Methylglyoxal activates Gcn2 to phosphorylate eIF2α independently of the TOR pathway in Saccharomyces cerevisiae

Methylglyoxal is a ubiquitous 2-oxoaldehyde derived from glycolysis. Previously, we have reported that methylglyoxal attenuates the rate of overall protein synthesis in Saccharomyces cerevisiae through phosphorylation of the α subunit of translation initiation factor 2 (eIF2α) in a Gcn2-dependent manner. Phosphorylation of eIF2α impedes the formation of a translation initiation complex, and subsequently, overall protein synthesis is reduced. Uncharged tRNA plays an important role in the activation of Gcn2, although we found that MG treatment did not elevate the levels of uncharged tRNA. Rapamycin, a potent inhibitor of TOR kinase, is known to induce phosphorylation of eIF2α without affecting the levels of uncharged tRNA. We determined the correlation between methylglyoxal and TOR kinase activity and found that phosphorylation of eIF2α by methylglyoxal occurred independently of the target of rapamycin (TOR) pathway.     

Improvement of cell wall digestibility in tall fescue by <Emphasis Type="Italic">Oryza sativa</Emphasis> SECONDARY WALL NAC DOMAIN PROTEIN2 chimeric repressor

Forage digestibility is one of the most important factors in livestock performance. As grasses grow and mature, dry matter increases but they become fibrous with secondary cell wall deposition and lignification of sclerenchyma cells, and forage quality drops. In rice (Oryza sativa), the SECONDARY WALL NAC DOMAIN PROTEIN2 fused with the modified EAR-like motif repression domain (OsSWN2-SRDX) reduces secondary cell wall thickening in sclerenchyma cells. We introduced OsSWN2-SRDX under the control of the OsSWN1 promoter into tall fescue (Festuca arundinacea Schreb.) to increase cell wall digestibility. Of 23 transgenic plants expressing OsSWN2-SRDX, nine had brittle internodes that were easily broken by bending. Their secondary cell walls were significantly thinner than those of the wild type in interfascicular fibers of internodes and in cortical fiber cells between leaf epidermal cells and vascular bundles. The dry matter digestibility increased by 11.8% in stems and by 6.8% in leaves compared with the wild type, and therefore forage quality was improved. In stem interfascicular fibers, acid detergent fiber and acid insoluble lignin were greatly reduced. Thus, the reduction of indigestible fiber composed of cellulose and lignin increased the degradability of sclerenchyma cell walls. OsSWN2-SRDX plants offer great potential in the genetic improvement of forage digestibility.     

Mechanical force-based probing of intracellular proteins from living cells using antibody-immobilized nanoneedles

We developed a method combining atomic force microscopy (AFM) and antibody-immobilized nanoneedles to discriminate living cells by probing intracellular cytoskeletal proteins without the need for cell labeling. The nanoneedles are ultra-thin AFM probes sharpened to 200 nm in diameter. While retracting a nanoneedle inserted into a cell, we measured the mechanical force needed to unbind the antibody-target protein complex. Using this method, the intermediate filament protein, nestin and neurofilament were successfully detected in mouse embryonic carcinoma P19 cells and rat primary hippocampal cells within a minute for a single cell and cell differentiation states could be determined. Additionally, the measured magnitude of the force detecting nestin was indicative of the malignancy of breast cancer cells. This method was shown to affect neither the doubling time of cells nor does it leave extrinsic antibodies within the examined cells, allowing to be used in subsequent analyses in their native state.     

Activation of Polycystic Kidney Disease-2-like 1 (PKD2L1)-PKD1L3 Complex by Acid in Mouse Taste Cells

Five basic tastes (bitter, sweet, umami, salty, and sour) are detected in the four taste areas where taste buds reside. Although molecular mechanisms for detecting bitter, sweet, and umami have been well clarified, those for sour and salty remain poorly understood. Several channels including acid-sensing ion channels have been proposed as candidate sour receptors, but they do not encompass all sour-sensing abilities in vivo. We recently reported a novel candidate for sour sensing, the polycystic kidney disease-2-like 1 (PKD2L1)-PKD1L3 channel complex. This channel is not a traditional ligand-gated channel and is gated open only after removal of an acid stimulus, called an off response. Here we show that off responses upon acid stimulus are clearly observed in native taste cells from circumvallate, but not fungiform papillae, of glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice, from which Type III taste cells can be visualized, using Ca²⁺ imaging and patch clamp methods. Off responses were detected in most cells where PKD2L1 immunoreactivity was observed. Interestingly, the pH threshold for acid-evoked intracellular Ca²⁺ increase was around 5.0, a value much higher than that observed in HEK293 cells expressing the PKD2L1-PKD1L3 complex. Thus, PKD2L1-PKD1L3-mediated acid-evoked off responses occurred both in HEK293 cells and in native taste cells, suggesting the involvement of the PKD2L1-PKD1L3 complex in acid sensing in vivo.     

A novel gnd mutation leading to increased L-lysine production in Corynebacterium glutamicum

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.     

Characterization of the Gut Microbiota of Papua New Guineans Using Reverse Transcription Quantitative PCR

There has been considerable interest in composition of gut microbiota in recent years, leading to a better understanding of the role the gut microbiota plays in health and disease. Most studies have been limited in their geographical and socioeconomic diversity to high-income settings, and have been conducted using small sample sizes. To date, few analyses have been conducted in low-income settings, where a better understanding of the gut microbiome could lead to the greatest return in terms of health benefits. Here, we have used quantitative real-time polymerase chain reaction targeting dominant and sub-dominant groups of microorganisms associated with human gut microbiome in 115 people living a subsistence lifestyle in rural areas of Papua New Guinea. Quantification of Clostridium coccoides group, C. leptum subgroup, C. perfringens, Bacteroides fragilis group, Bifidobacterium, Atopobium cluster, Prevotella, Enterobacteriaceae, Enterococcus, Staphylococcus, and Lactobacillus spp. was conducted. Principle coordinates analysis (PCoA) revealed two dimensions with Prevotella, clostridia, Atopobium, Enterobacteriaceae, Enterococcus and Staphylococcus grouping in one dimension, while B. fragilis, Bifidobacterium and Lactobacillus grouping in the second dimension. Highland people had higher numbers of most groups of bacteria detected, and this is likely a key factor for the differences revealed by PCoA between highland and lowland study participants. Age and sex were not major determinants in microbial population composition. The study demonstrates a gut microbial composition with some similarities to those observed in other low-income settings where traditional diets are consumed, which have previously been suggested to favor energy extraction from a carbohydrate rich diet.     

Removal of estrogenic activity of iso-butylparaben and n-butylparaben by laccase in the presence of 1-hydroxybenzotriazole

In the presence of a redox mediator, 1-hydroxybenzotriazole (HBT), iso-butylparaben (iso-BP) and n-butylparaben (n-BP) were treated with laccase from white rot fungus Trametes versicolor. HPLC analysis demonstrated that iso-BP and n-BP almost completely disappeared from the reaction mixture after 4 h of treatment with the laccase-HBT system. Using the yeast two-hybrid assay system, it was also confirmed that the laccase-HBT system substantially removed the estrogenic activity of iso-BP and n-BP after 4 h of treatment. Furthermore, there was a linear relationship between the removal of estrogenic activity of both parabens and the decrease in their concentrations. These results demonstrate that the laccase-HBT system is effective in eliminating iso-BP and n-BP, and removing the estrogenic activity of both parabens.