1,8-Cineole inhibits root growth and DNA synthesis in the root apical meristem ofBrassica campestris L.

1,8-cineole is a volatile growth inhibitor produced bySalvia species. We examined the effect of this allelopathic compound on the growth of other plants usingBrassica campestris as the test plant. Cineole inhibited germination and growth ofB. campestris in a dosedependent manner. WhenB. campestris was grown for 5 days with various concentrations of cineole, the length of the roots was found to be shorter as the concentration of cineole increased, whereas the length of the hypocotyl remained constant up to 400 μM cineole, indicating that cineole specifically inhibited growth of the root. The mitotic index in the root apical meristem of 3-day-old seedlings decreased from 5.6% to 1.6% when exposed to 400 μM cineole, showing that cineole inhibits the proliferation of root cells. We then examined the effect of cineole on DNA synthesis by indirect immunofluorescence microscopy using antibody raised against 5-bromo-2′-deoxyuridine (BrdU, an analogue of thymidine) in thin sections of samples embedded in Technovit 7100 resin. The results clearly demonstrated that cineole inhibits DNA synthesis in both cell nuclei and organelles in root apical meristem, suggesting that cineole may interfere with the growth of other plant species by inhibiting DNA synthesis in the root apical meristem.     

An Improved Fluorometric High-Performance Liquid Chromatography Method for Sialic Acid Determination: An Internal Standard Method and Its Application to Sialic Acid Analysis of Human Apolipoprotein E

An improved fluorometric HPLC method for sialic acid determination was developed by employing synthetic N-propionylneuraminic acid (NPNA) as an internal standard. A fixed amount of NPNA was added to a sialoglycoconjugate sample. After hydrolyzing sialioglycoconjugates with diluted sulfuric acid, the released sialic acids and NPNA were derivatized with a fluorogenic compound, 1,2-diamino-4,5-(methylenedioxy)benzene (DMB), followed by fluorometric HPLC. The fluorescent derivative of NPNA was separated from those of N-acetylneuraminic acid, N-glycolylneuraminic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nonoic acid, and 2-keto-3-deoxyoctanoate on HPLC. The separation of NPNA derivative on HPLC was not interfered by components of biological samples such as human sera. Using this internal standard method, low amounts of NANA (0.15-1.0 ng) were quantified with the coefficient of variation values below 4%. Using this method, the sialic acid content of human apolipoprotein E was successfully determined. The present method is useful for sensitive and accurate quantification of sialic acids of different molecular species in biological samples.     

Microwave‐assisted solid‐phase peptide synthesis of neurosecretory protein GL composed of 80 amino acid residues

We recently identified a novel cDNA encoding a small secretory protein of 80 amino acid residues, termed neurosecretory protein GL (NPGL), from the chicken hypothalamus. Homologs of NPGL have been reported to be present in mammals, such as human and rat. NPGL is amidated at its C‐terminus, contains an intramolecular disulfide bond, and is hydrophobic in nature. In this study, we have optimized the synthesis of the entire 80‐amino acid peptide sequence of rat NPGL by microwave‐assisted solid‐phase peptide synthesis. NPGL was obtained with a 10% yield when the coupling reactions were performed using 1‐[Bis(dimethylamino)methylene]‐1H‐1,2,3‐triazolo[4,5‐b]pyridinium‐3‐oxid hexafluorophosphate (HATU) at 50 °C for 5 min, and Fmoc deprotections were performed using 40% piperidine containing 0.1 M HOBt. Furthermore, the disulfide bond of NPGL was formed with 20% yield with the use of glutathione‐containing redox buffer and 50% acetonitrile. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

A novel anti-inflammatory role for spleen-derived interleukin-10 in obesity-induced hypothalamic inflammation

Obesity can be associated with systemic low-grade inflammation that contributes to obesity-related metabolic disorders. Recent studies raise the possibility that hypothalamic inflammation contributes to the pathogenesis of diet-induced obesity (DIO), while another study reported that obesity decreases the expression of pro-inflammatory cytokines in spleen. The following study examines the hypothesis that obesity suppresses the splenic synthesis of the anti-inflammatory cytokine, interleukin (IL)-10, thereby resulting in chronic hypothalamic inflammation. The results showed that due to oxidative stress or apoptosis, the synthesis of splenic IL-10 was decreased in DIO when compared with non-obesity rats. Splenectomy (SPX) accelerated DIO-induced inflammatory responses in the hypothalamus. Interestingly, SPX suppressed the DIO-induced increases in food intake and body weight and led to a hypothalamic pro-inflammatory state that was similar to that produced by DIO, indicating that hypothalamic inflammation exerts a dual effect on energy metabolism. These SPX-induced changes were inhibited by the systemic administration of IL-10. Moreover, SPX had no effect on hypothalamic inflammatory responses in IL-10-deficient mice. These data suggest that spleen-derived IL-10 plays an important role in the prevention of hypothalamic inflammation and may be a therapeutic target for the treatment of obesity and hypothalamic inflammation.     

A retrospective analysis of germline competence in rat embryonic stem cell lines

The factors responsible for conferring germline competence in embryonic stem (ES) cell lines remain unidentified. In the present study, rat ES cell lines (n = 17) were established with 3i medium (SU5402, PD0325901, CHIR99021), 2i medium (PD0325901, CHIR99021) or 2iF medium (PD0325901, CHIR99021, forskolin), and their potential for germline transmission to the G1 generation was examined. Rat strains were divided into an albino group (F344, Wistar or CAG/Venus transgenic rats with the Wistar background) or a colored coat group (Brown-Norway, Dark-Agouti, or BLK rats selected from >F3 generations of Wistar × Dark-Agouti rats based on their black coat color). Successful germline transmission was observed in 57 % (4/7), 40 % (2/5) and 100 % (5/5) of the ES cells established with 3i, 2i and 2iF media, respectively. ES cell lines from the homozygous CAG/Venus transgenic rats were established in all three media, but only the lines established with the 2iF medium were germline-competent. Neither coat-color (albino: 64 %, 7/11; colored: 67 %, 4/6) nor gender of the ES cell lines (XX: 67 %, 2/3; XY: 64 %, 9/14) were likely to affect germline transmission.     

Transgenic animal production and animal biotechnology

Considerable progress has been made in methods for production of transgenic livestock; beginning with pronuclear microinjection over 20 years ago. New methods, including the use of viral vectors, sperm-mediated gene transfer and somatic cell cloning, have overcome many of the limitations of pronuclear microinjection. It is now possible to not only readily make simple insertional genetic modifications, but also to accomplish, more complex, homozygous gene targeting and artificial chromosome transfer in livestock.     

Purification and characterization of macrolide 2''-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics

Hyperimmune and high-titered polyclonal pneumococcal antisera, specific for cross-reactive types within groups, were produced in adult rabbits. Purified capsular polysaccharide was injected intravenously into adult rabbits. One week later, these rabbits were given multiple intravenous injections of formalin-inactivated pneumococci of the cross-reactive type by an established method. Each of the resultant antisera were specific for the cross-reactive type indicating that the previous injection of the polysaccharide had induced epitope-specific tolerance. This method was successful for production of antisera against pneumococcal types 6A, 6B, 9N, 9V, 19F and 19A. Polyclonal rabbit pneumococcal antisera have some advantages over murine monoclonal antibodies for serologic studies and this method should be applicable for producing type-specific antibodies to cross-reactive polysaccharides of clinical interest. Further, this method is simpler and generally produces higher titered monovalent (factor) reagents than absorbed antisera.