Glycosylation of hesperetin by plant cell cultures

The biotransformation of hesperetin by cultured cells of Ipomoea batatas and Eucalyptus perriniana was investigated. Three glycosides, hesperetin 3'-O-beta-D-glucopyranoside (33 microg/g fr. wt of cells), hesperetin 3',7-O-beta-D-diglucopyranoside (217 microg/g fr. wt of cells), and hesperetin 7-O-[6-O-(beta-D-glucopyranosyl)]-beta-d-glucopyranoside (beta-gentiobioside, 22 microg/g fr. wt of cells), together with three hitherto known glycosides, hesperetin 5-O-beta-d-glucopyranoside (23 microg/g fr. wt of cells), hesperetin 7-O-beta-D-glucopyranoside (57 microg/g fr. wt of cells), and hesperetin 7-O-[6-O-(alpha-L-rhamnopyranosyl)]-beta-D-glucopyranoside (beta-rutinoside, hesperidin, 13 microg/g fr. wt of cells), were isolated from cultured suspension cells of E. perriniana that had been treated with hesperetin. Oligosaccharide chains were regioselectively formed at the C-7 position of hesperetin to afford beta-gentiobioside and beta-rutinoside. On the other hand, cultured I. batatas cells converted hesperetin into hesperetin 3'-O-beta-D-glucopyranoside (60 microg/g fr. wt of cells), hesperetin 5-O-beta-D-glucopyranoside (23 microg/g fr. wt of cells), and hesperetin 7-O-beta-D-glucopyranoside (110 microg/g fr. wt of cells).     

Effect of pre-treatment with St John's Wort on nephrotoxicity of cisplatin in rats

An herbal health care supplement, St John's Wort (SJW, Hypericum perforatum) has become widely used in the treatment of depression, and is known to interact with therapeutic drugs. Here we report a preventive effect of SJW on cisplatin nephrotoxicity in rats. Rats were given SJW (400 mg/kg/day, p.o.) for 10 consecutive days, and were injected with cisplatin (5 mg/kg, i.v.) on the day after the final SJW treatment. Cisplatin treatment increased the serum creatinine level, which is an index of nephrotoxicity, to 1.51+/-0.22 mg/dl (mean+/-SE) from 0.28+/-0.05 mg/dl (control) on day 5 after the cisplatin injection. This increase fell significantly to 0.86+/-0.13 mg/dl by pre-treatment with SJW. Cisplatin-induced histological abnormality of the kidney was blocked by pre-treatment with SJW. When SJW was administered for 10 days, the amounts of renal metallothionein (MT) and hepatic multidrug resistance protein 2 (Mrp2) were increased to 164.8+/-13.0% and 220.8+/-39.3% (mean+/-SE) of controls, respectively. GSH levels in the kidney and liver were not changed. Total and free cisplatin concentration in serum was not influenced by SJW treatment. In conclusion, the results suggest that pre-treatment with SJW may diminish cisplatin nephrotoxicity.     

Difference in response by two cyprinid species to predatory threat from the nocturnal catfish Silurus asotus

Response to predators may not be identical between different prey species with different life histories and body sizes, particularly when the threat of predation is not great. To clarify this hypothesis, we introduced two prey species (10 Japanese dace, Tribolodon hakonensis, and 10 pale chub, Zacco platypus) into each experimental pond (in total, 8 ponds×4 trials) in which benthic algae had been allowed to grow. The presence or absence of Far Eastern catfish, Silurus asotus, and a refuge for prey fish was used to produce four treatments. The presence of catfish and/or a refuge did not affect either the feeding behavior or growth rate of Japanese dace. In contrast, when catfish were present and no refuge was available, the incidence of bottom feeding for pale chub greatly decreased. Pale chub growth rate was low when catfish were present and a refuge was available, indicating that pale chub spent more of their time in the refuge and lost opportunities of acquiring food. Japanese dace can reach a threshold size at which the prey are safe from predation, but pale chub cannot, and this may explain the differences in response to predators of the two species.     

FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells

Autophagy is a membrane-mediated intracellular degradation system. The serine/threonine kinase Atg1 plays an essential role in autophagosome formation. However, the role of the mammalian Atg1 homologues UNC-51-like kinase (ULK) 1 and 2 are not yet well understood. We found that murine ULK1 and 2 localized to autophagic isolation membrane under starvation conditions. Kinase-dead alleles of ULK1 and 2 exerted a dominant-negative effect on autophagosome formation, suggesting that ULK kinase activity is important for autophagy. We next screened for ULK binding proteins and identified the focal adhesion kinase family interacting protein of 200 kD (FIP200), which regulates diverse cellular functions such as cell size, proliferation, and migration. We found that FIP200 was redistributed from the cytoplasm to the isolation membrane under starvation conditions. In FIP200-deficient cells, autophagy induction by various treatments was abolished, and both stability and phosphorylation of ULK1 were impaired. These results suggest that FIP200 is a novel mammalian autophagy factor that functions together with ULKs.     

A new tandem repeat in bovine fibrinogen Aalpha gene

In this study, we describe intraspecies variation in the alphaC connector region of the bovine fibrinogen Aalpha gene. Sequencing and genotyping of six bovine breeds revealed 7 to 10 tandem repeats in the alphaC connector region. In addition, we observed length differences between B. indicus and B. taurus, with the B. indicus having longer fibrinogen alphaC connectors (10-repeat alleles) than B. taurus (7- and 9-repeats). The difference in tandem repeats may be related to the function of blood coagulation system.     

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp⁰ strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp⁰ strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp⁰ suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp⁰ backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.     

Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.     

Functional genetic selection of Helix 66 in Escherichia coli 23S rRNA identified the eukaryotic-binding sequence for ribosomal protein L2

Ribosomal protein L2 is a highly conserved primary 23S rRNA-binding protein. L2 specifically recognizes the internal bulge sequence in Helix 66 (H66) of 23S rRNA and is localized to the intersubunit space through formation of bridge B7b with 16S rRNA. The L2-binding site in H66 is highly conserved in prokaryotic ribosomes, whereas the corresponding site in eukaryotic ribosomes has evolved into distinct classes of sequences. We performed a systematic genetic selection of randomized rRNA sequences in Escherichia coli, and isolated 20 functional variants of the L2-binding site. The isolated variants consisted of eukaryotic sequences, in addition to prokaryotic sequences. These results suggest that L2/L8e does not recognize a specific base sequence of H66, but rather a characteristic architecture of H66. The growth phenotype of the isolated variants correlated well with their ability of subunit association. Upon continuous cultivation of a deleterious variant, we isolated two spontaneous mutations within domain IV of 23S rRNA that compensated for its weak subunit association, and alleviated its growth defect, implying that functional interactions between intersubunit bridges compensate ribosomal function.