Mass propagation of shoots of Stevia rebaudiana using a large scale bioreactor

A procedure for the mass propagation of multiple shoots of Stevia rebaudiana is described. Isolated shoot primordia were used as the inoculum to obtain clusters of shoot primordia. Such clusters were grown in a 500 liter bioreactor to obtain shoots. A total of 64.6 Kg of shoots were propagated from 460 g of the inoculated shoot primordia. These shoots were easily acclimatized in soil.     

Comparison of Barium and Arsenic Concentrations in Well Drinking Water and in Human Body Samples and a Novel Remediation System for These Elements in Well Drinking Water

Health risk for well drinking water is a worldwide problem. Our recent studies showed increased toxicity by exposure to barium alone (≤700 µg/L) and coexposure to barium (137 µg/L) and arsenic (225 µg/L). The present edition of WHO health-based guidelines for drinking water revised in 2011 has maintained the values of arsenic (10 µg/L) and barium (700 µg/L), but not elements such as manganese, iron and zinc. Nevertheless, there have been very few studies on barium in drinking water and human samples. This study showed significant correlations between levels of arsenic and barium, but not its homologous elements (magnesium, calcium and strontium), in urine, toenail and hair samples obtained from residents of Jessore, Bangladesh. Significant correlation between levels of arsenic and barium in well drinking water and levels in human urine, toenail and hair samples were also observed. Based on these results, a high-performance and low-cost adsorbent composed of a hydrotalcite-like compound for barium and arsenic was developed. The adsorbent reduced levels of barium and arsenic from well water in Bangladesh and Vietnam to <7 µg/L within 1 min. Thus, we have showed levels of arsenic and barium in humans and propose a novel remediation system.     

Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts

Fucosylated alpha-fetoprotein (AFP) is a highly specific tumor marker for hepatocellular carcinoma (HCC). However, the molecular mechanism by which serum level of fucosylated AFP increases in patients with HCC remains largely unknown. Here, we report that the fucosylation of glycoproteins could be a possible signal for secretion into bile ducts in the liver. We compared oligosaccharide structures on glycoproteins in human bile with those in serum by several types of lectin blot analyses. Enhanced binding of biliary glycoproteins to lectins that recognize a fucose residue was observed over a wide range of molecular weights compared with serum glycoproteins. A structural analysis of oligosaccharides by two-dimensional mapping high performance liquid chromatography and matrix-assisted laser desorption ionization time-of flight mass spectrometry confirmed the increases in the fucosylation of biliary glycoproteins. Purification followed by structural analysis on alpha1-antitrypsin, alpha1-acid glycoprotein and haptoglobin, which are synthesized in the liver, showed higher fucosylation in bile than in serum. To find direct evidence for fucosylation and sorting signal into bile ducts, we used alpha1-6 fucosyltransferase (Fut8)-deficient mice because fucosylation of glycoproteins produced in mouse liver was mainly an alpha1-6 linkage. Interestingly, the levels of alpha1-antitrypsin and alpha1-acid glycoprotein were quite low in bile of Fut8-deficient mice as compared with wild-type mice. An immunohistochemical study showed dramatic changes in the localization of these glycoproteins in the liver of Fut8-deficient mice. Taken together, these results suggest that fucosylation is a possible signal for the secretion of glycoproteins into bile ducts in the liver. A disruption in this system might involve an increase in fucosylated AFP in the serum of patients with HCC.     

Evolution of Mhc–DRB Introns: Implications for the Origin of Primates

Introns are generally believed to evolve too rapidly and too erratically to be of much use in phylogenetic reconstructions. Few phylogenetically informative intron sequences are available, however, to ascertain the validity of this supposition. In the present study the supposition was tested on the example of the mammalian class II major histocompatibility complex (Mhc) genes of the DRB family. Since the Mhc genes evolve under balancing selection and are believed to recombine or rearrange frequently, the evolution of their introns could be expected to be particularly rapid and subject to scrambling. Sequences of intron 4 and 5 DRB genes were obtained from polymerase chain reaction-amplified fragments of genomic DNA from representatives of six eutherian orders—Primates, Scandentia, Chiroptera, Dermoptera, Lagomorpha, and Insectivora. Although short stretches of the introns have indeed proved to be unalignable, the bulk of the intron sequences from all six orders, spanning >85 million years (my) of evolution, could be aligned and used in a study of the tempo and mode of intron evolution. The analysis has revealed the Mhc introns to evolve at a rate similar to that of other genes and of synonymous sites of non-Mhc genes. No evidence of homogenization or large-scale scrambling of the intron sequences could be found. The Mhc introns apparently evolve largely by point mutations and insertions/deletions. The phylogenetic signals contained in the intron sequences could be used to identify Scandentia as the sister group of Primates, to support the existence of the Archonta superorder, and to confirm the monophyly of the Chiroptera. Received: 26 October 1998 / Accepted: 21 December 1998     

Nucleocytoplasmic shuttling of the zinc finger protein EZI Is mediated by importin-7-dependent nuclear import and CRM1-independent export mechanisms

Nucleocytoplasmic translocation constitutes a foundation for nuclear proteins to exert their proper functions and hence for various biological reactions to occur normally in eukaryotic cells. We reported previously that EZI/Zfp467, a 12 zinc finger motif-containing protein, localizes predominantly in the nucleus, yet the underlying mechanism still remains elusive. Here we constructed a series of mutant forms of EZI and examined their subcellular localization. The results delineated a non-canonical nuclear localization signal in the region covering the 9th to the 12th zinc fingers, which was necessary for nuclear accumulation of EZI as well as sufficient to confer nuclear localizing ability to a heterologous protein. We also found that the N-terminal domain of EZI is necessary for its nuclear export, the process of which was not sensitive to the CRM1 inhibitor leptomycin B. An interaction proteomics approach and the following co-immunoprecipitation experiments identified the nuclear import receptor importin-7 as a molecule that associated with EZI and, importantly, short interfering RNA-mediated knockdown of importin-7 expression completely abrogated nuclear accumulation of EZI. Taken together, these results identify EZI as a novel cargo protein for importin-7 and demonstrate a nucleocytoplasmic shuttling mechanism that is mediated by importin-7-dependent nuclear localization and CRM1-independent nuclear export.     

A combination effect of epigallocatechin gallate, a major compound of green tea catechins, with antibiotics on Helicobacter pylori growth in vitro

Since green tea catechins are known to have antimicrobial activity against a variety of microorganisms, their possible effects on Helicobacter pylori in combination with antibiotics were examined. Fifty-six clinical isolates of H. pylori, including 19 isolates highly resistant to metronidazole (MTZ) and/or clarithromycin (CLR), were used to determine in vitro sensitivity to tea catechins. The MIC90 of both epigallocatechin gallate (EGCg) and epicatechin gallate (ECg) was 100 microg/ml. However, other tea catechins tested did not show any anti-H. pylori activity. Highly antibiotic-resistant clinical isolates showed a similar sensitivity to both EGCg and ECg. The kinetic study of antibacterial activity in liquid cultures revealed a relatively slow but strong activity on the growth of H. pylori. In combination with sub-MIC of amoxicillin (AMX), the antibacterial activity of AMX was significantly enhanced by the presence of EGCg. To estimate the general combination effect between EGCg and other antibiotics, such as MTZ and CLR, on the antibacterial activity against clinical isolates, the fraction inhibitory concentration (FIC) was determined by checkerboard study. The FIC indexes showed additive effects between EGCg and antibiotics tested. These results indicatethat EGCg may be a valuable therapeutic agent against H. pylori infection.