ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element

Arginine rich, mutated in early stage of tumors (ARMET) was first identified as a human gene highly mutated in a variety of cancers. However, little is known about the characteristics of the ARMET protein and its expression. We identified ARMET as a gene upregulated by endoplasmic reticulum (ER) stress. Here, we show that the mouse homologue of ARMET is an 18-kDa soluble ER protein that is mature after cleavage of a signal sequence and has four intramolecular disulfide bonds, including two in CXXC sequences. ER stress stimulated ARMET expression, and the expression patterns of ARMET mRNA and protein in mouse tissues were similar to those of Grp78, an Hsp70-family protein required for quality control of proteins in the ER. A reporter gene assay using a mouse ARMET promoter revealed that the unfolded protein response of the ARMET gene is regulated by an ERSE-II element whose sequence is identical to that of the HERP gene. ARMET is the second fully characterized ERSE-II-dependent gene and likely contributes to quality control of proteins in the ER.     

Different Impacts of Neck Circumference and Visceral Obesity on the Severity of Obstructive Sleep Apnea Syndrome

Our aim was to investigate the significance of neck circumference (NC) on the presence and severity of obstructive sleep apnea (OSA) syndrome independent of visceral fat (VF) obesity. A total of 219 subjects with suspected OSA underwent a complete polysomnography (PSG) study, along with the measurement of NC, and total body fat (TF) and VF levels (VFLs) measured by bioelectrical impedance analysis. We proposed NC divided by height (NC/H) as the simple index for height‐corrected NC in Japanese subjects. NC/H exhibited a significantly stronger correlation than NC per se with BMI (r = 0.781 vs. 0.675, P = 0.0178), TF (r = 0.531 vs. 0.156, P < 0.0001), and VF (r = 0.819 vs. 0.731, P = 0.0203), indicating that NC/H is a better indicator of visceral obesity than NC per se. Interestingly, despite the strong correlation between NC/H and VFL, VFL was significantly associated with the apnea‐hypopnea index (AHI) ≥5, ≥15, and ≥30, but not with ≥40 or ≥50, whereas NC/H was significantly associated with higher AHI values, i.e., AHI ≥50 but not with lower AHI value. Furthermore, multiple regression analyses revealed that VFL and NC/H were independently associated with the square root of AHI (AHI^0.5) levels in obese and nonobese patients, respectively. In conclusion, NC is associated with the severity of OSA independently of visceral obesity, especially in nonobese patients.     

Expression, purification and characterization of isoforms of peripheral stalk subunits of human V-ATPase

The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase.     

Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts

The production of human therapeutic proteins in plants provides opportunities for low-cost production, and minimizes the risk of contamination from potential human pathogens. Chloroplast genetic engineering is a particularly promising strategy, because plant chloroplasts can produce large amounts of foreign target proteins. Oxidative stress is a key factor in various human diseases. Human thioredoxin 1 (hTrx1) is a stress-induced protein that functions as an antioxidant against oxidative stress, and overexpression of hTrx1 has been shown to suppress various diseases in mice. Therefore, hTrx1 is a prospective candidate as a new human therapeutic protein. We created transplastomic lettuce expressing hTrx1 under the control of the psbA promoter. Transplastomic plants grew normally and were fertile. The hTrx1 protein accumulated to approximately 1% of total soluble protein in mature leaves. The hTrx1 protein purified from lettuce leaves was functionally active, and reduced insulin disulfides. The purified protein protected mouse insulinoma line 6 cells from damage by hydrogen peroxide, as reported previously for a recombinant hTrx1 expressed in Escherichia coli. This is the first report of expression of the biologically active hTrx1 protein in plant chloroplasts. This research opens up possibilities for plant-based production of hTrx1. Considering that this expression host is an edible crop plant, this transplastomic lettuce may be suitable for oral delivery of hTrx1.     

Sequence analyses of the ITS regions and the matK gene for determining phylogenetic relationships of Diospyros kaki (persimmon) with other wild Diospyros (Ebenaceae) species

To elucidate the relationships among Diospyros kaki and species closely related in previous studies, the nuclear ribosomal internal transcribed spacer (ITS) DNA sequence and the chloroplast matK gene were sequenced and compared with those of nine Diospyros species from Thailand, four species from temperate regions, and one species of southern Africa, D. lycioides. Maximum parsimony, maximum likelihood, and neighbor joining analyses of the matK and ITS data sets revealed that D. kaki is closely related to two diploid species, D. oleifera and D. glandulosa. D. kaki, D. glandulosa, and D. oleifera were placed differently in the trees obtained from ITS and matK data sets, suggesting that hybridization and/or introgression may have occurred during the development of these species. D. kaki was not found to be closely related to D. ehretioides, a diploid species from Thailand. These results differed from a prior analysis of this genus performed with chloroplast DNA (cpDNA) restriction site mutations in 3.2- and 2.1-kb amplified sequences. The results supported Ng’s hypothesis that D. glandulosa and D. kaki may share a common ancestor. D. oleifera was also closely associated with D. kaki.     

Induced pluripotent stem cells: past, present, and future

The development of iPSCs reflected the merging of three major scientific streams and has in turn led to additional new branches of investigation. However, there is still debate about whether iPSCs are functionally equivalent to ESCs. This question should be answered only by science, not by politics or business.     

In vivo receptor binding of benidipine and amlodipine in mesenteric arteries and other tissues of spontaneously hypertensive rats

The present study was undertaken to characterize the in vivo 1,4-dihydropyridine (DHP) receptor binding of long-acting 1,4-DHP calcium channel antagonists in the mesenteric artery and other tissues of SHR. In vivo specific binding of (+)-[3H]PN 200-110 in the SHR mesenteric artery was significantly (36.6-49.7 %) reduced 1-8 h after oral administration of benidipine (1.84 micromol/kg). A greater reduction in (+)-[3H]PN 200-110 binding in the mesenteric artery was observed at a higher dose (5.53 micromol/kg) of this drug. This dose of benidipine also reduced significantly the in vivo specific (+)-[3H]PN 200-110 binding in the aorta but not in the myocardium and cerebral cortex. Following oral administration of amlodipine (17.6 micromol/kg), a significant (51.7-94.2 %) reduction in (+)-[3H]PN 200-110 binding was seen at 1-18 h in the mesenteric artery and at 1-12 h in the aorta. Only a slight reduction in myocardial and cerebral cortical (+)-[3H]PN 200-110 binding was seen following amlodipine administration. In contrast, oral administration of nifedipine (28.9 micromol/kg) reduced markedly in vivo (+)-[3H]PN 200-110 binding in all the tissues of SHR at 1-6 h, and the degree and time-course of the reduction did not differ significantly among the tissues. The area under the curve (AUC) for the receptor occupancy vs time was calculated from the reduction rate (%) of in vivo specific (+)-[3H]PN 200-110 binding. The ratios of the AUCmesenteric artery to AUCaorta or AUCmesenteric artery to AUCmyocardium after oral administration of benidipine and amlodipine were greater than the corresponding value for nifedipine. The degree and time-course of arterial receptor occupancy by benidipine and amlodipine agreed well with those of their hypotensive effects in the conscious SHR. In conclusion, the present study demonstrates that benidipine and amlodipine may occupy, in a more selective and sustained manner, 1,4-DHP receptors in arterial tissues than in other tissues of SHR, and thus, such receptor binding specificity may be responsible for the long-lasting hypotensive effects of these drugs.     

Characterization of the catabolic pathway for a phenylcoumaran-type lignin-derived biaryl in Sphingobium sp. strain SYK-6

Sphingobium sp. strain SYK-6 is capable of degrading various lignin-derived biaryls. We determined the catabolic pathway of a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA) in SYK-6, and identified some of the DCA catabolism genes. In SYK-6 cells, the alcohol group of DCA was oxidized to the carboxyl group, first at the B-ring side chain and then at the A-ring side chain. The resultant metabolite was degraded to 5-formylferulate and vanillin through the decarboxylation and the Cα–Cβ cleavage of the A-ring side chain. Based on the DCA catabolic pathway, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) genes are thought to be involved in the conversion of DCA into an aldehyde intermediate (DCA-L) and the conversion of DCA-L into a carboxylic acid intermediate (DCA-C), respectively. SLG_05620 and SLG_24930, which belong to quinohemoprotein ADH and aryl ADH, respectively, were isolated as the genes responsible for the oxidation of DCA. In addition to these genes, multiple genes similar to SLG_05620 and SLG_24930 were found to confer DCA oxidation activities on Escherichia coli cells. In order to identify the DCA-L dehydrogenase genes, the DCA-L oxidation activities of the SYK-6 gene products of putative twenty-one ALDH genes were examined. Significant activities were observed in the four ALDH gene products, including the SLG_27910 product, which showed the highest activity. The disruption of SLG_27910 caused a decreased conversion of DCA-L, suggesting that SLG_27910 plays an important role in the DCA-L oxidation. In conclusion, no specific gene seems to be solely responsible for the conversion of DCA and DCA-L, however, the multiple genes encoding quinohemoprotein ADH and aryl ADH genes, and four ALDH genes are probably involved in the conversion processes.     

Characterization of Oseltamivir-Resistant 2009 H1N1 Pandemic Influenza A Viruses

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.