UCP1-dependent and UCP1-independent metabolic changes induced by acute cold exposure in brown adipose tissue of mice

Background: Brown adipose tissue (BAT) is a site of metabolic thermogenesis mediated by mitochondrial uncoupling protein 1 (UCP1) and represents a target for a therapeutic intervention in obesity. Cold exposure activates UCP1-mediated thermogenesis in BAT and causes drastic changes in glucose, lipid, and amino acid metabolism; however, the relationship between these metabolic changes and UCP1-mediated thermogenesis is not fully understood. Methods: We conducted metabolomic and GeneChip array analyses of BAT after 4-h exposure to cold temperature (10 °C) in wild-type (WT) and UCP1-KO mice. Results: Cold exposure largely increased metabolites of the glycolysis pathway and lactic acid levels in WT, but not in UCP1-KO, mice, indicating that aerobic glycolysis is enhanced as a consequence of UCP1-mediated thermogenesis. GeneChip array analysis of BAT revealed that there were 2865 genes upregulated by cold exposure in WT mice, and 838 of these were upregulated and 74 were downregulated in UCP1-KO mice. Pathway analysis revealed the enrichment of genes involved in fatty acid (FA) β oxidation and triglyceride (TG) synthesis in both WT and UCP1-KO mice, suggesting that these metabolic pathways were enhanced by cold exposure independently of UCP1-mediated thermogenesis. FA and cholesterol biosynthesis pathways were enhanced only in UCP1-KO mice. Cold exposure also significantly increased the BAT content of proline, tryptophan, and phenylalanine amino acids in both WT and UCP1-KO mice. In WT mice, cold exposure significantly increased glutamine content and enhanced the expression of genes related to glutamine metabolism. Surprisingly, aspartate was almost completely depleted after cold exposure in UCP1-KO mice. Gene expression analysis suggested that aspartate was actively utilized after cold exposure both in WT and UCP1-KO mice, but it was replenished from intracellular N-acetyl-aspartate in WT mice. Conclusions: These results revealed that cold exposure induces UCP1-mediated thermogenesis-dependent glucose utilization and UCP1-independent active lipid metabolism in BAT. In addition, cold exposure largely affects amino acid metabolism in BAT, especially UCP1-dependently enhances glutamine utilization. These results contribute a comprehensive understanding of UCP1-mediated thermogenesis-dependent and thermogenesis-independent metabolism in BAT

Transcellular transport of West Nile virus-like particles across human endothelial cells depends on residues 156 and 159 of envelope protein

<p>Abstract</p> <p>Background</p> <p>West Nile virus (WNV) causes viremia after invasion to the hosts by mosquito bite. Endothelial cells could play an important role in WNV spread from the blood stream into the central nervous system and peripheral tissues. Here, we analyzed the capacity of virus-like particles (VLPs) of the highly virulent NY99 6-LP strain (6-LP VLPs) and the low virulence Eg101 strain (Eg VLPs) to cross cultured human endothelial cells.</p> <p>Results</p> <p>6-LP VLPs were transported from the apical to basolateral side of endothelial cells, whereas Eg VLPs were hardly transported. The localization of tight junction marker ZO-1 and the integrity of tight junctions were not impaired during the transport of 6-LP VLPs. The transport of 6-LP VLPs was inhibited by treatment with filipin, which prevents the formation of cholesterol-dependent membrane rafts, suggesting the involvement of raft-associated membrane transport. To determine the amino acid residues responsible for the transport of VLPs, we produced mutant VLPs, in which residues of E protein were exchanged between the 6-LP and Eg strains. Double amino acid substitution of the residues 156 and 159 greatly impaired the transport of VLPs.</p> <p>Conclusion</p> <p>Our results suggest that a transcellular pathway is associated with 6-LP VLPs transport. We also showed that the combination of the residues 156 and 159 plays an important role in the transport of VLPs across endothelial cells.</p

Dehydrogenation of Conjugated Cholic Acid by Escherichia coli

7α-Dehydrogenation of taurocholic acid and glycocholic acid by Escherichia coli (E. coli) was examined in aerobic and anaerobic culture conditions. Bile acids in the culture medium of E. coli were extracted, separated into free, glycine-conjugate and taurine-conjugate fractions by piperidinohydroxypropyl dextran gel column chromatography, hydrolyzed in alkali and analyzed by gas-liquid chromatography. Both conjugated cholic acids were dehydrogenated to the corresponding 3α,12α-dihydroxy-7-oxo-5β-cholan oic acid without deconjugation and no deconjugation of both conjugated cholic acid was detected in aerobic cultures. But there was little transformation in anaerobic cultures. These data suggest that conjugated cholic acids are taken up by E. coli in an aerobic culture as conjugate forms, dehydrogenated without deconjugation and excreted from the cell as conjugate forms

A novel high-throughput (HTP) cloning strategy for site-directed designed chimeragenesis and mutation using the Gateway cloning system

There is an increasing demand for easy, high-throughput (HTP) methods for protein engineering to support advances in the development of structural biology, bioinformatics and drug design. Here, we describe an N- and C-terminal cloning method utilizing Gateway cloning technology that we have adopted for chimeric and mutant genes production as well as domain shuffling. This method involves only three steps: PCR, in vitro recombination and transformation. All three processes consist of simple handling, mixing and incubation steps. We have characterized this novel HTP method on 96 targets with >90% success. Here, we also discuss an N- and C-terminal cloning method for domain shuffling and a combination of mutation and chimeragenesis with two types of plasmid vectors

Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization

RBM20 is a major regulator of heart-specific alternative pre-mRNA splicing of TTN encoding a giant sarcomeric protein titin. Mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), yet most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. In the present study, we identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. We demonstrate that the two serine residues in the RSRSP stretch are constitutively phosphorylated and mutations in the stretch disturb nuclear localization of RBM20. Rbm20 S637A knock-in mouse mimicking an S635A mutation reported in a familial case showed a remarkable effect on titin isoform expression like in a patient carrying the mutation. These results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20 S637A mouse as a good model for in vivo study

長期的な酸化脂質摂取が線虫の生殖能力と寿命に与える影響

Fatty acids, which are biologically and functionally crucial components of triacylglycerol and phospholipid,are very sensitive to oxygen attack, and subsequently produce substances such as hydroperoxides and aliphaticlong-chain aldehydes. Although these compounds, collectively called oxidized lipids, are known to be toxicto organism, long-term effects of the oxidized lipids uptake upon organism have much to be uncovered. Inthis study, the effects of long-term uptake of oxidized lipids upon fecundity and life-span were analyzed usingmodel organism, Caenorhabditis elegans. Although oxidized lipids showed little effect on fecundity, mean lifespanof the worms were shortened when they were fed with oxidized fatty acids. However, maximum life span was not affected by oxidized lipid indicating the toxic effect of the oxidized lipids were not uniform throughoutthe life cycle; relatively strong in the early stage of life and gradually become weak as aging proceeds. The toxiceffects of oxidized lipids were diminished by addition of anti-oxidative vitamin α-tocopherol. The expressionof anti-oxidative stress enzyme gene, such as superoxide dismutases and catalases, were analyzed by RT-PCR.The expression of these genes were increased upon long-term oxidized lipids exposure. This indicates that antioxidantprotection system indeed responded to the oxidative stress caused by the oxidized lipids