Introducing site-specific glycosylation using protein engineering techniques reduces the immunogenicity of β-lactoglobulin

To reduce the immunogenicity of β-lactoglobulin (BLG), we prepared wild-type bovine BLG variant A (wt) and three site-specifically glycosylated BLGs (D28N, D137N/A139S, and P153A), and expressed them in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the glycosylated BLGs were conjugated with a ~4 kDa high-mannose chain. Each glycosylated BLG retained ～80% of the retinol-binding activity of BLG. Structural analyses by intrinsic fluorescence, CD spectra, and ELISA with monoclonal antibodies indicated that the surface structure was slightly changed by using protein engineering techniques, but that the site-specifically glycosylated BLGs were covered by high-mannose chains without substantial disruption of wt conformation. Antibody responses to the glycosylated BLGs tended to be weaker in BALB/c, C57BL/6, and C3H/He mice. We conclude that site-specific glycosylation is an effective method to reduce the immunogenicity of BLG, and that masking of epitopes by high-mannose chains is effective to reduce immunogenicity.     

Novel cysteine-rich secretory protein in the buccal gland secretion of the parasitic lamprey, Lethenteron japonicum

Lampreys are one of the most primitive vertebrates diverged some 500 million years ago. It has long been known that parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We found two major protein components of 160 and 26 kDa in the buccal gland secretion of parasitic river lamprey, Lethenteron japonicum. The larger protein was identified as river lamprey plasma albumin. The complete primary structure of the 26-kDa protein was determined by protein and cDNA analysis. It belonged to the cysteine-rich secretory protein (CRISP) superfamily that includes recently identified reptile venom ion-channel blockers. Lamprey CRISP blocked depolarization-induced contraction of rat-tail arterial smooth muscle, but showed no effect on caffeine-induced contraction. The result suggests that lamprey CRISP is an L-type Ca(2+)-channel blocker and may act as a vasodilator, which facilitates the parasite to feed on the host's blood. The lamprey CRISP protein contains a number of short insertions throughout the sequence, when aligned with reptilian venom CRISP proteins, probably due to the large evolutionary distance between the Agnatha and the Reptilia, and may represent a novel class of venom CRISP family proteins.     

Genetic analysis of Anatolian pear germplasm by simple sequence repeats

The pear (Pyrus communis L.) is a fruit species grown in many temperate regions of the world. Turkey harbours a rich and ancient pear germplasm adapted to diverse ecological regions of the country. The aim of this study was to genetically characterise locally grown Anatolian pear germplasm. We have analysed large numbers (228) of pear accessions originated from six eco‐geographically diverse regions using 18 simple sequence repeat (SSR) markers and identified 308 SSR alleles. Genetic similarities among the accessions examined were generally below 80%. The highest heterozygosity rate was obtained for the SSR locus ‘CH02D11’ derived from apples and ‘KA16’ and ‘NH0021a’ derived from pears. No identical or synonymous genotypes were found, while five homonymous genotypes were identified. Factorial correspondence analysis could not clearly separate different pear accession groups studied, suggesting that Anatolian pear accessions were intermixed possibly due to gene flow and/or germplasm movements between different eco‐geographical regions. However, most pear accessions were grouped according to their collection sites in structure analyses. The SSR data reported here for Anatolian pear accessions will be valuable for future germplasm management efforts as well as for comparative studies that investigate genetic relationships of pears from Anatolia and the surrounding regions.     

Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrial dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ⁰ cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.     

Repeated exposures to daytime bright light increase nocturnal melatonin rise and maintain circadian phase in young subjects under fixed sleep schedule

Effects of two different light intensities during daytime were examined on human circadian rhythms in plasma melatonin, core body temperature, and wrist activity under a fixed sleep schedule. Sleep qualities as indicated by polysomnography and subjective sleepiness were also measured. In the first week, under dim light conditions ( approximately 10 lx), the onset and peak of nocturnal melatonin rise were significantly delayed, whereas the end of melatonin rise was not changed. The peak level of melatonin rise was not affected. As a result, the width of nocturnal melatonin rise was significantly shortened. In the second week, under bright light conditions ( approximately 5,000 lx), the phases of nocturnal melatonin rise were not changed further, but the peak level was significantly increased. Core body temperature at the initial sleep phase was progressively elevated during the course of dim light exposure and reached the maximum level at the first night of bright light conditions. Subjective sleepiness gradually declined in the course of dim light exposure and reached the minimum level at the first day of bright light. These findings indicate that repeated exposures to daytime bright light are effective in controlling the circadian phase and increasing the peak level of nocturnal melatonin rise in plasma and suggest a close correlation between phase-delay shifts of the onset of nocturnal melatonin rise or body temperature rhythm and daytime sleepiness.     

The C-terminal residues of the 2b protein of Cucumber mosaic virus are important for efficient expression in Escherichia coli and DNA-binding

The RNA silencing suppressor 2b protein of Cucumber mosaic virus (CMV) is difficult to produce in Escherichia coli. We compared two CMV 2b proteins that differ in their toxicity against E. coli and found that the acidic amino acid residues in the C-terminal significantly affected the toxicity and expression level of the protein in E. coli. In addition, in a DNA-binding assay, 2b had the ability to bind to DNA, and this ability was affected by the charge on the C-terminal residues of 2b. We concluded that the C-terminal residues were important for 2b’s DNA-binding ability, which may partly explain the toxicity of the protein.     

Analogs of unusual sponge phospholipids. Synthesis and thermotropic properties of 1,2-di-(6Z,9Z)-6,9-hexacosadienoyl phosphatidylcholine and phosphatidylethanolamine

The major marine sponge phospholipids 1,2-di-(5Z,9Z)-5,9-hexacosadienoyl phosphatidylcholine (PC) and phosphatidyl-ethanolamine (PE) hardly incorporate cholesterol into their liposomal bilayers, as reported earlier. Our previous studies indicated that their synthetic short chain (C18-C24) analogs with the same double bond pattern readily incorporated cholesterol, thus demonstrating the importance of the chain length. In order to investigate the possible role of the unusual delta 5,9 diunsaturation 1,2-di-(6Z,9Z)-6,9-hexacosadienoyl phosphatidylcholine and phosphatidylethanolamine were synthesized and their thermotropic behavior studied. Both analogs shows a transition endoterm at 45 degrees C, while the natural 1,2-di-(5Z,9Z)-5,9-hexacosadienoyl PC and its PE counterpart exhibited it at 42 degrees C. A partial incorporation of cholesterol into liposomal bilayers of 1,2-di-(6Z,9Z)-6,9-hexacosadienoyl PC was observed. Our results suggest that while the chain length is the predominant factor in the interactions of these phospholipids with sterols, the double bond location may also play a contributing role.     

Vergatic acid,a new pentacyclic triterpene from Salvia virgata

A new pentacyclic triterpene acid was isolated from the arial parts of Salvia virgata and its constitution was established as 3β-hydroxy-1-oxo-olean-12-en-28-oic acid and named virgatic acid.