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191.
Yamamoto Y Tsutsumi Y Yoshioka Y Nishibata T Kobayashi K Okamoto T Mukai Y Shimizu T Nakagawa S Nagata S Mayumi T 《Nature biotechnology》2003,21(5):546-552
Addition of polyethylene glycol to protein (PEGylation) to improve stability and other characteristics is mostly nonspecific and may occur at all lysine residues, some of which may be within or near an active site. Resultant PEGylated proteins are heterogeneous and can show markedly lower bioactivity. We attempted to develop a strategy for site-specific mono-PEGylation using tumor necrosis factor-alpha (TNF-alpha). We prepared phage libraries expressing TNF-alpha mutants in which all the lysine residues were replaced with other amino acids. A fully bioactive lysine-deficient mutant TNF-alpha (mTNF-alpha-Lys(-)) was isolated by panning against TNF-alpha-neutralizing antibody despite reports that some lysine residues were essential for its bioactivity. mTNF-alpha-Lys(-) was site-specifically mono-PEGylated at its N terminus. This mono-PEGylated mTNF-alpha-Lys(-), with superior molecular uniformity, showed higher bioactivity in vitro and greater antitumor therapeutic potency than randomly mono-PEGylated wild-type TNF-alpha. These results suggest the usefulness of the phage display system for creating functional mutant proteins and of our site-specific PEGylation approach. 相似文献
192.
Isolation and characterization of novel tachykinins from the posterior salivary gland of the common octopus Octopus vulgaris 总被引:1,自引:0,他引:1
Two novel tachykinins (OctTK-I: Lys-Pro-Pro-Ser-Ser-Ser-Glu-Phe-Ile-Gly-Leu-Met-NH(2) and OctTK-II: Lys-Pro-Pro-Ser-Ser-Ser-Glu-Phe-Val-Gly-Leu-Met-NH(2)) were isolated from the posterior salivary gland of the octopus (Octopus vulgaris) using a contraction assay of the carp rectum. These peptides had in common the pentapeptide sequence -Phe-X-Gly-Leu-Met-NH(2) at the C-terminal and induced immediate contractions on the carp rectum and the guinea-pig ileum. cDNAs encoding their precursor proteins were cloned. The OctTK gene was expressed in the posterior salivary gland and the expression was localized in mucus-secreting cells of the gland. The results suggested that OctTKs might be secreted as a venomous substance acting on vertebrates such as fishes, which are the prey or natural enemies of the octopus. 相似文献
193.
Tomita K Okuhara Y Shigematsu N Suh H Lim K 《Bioscience, biotechnology, and biochemistry》2003,67(9):1999-2001
We examined the effects of (-)-Hydroxycitrate (HCA) ingestion on fat oxidation during moderate intensity exercise in untrained men. Six subjects ingested 500 mg of HCA or a placebo for 5 days and did endurance exercise. Blood FFA concentrations were significantly increased and respiratory exchange ratio (RER) decreased by HCA ingestion. These results suggested short-term HCA ingestion increases fat oxidation in untrained men. 相似文献
194.
Identification of the lantibiotic nisin Q,a new natural nisin variant produced by Lactococcus lactis 61-14 isolated from a river in Japan 总被引:1,自引:0,他引:1
Zendo T Fukao M Ueda K Higuchi T Nakayama J Sonomoto K 《Bioscience, biotechnology, and biochemistry》2003,67(7):1616-1619
Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria. N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q. Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence. 相似文献
195.
For the purpose of phylogeographic study of lucidophyllous (evergreen broad-leaved) forests in Japan, we surveyed intraspecific chloroplast DNA (cpDNA) variation in 41 component species of such forests. Intraspecific cpDNA variations were detected in 14 species. In 15 species and one species group, 16 non-coding cpDNA regions were examined to find intraspecific sequence variation. The extent of variation in these regions was compared. The largest amount of intraspecific variation was detected in the rps16 region. A relatively large amount of intraspecific variation was detected in the petD-rpoA, rpl16, and trnL-F regions. It is suggested that these regions of cpDNA would be useful for detecting intraspecific variation in plant species, and could provide valuable information for various research purposes. 相似文献
196.
Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), 13C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of 13C=O isotope-labeled (Ala-Gly-Gly)10. The torsion angles were determined to be (, ) = (–90°, 150° ) within an experimental error of ±10° for each residue. This conformation is characterized as 31 helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 31 helix of (Ala-Gly-Gly)10 does not change by formic acid treatment although (Ala-Gly)15 easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 31 helix conformation of (Ala-Gly-Gly)10 is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)10 were also determined as (, ) = (–90°, 150° ) and this peptide is also considered to take 31 helix conformation. 相似文献
197.
198.
Matsuura S Komatsu J Hirano K Yasuda H Takashima K Katsura S Mizuno A 《Nucleic acids research》2001,29(16):E79
A fluorescence microscopy technique has been developed to visualize the behavior of individual DNA and protein molecules. Real-time direct observation of a single DNA molecule can be used to investigate the dynamics of DNA-protein interactions, such as the DNA digestion reaction by lambda exonuclease. In conventional methods it is impossible to analyze the dynamics of an individual lambda exonuclease molecule on a DNA because they can only observe the average behavior of a number of exonuclease molecules. Observation of a single molecule, on the other hand, can reveal processivity and binding rate of an individual exonuclease molecule. To evaluate the dynamics of lambda exonuclease, a stained lambda DNA molecule with one biotinylated terminal was fixed on an avidin-coated coverslip and straightened using a d.c. electric field. Microscopic observation of digestion of a straightened DNA molecule by lambda exonuclease revealed that the DNA digestion rate was approximately 1000 bases/s and also demonstrated high processivity. 相似文献
199.
Komatsu S Berg RD Russell JM Nimura Y Granger DN 《American journal of physiology. Gastrointestinal and liver physiology》2000,279(1):G186-G191
Quantitative estimates of endothelial cell adhesion molecule expression have revealed that some adhesion molecules [e.g., intercellular adhesion molecule-1 (ICAM-1)] are abundantly expressed in different vascular beds under normal conditions. The objective of this study was to determine whether the enteric microflora contribute to the constitutive expression of ICAM-1 and other endothelial cell adhesion molecules in the gastrointestinal tract and other regional vascular beds. The dual radiolabeled monoclonal antibody technique was used to measure endothelial expression of ICAM-1, ICAM-2, vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in conventional, germ-free mice and germ-free mice receiving the cecal contents of conventional mice to reestablish the enteric microflora (total association). Constitutive ICAM-1 expression was significantly lower in the splanchnic organs (pancreas, stomach, small and large intestine, mesentery, and liver), kidneys, skeletal muscle, and skin of germ-free mice compared with their conventional counterparts. These differences were abolished after total association of germ-free mice with the indigenous gastrointestinal flora. The expression of ICAM-2, VCAM-1, and E-selectin in the various tissues studied did not differ between conventional and germ-free mice. These findings indicate that the indigenous gastrointestinal microflora are responsible for a significant proportion of the basal ICAM-1 expression detected in both intestinal and extraintestinal tissues. 相似文献
200.