Hot-water extract from mycelia of Cordyceps sinensis as a substitute for antibiotic growth promoters

Broiler chicks were orally dosed with a hot-water extract of mycelia from Cordyceps sinensis (CS-HW) to assess possible substitution of Avilamycin as an antibiotic growth promoter (AGP). The growth performance (body weight gain and survivability) and the health index (the microflora in the small intestines and the antibody titer to Newcastle disease virus) of chicks were significantly improved in the CS-HW (600 mg/kg diet) and the Avilamycin (20 mg/kg diet) fed group in comparison with the control group (p<0.05). The Avilamycin-fed group and the CS-HW-fed group had similar growth performances but the latter gave a better microbial flora in the small intestines. These results indicate that CS-HW enhances the physiological activity in chicks and can be used as a substitute for AGPs.     

Enhanced purification of plasmid DNA using Q-Sepharose by modulation of alcohol concentrations

Ion-exchange chromatography is one of the most commonly used methods for plasmid preparation. In this study a modified method was used to purify plasmid from bacterial lysate using Q-Sepharose. Incorporation of alcohols into the washing buffers enhanced the separation of plasmid from RNA and proteins. The use of isopropanol and ethanol achieved a high yield and purity whereas the use of methanol failed to improve the plasmid purification using Q-Sepharose by batch adsorption-desorption. Stepwise elution containing various concentrations of isopropanol and NaCl was used in preparative chromatography to enhance the plasmid purification. The same stepwise elution was applied to the chromatography columns packed with 0.5, 20, and 200 ml of Q-Sepharose for plasmid purification from 7.5, 300, and 3000 ml bacterial broth, respectively. Complete separation of DNA from RNA and proteins was achieved under gravity flow by modulation of the alcohol concentrations in the stepwise elution. These three scales of chromatography maintained an approximate plasmid yield and the purified plasmid contained undetectable levels of RNA and protein.     

Improvement and validation of a liquid chromatographic method for the determination of levosulpiride in human serum and urine

A rapid, selective and highly sensitive reversed-phase high-performance liquid chromatography (HPLC) method was developed for the determination of levosulpiride, 5-(aminosulfonyl)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxy benzamide, in human serum and urine. The method involved the extraction with a dichloromethane followed by back-extraction into 0.025 M sulfuric acid. HPLC analysis was carried out using reversed-phase isocratic elution with a Luna C(18)(2) 5 microm column, a mobile phase of acetonitrile-0.01 M potassium hydrogen phosphate (30:70, v/v, adjusted to pH 8.5 with triethylamine), and a fluorescence detector with excitation at 300 nm and emission at 365 nm. The chromatograms showed good resolution and sensitivity and no interference of human serum and urine. The calibration curves were linear over the concentration range 0.25-200 ng/ml for serum and 0.2-20 microg/ml for urine with correlation coefficients greater than 0.997. Intra- and inter-day assay precision and accuracy fulfilled the international requirements. The mean absolute recovery for human serum was 89.8+/-3.7%. The lower limits of quantitation in human serum and urine were 0.25 ng/ml and 0.2 microg/ml, respectively, which were sensitive enough for pharmacokinetic studies. Stability studies showed that levosulpiride in human serum and urine was stable during storage, or during the assay procedure. This method was successfully applied to the study of pharmacokinetics of levosulpiride in human volunteers following a single oral administration of levosulpiride (25 mg) tablet.     

Proteome analysis of hairy root from Panax ginseng C.A. Meyer using peptide fingerprinting, internal sequencing and expressed sequence tag data

As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization quadrupole-time of flight mass spectrometry (ESI Q-TOF MS), respectively. More than 300 protein spots were detected on silver stained two-dimensional (2-D) gels using pH 3-10, 4-7, and 4.5-5.5 gradients. Major protein spots (159) were analyzed by peptide fingerprinting or de novo sequencing and the functions of 91 of these proteins were identified. Protein identification was achieved using the expressed sequence tag (EST) database from Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-TOF MS alone was insufficient for protein identification because of the lack of a genome database for Panax ginseng. Only 17 of the 159 protein spots were verified by peptide mass fingerprinting using MALDI-TOF MS whereas 87 out of 102 protein spots, which included 13 of the 17 proteins identified by MALDI-TOF MS, were identified by internal amino acid sequencing using tandem mass spectrometry analysis by ESI Q-TOF MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that a combination of internal sequencing and EST data analysis was an efficient identification method for proteome analysis of plants having incomplete genome data like ginseng. The 2-D patterns of the main root and leaves of Panax ginseng differed from that of the cultured hairy root, suggesting that some proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax ginseng.     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.     

High transfection efficiency of poly(4-vinylimidazole) as a new gene carrier

Poly(4-vinylimidazole) (P4V) was obtained by free radical polymerization of 4-vinylimidazole (4V) prepared by decarboxylation of urocanic acid. P4V formed a complex with DNA that exhibited higher transfection effiency on Hela cells than polyethylenimine (PEI), through the proton sponge mechanism of the imidazole groups in the side chain of the P4V, and low cell toxicity.     

Regulated expression of the centrosomal protein DdCP224 affects microtubule dynamics and reveals mechanisms for the control of supernumerary centrosome number

The Dictyostelium XMAP215 family member DdCP224 is involved in centrosome duplication and cytokinesis and is concentrated at the centrosome and microtubule tips. Herein, we have created a DdCP224 promoter replacement mutant that allows both over- and underexpression. Overexpression led to supernumerary microtubule-organizing centers and, independently, an increase of the number of multinuclear cells. Electron microscopy demonstrated that supernumerary microtubule-organizing centers represented bona fide centrosomes. Live cell imaging of DdCP224-green fluorescent protein mutants also expressing green fluorescent protein-histone2B as a DNA label revealed that supernumerary centrosomes were also competent of cell cycle-dependent duplication. In contrast, underexpression of DdCP224 inhibited cell growth, reduced the number and length of astral microtubules, and caused nocodazole hypersensitivity. Moreover, microtubule regrowth after nocodazole removal was dependent on DdCP224. Underexpression also resulted in a striking disappearance of supernumerary centrosomes and multinuclear cells caused by previous overexpression. We show for the first time by live cell observation that the number of supernumerary centrosomes can be reduced either by centrosome fusion (coalescence) or by the formation of cytoplasts containing supernumerary centrosomes during cytokinesis.