Genome Mining of Oxidation Modules in trans-Acyltransferase Polyketide Synthases Reveals a Culturable Source for Lobatamides

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are multimodular megaenzymes that biosynthesize many bioactive natural products. They contain a remarkable range of domains and module types that introduce different substituents into growing polyketide chains. As one such modification, we recently reported Baeyer–Villiger-type oxygen insertion into nascent polyketide backbones, thereby generating malonyl thioester intermediates. In this work, genome mining focusing on architecturally diverse oxidation modules in trans-AT PKSs led us to the culturable plant symbiont Gynuella sunshinyii, which harbors two distinct modules in one orphan PKS. The PKS product was revealed to be lobatamide A, a potent cytotoxin previously only known from a marine tunicate. Biochemical studies show that one module generates glycolyl thioester intermediates, while the other is proposed to be involved in oxime formation. The data suggest varied roles of oxygenation modules in the biosynthesis of polyketide scaffolds and support the importance of trans-AT PKSs in the specialized metabolism of symbiotic bacteria.     

Interlaboratory evaluation of two enzyme-linked immunosorbent assay kits for the determination of crustacean protein in processed foods

The labeling of foods containing material derived from crustaceans such as shrimp and crab is to become mandatory in Japan because of increases in the number of allergy patients. To ensure proper labeling, 2 novel sandwich enzyme-linked immunosorbent assay (ELISA) kits for the determination of crustacean protein in processed foods, the N kit (Nissui Pharmaceutical Co., Ltd, Ibaraki, Japan) and the M kit (Maruha Nichiro Holdings, Inc., Ibaraki, Japan), have been developed. Five types of model processed foods containing 10 and/or 11.9 microg/g crustacean soluble protein were prepared for interlaboratory evaluation of the performance of these kits. The N kit displayed a relatively high level of reproducibility relative standard deviation (interlaboratory precision; 4.0-8.4% RSDR) and sufficient recovery (65-86%) for all the model processed foods. The M kit displayed sufficient reproducibility (17.6-20.5% RSDR) and a reasonably high level of recovery (82-103%). The repeatability relative standard deviation (RSDr) values regarding the detection of crustacean proteins in the 5 model foods were mostly < 5.1% RSDr for the N kit and 9.9% RSDr for the M kit. In conclusion, the results of this interlaboratory evaluation suggest that both these ELISA kits would be very useful for detecting crustacean protein in processed foods.     

Visualization of phosphatidylcholine,lysophosphatidylcholine and sphingomyelin in mouse tongue body by matrix-assisted laser desorption/ionization imaging mass spectrometry

The mammalian tongue is one of the most important organs during food uptake because it is helpful for mastication and swallowing. In addition, taste receptors are present on the surface of the tongue. Lipids are the second most abundant biomolecules after water in the tongue. Lipids such as phosphatidylcholine (PC), lysophosphatidylcholine (LPC) and sphingomyelin (SM) are considered to play fundamental roles in the mediation of cell signaling. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of lipids across sections of dissected tissue. In this study, we identified and visualized the PC, LPC, and SM species in a mouse tongue body section with matrix-assisted laser desorption/ionization (MALDI)-IMS. The ion image constructed from the peaks revealed that docosahexaenoic acid (DHA)-containing PC, LPC, linoleic acid-containing PC and SM (d18:1/16:0), and oleic acid-containing PC were mainly distributed in muscle, connective tissue, stratified epithelium, and the peripheral nerve, respectively. Furthermore, the distribution of SM (d18:1/16:0) corresponded to the distribution of nerve tissue relating to taste in the stratified epithelium. This study represents the first visualization of PC, LPC and SM localization in the mouse tongue body.     

Synthesis of tri- or tetrasubstituted pyrimidine derivatives through the [5+1] annulation of enamidines with either N,N-dimethylformamide dialkyl acetals or orthoesters and their application in a ring transformation of pyrimidines to pyrido[2,3-d]pyrimidin-5-one derivatives

The [5+1] annulation of enamidines, which were prepared from functionalized silanes, organolithium compounds and two nitriles, with N,N-dimethylformamide dialkyl acetals as the C1 unit is described, leading to the synthesis of tri- and tetrasubstituted pyrimidine derivatives under catalyst- and solvent-free reaction conditions. Furthermore, the [5+1] annulation of enamidines by using orthoesters as the C1 unit is described, in which catalytic amounts of ZnBr(2) catalyze the annulation to produce polysubstituted pyrimidines under toluene or xylene reflux conditions. Moreover, the combination of a reductive ring-opening reaction with [Mo(CO)(6)] and a subsequent intramolecular cyclization with tBuOK effectively causes a skeletal transformation from the pyrimidines containing an isoxazolyl and an ethoxy substituent to form pyrido[2,3-d]pyrimidin-5-one frameworks in excellent yield.     

A tertiary amine as a hydride donor: trichlorosilyl triflate-mediated conjugate reduction of unsaturated ketones

Bulky tertiary amines, especially dicyclohexylisobutylamine, smoothly reduced α,β-unsaturated ketones in the presence of trichlorosilyl triflate to give the corresponding saturated ketones in excellent yields. Isotope-labeling studies revealed that an α-hydrogen of the amine was transferred to the enones during reduction.     

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.     

Formation of double helix self-assembled monolayers of ethynylhelicene oligomer disulfides on gold surfaces

Optically active ethynylhelicene pentamers and hexamers linked by disulfide bonds were synthesized. They formed self-assembled monolayers (SAMs) with double helix structure on gold surfaces, which were analyzed by infrared reflection-absorption spectroscopy (IR-RAS), quartz crystal microbalance (QCM), surface plasmon resonance (SPR), and circular dichroism (CD). Double helix SAMs could be formed on gold surfaces either from double helices or random coils in solution. The double helices on the surface were more stable than in solution. This result suggested the presence of strong intercomplex interactions between double helix complexes on the surface.     

Abnormal phospholipids distribution in the prefrontal cortex from a patient with schizophrenia revealed by matrix-assisted laser desorption/ionization imaging mass spectrometry

Schizophrenia is one of the major psychiatric disorders, and lipids have focused on the important roles in this disorder. In fact, lipids related to various functions in the brain. Previous studies have indicated that phospholipids, particularly ones containing polyunsaturated fatty acyl residues, are deficient in postmortem brains from patients with schizophrenia. However, due to the difficulties in handling human postmortem brains, particularly the large size and complex structures of the human brain, there is little agreement regarding the qualitative and quantitative abnormalities of phospholipids in brains from patients with schizophrenia, particularly if corresponding brain regions are not used. In this study, to overcome these problems, we employed matrix-assisted laser desorption/ionization imaging mass spectrometry (IMS), enabling direct microregion analysis of phospholipids in the postmortem brain of a patient with schizophrenia via brain sections prepared on glass slides. With integration of traditional histochemical examination, we could analyze regions of interest in the brain at the micrometric level. We found abnormal phospholipid distributions within internal brain structures, namely, the frontal cortex and occipital cortex. IMS revealed abnormal distributions of phosphatidylcholine molecular species particularly in the cortical layer of frontal cortex region. In addition, the combined use of liquid chromatography/electrospray ionization tandem mass spectrometry strengthened the capability for identification of numerous lipid molecular species. Our results are expected to further elucidate various metabolic processes in the neural system.