Cloning and Expression of the Bacillus subtilis No. 313 γ-Cyclodextrin Forming CGTase Gene in Escherichia coli

All four stereoisomers of pyriculol were synthesized to assist in forming a correlation between their chemical structure and biological activity. The (R,E)-2-hydroxy-3-pentenal derivative was coupled with a lithium acetylide derivative to give a diastereomeric mixture of the acetylenic alcohol, which led to the antipode of pyriculol and its 3′-epimer. Similarly obtained were the natural pyriculol and its 3′-epimer from the (S)-isomer of this aldehyde.     

Individualized Mutation Detection in Circulating Tumor DNA for Monitoring Colorectal Tumor Burden Using a Cancer-Associated Gene Sequencing Panel

Background

Circulating tumor DNA (ctDNA) carries information on tumor burden. However, the mutation spectrum is different among tumors. This study was designed to examine the utility of ctDNA for monitoring tumor burden based on an individual mutation profile.

Methodology

DNA was extracted from a total of 176 samples, including pre- and post-operational plasma, primary tumors, and peripheral blood mononuclear cells (PBMC), from 44 individuals with colorectal tumor who underwent curative resection of colorectal tumors, as well as nine healthy individuals. Using a panel of 50 cancer-associated genes, tumor-unique mutations were identified by comparing the single nucleotide variants (SNVs) from tumors and PBMCs with an Ion PGM sequencer. A group of the tumor-unique mutations from individual tumors were designated as individual marker mutations (MMs) to trace tumor burden by ctDNA using droplet digital PCR (ddPCR). From these experiments, three major objectives were assessed: (a) Tumor-unique mutations; (b) mutation spectrum of a tumor; and (c) changes in allele frequency of the MMs in ctDNA after curative resection of the tumor.

Results

A total of 128 gene point mutations were identified in 27 colorectal tumors. Twenty-six genes were mutated in at least 1 sample, while 14 genes were found to be mutated in only 1 sample, respectively. An average of 2.7 genes were mutated per tumor. Subsequently, 24 MMs were selected from SNVs for tumor burden monitoring. Among the MMs found by ddPCR with > 0.1% variant allele frequency in plasma DNA, 100% (8 out of 8) exhibited a decrease in post-operation ctDNA, whereas none of the 16 MMs found by ddPCR with < 0.1% variant allele frequency in plasma DNA showed a decrease.

Conclusions

This panel of 50 cancer-associated genes appeared to be sufficient to identify individual, tumor-unique, mutated ctDNA markers in cancer patients. The MMs showed the clinical utility in monitoring curatively-treated colorectal tumor burden if the allele frequency of MMs in plasma DNA is above 0.1%.     

Finlay–Wilkinson's regression coefficient as a pre‐screening criterion for yield responsiveness to elevated atmospheric CO2 concentration in crops

The rising atmospheric CO₂ concentration ([CO₂]) can increase crop productivity, but there are likely to be intraspecific variations in the response. To meet future world food demand, screening for genotypes with high [CO₂] responsiveness will be a useful option, but there is no criterion for high [CO₂] responsiveness. We hypothesized that the Finlay–Wilkinson regression coefficient (RC) (for the relationship between a genotype's yield versus the mean yield of all genotypes in a specific environment) could serve as a pre‐screening criterion for identifying genotypes that respond strongly to elevated [CO₂]. We collected datasets on the yield of 6 rice and 10 soybean genotypes along environmental gradients and compared their responsiveness to elevated [CO₂] based on the regression coefficients (i.e. the increases of yield per 100 µmol mol^?1 [CO₂]) identified in previous reports. We found significant positive correlations between the RCs and the responsiveness of yield to elevated [CO₂] in both rice and soybean. This result raises the possibility that the coefficient of the Finlay–Wilkinson relationship could be used as a pre‐screening criterion for [CO₂] responsiveness.     

A high-molecular-weight,alkaline, and thermostable β-1,4-xylanase of a subseafloor <Emphasis Type="Italic">Microcella alkaliphila</Emphasis>

An endo β-1,4-xylanase (XynE15) from a culture broth of a deep subseafloor microorganism, Microcella alkaliphila JAM-AC0309, was purified to homogeneity. The molecular mass of XynE15 was approximately 150 kDa as judged by SDS-PAGE. The optimal pH and temperature for hydrolysis of xylan were pH 8 and 65 °C. The enzyme was stable to incubation for 30 min at up to 75 °C, and the half-life at 50 °C was 48 h. XynE15 hydrolyzed arabinoxylan, oat spelt xylan, and birchwood xylan well, but not avicel, carboxymethylcellulose, or arabinan. Xylooligosaccharides were hydrolyzed to mainly xylobiose from higher than xylotetraose. The genome sequencing analysis of strain JAM-AC03039 revealed that XynE15 was composed of 1,319 amino acids with one catalytic domain and three carbohydrate-binding domains belonging to glycoside hydrolase (GH) family 10 and carbohydrate-binding module (CBM) family 4, respectively.     

An improved methodology for the quantification of uronic acid units in xylans and other polysaccharides

Uronic acids can be quantified either by a colorimetric determination after treatment with concentrated sulfuric acid and carbazole or by gas chromatography after methanolysis and subsequent acetylation. Both methods suffer from incomplete hydrolysis, an unavoidable degradation of the products to be analysed, and an inability to separate and quantify different types of uronic acids. In the present work, the fundamental chemistry involved in the two methods has been evaluated, and some modifications to increase their accuracy are suggested. By combining the two methods, a complete quantification of all individual types of uronic acids present in a sample can be achieved.     

Thy-1-Mediated phosphatidylinositol turnover in cultured rat glomerular mesangial cell

Thy-1 glycoprotein is expressed in rat glomerular mesangial cells, and anti-Thy-1 nephritis induced by anti-Thy-1 antibodies is a model of human renal diseases. In this study, we examined Thy-1-mediated biological reactions in cultured rat glomerular mesangial cells utilizing two anti-Thy-1 monoclonal antibodies (mAbs), 1-22-3 and OX-7. Incubation of the cells with these mAbs resulted in increased inositol trisphosphate (IP₃) levels. The rise in IP₃ produced by mAb 1-22-3 was greater than that produced by mAb OX-7 at the same dose. Incubation of mesangial cells with these mAbs resulted in an increase in the intracellular free calcium concentration ([Ca²⁺]i). mAb 1-22-3 induced a sustained increase in [Ca²⁺]i, while that induced by mAb OX-7 lasted 1-2 min, then decreased to the basal level. An transient increase in [Ca²⁺]i was also observed in Ca²⁺-free medium, indicating that these [Ca²⁺]i increases are due to release of Ca²⁺ from internal stores by IP₃ without calcium flux across cell membrane. When cells were pretreated with protein tyrosine kinase (PTK) inhibitors (herbimycin A or genistein), Thy-1-mediated increases in [Ca²⁺]i were inhibited. These data suggest that Thy-1 induces the production of IP₃ (including inositol 1,4,5-triphosphate, an intracellular Ca²⁺-releasing factor) and that PTKs may contribute to the Thy-1-mediated elevation of [Ca²⁺]i which presumably results from phospholipase C activation following Thy-1-mediated signaling in rat mesangial cells. © 1996 Wiley-Liss, Inc.     

Complex gangliosides as cell surface inhibitors for the ecto-NAD+ glycohydrolase of CD38

Leukocyte cell surface antigen CD38 is a single-transmembrane protein whose extracellular domain has catalytic activity for NAD(+) glycohydrolase (NADase). We previously reported that b-series gangliosides inhibit the NADase activity of the extracellular domain of CD38 expressed as a fusion protein [Hara-Yokoyama, M., Kukimoto, I., Nishina, H., Kontani, K., Hirabayashi, Y., Irie, F., Sugiya, H., Furuyama, S., and Katada, T. (1996) J. Biol. Chem. 271, 12951-12955]. In the present study, we examined the effect of exogenous gangliosides on the NADase activity of CD38 on the surface of retinoic acid-treated human leukemic HL60 cells and CD38-transfected THP-1 cells. After incubation of the cells with G(T1b), inhibition of NADase activity was observed. The time course of inhibition was slower than that of the incorporation of G(T1b) into the cells, suggesting that incorporation into the cell membranes is a prerequisite for inhibition. Inhibition occurred efficiently when G(T1b) and CD38 were present on the same cells (cis interaction) rather than on different cells (trans interaction). Although gangliosides may affect localization of cell surface proteins, indirect immunofluorescence intensity due to CD38 was not affected after G(T1b) treatment. Comparison of the effect of G(T1b) and G(D1a) indicates that the tandem sialic acid residues linked to the internal galactose residue of the gangliotetraose core are crucial to the inhibition. These results suggest a novel role of complex gangliosides for the first time as cell surface inhibitors of CD38 through specific and cis interaction between the oligosaccharide moiety and the extracellular domain.     

Sequencing of three lambda clones from the genome of alkaliphilic Bacillus sp. strain C-125

The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15–20 kb in size) of the genome of alkaliphilic Bacillus sp. C-125 cloned in a λ phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis. Received: April 17, 1998 / Accepted: June 23, 1998     

Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27°32.7′N, 126°58.5′E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 μm in diameter. Growth was observed at temperatures between 50° and 93°C, with an optimum at 85°C. The pH range for growth is 5.0–9.0, with an optimum around 7.0. Strain RG-20 requires 1%–4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 μg/ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si′cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349). Received: May 11, 1998 / Accepted: July 24, 1998