanamycin medium, respectively. Transgenic plants were regenerated through somatic embryogenesis in 4 lines out of these 11 lines. Green fluorescent protein fluorescence was observed under fluorescent microscopy. Integration of the genes into the genome was confirmed by polymerase chain reaction analysis of embryogenic tissues and Southern blot analysis of regenerated plantlets.     

Metabolism of hydroxylated PCB congeners by cloned laccase isoforms

The white-rot fungus T. versicolor UAMH 8272 produced two groups of laccases, each of which included several isoforms showing different isoelectric points (pI). Group 1 and group 2 laccases, respectively, displayed higher pI 5–6 and lower pI 3–4. Of the four cloned full-length laccase cDNAs, Lac 1 and Lac 4 were expressed in the heterologous protein expression system using Aspergillus oryzae. The measured pI of each Lac 1 and Lac 4 expressed in A. oryzae was lower than that of pI predicted from the amino acid composition. With this regard, isoelectric focusing of Lac 1 showed the presence of multiple protein bands in the 3.0–4.0 pI range, although the predicted pI value of Lac 1 was 4.7. Similarly, Lac 4 exhibited a pI value which was lower than that predicted (3.6 vs. 4.3, respectively). In all tested hydroxyPCBs, higher chlorinated hydroxyPCBs were less susceptible to in vitro degradation by laccase than lower chlorinated hydroxyPCBs. Although Lac 4 showed a generally higher activity than Lac 1, the two laccases were characterized by quite different substrate specificity toward two hydroxy-tetrachlorobiphenyl congeners. Two metabolites were obtained from the metabolism of hydroxy-pentachlorobiphenyl: a ten chlorine-substituted dimer with a C–O bond, and one with a C–C bond.     

A bioluminescent enzyme immunoassay for prostaglandin E2 using Cypridina luciferase

Prostaglandin E₂ is one of the major cyclooxygenase metabolites of arachidonic acid. We developed a competitive immunosorbent assay for prostaglandin E₂ utilizing a bioluminescent enzyme Cypridina luciferase. The prostaglandin E₂ amount could be quantified over the concentration ranging from 7.8 to 500 pg/mL. The amount of unlabeled prostaglandin E₂ required to displace 50% of the maximal binding of Cypridina luciferase‐labeled prostaglandin E₂ (B/B₀) was approximately 35 pg/mL. The results show a great potential of Cypridina luciferase as a new labeling enzyme for enzyme‐linked immunosorbent assay. Copyright © 2009 John Wiley & Sons, Ltd.     

High-Sensitivity Real-Time Imaging of Dual Protein-Protein Interactions in Living Subjects Using Multicolor Luciferases

Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects.