Impact of Cationic Amino Acid Transporter 1 on Blood- Retinal Barrier Transport of L-Ornithine

PURPOSE. To elucidate L-ornithine transport at the blood-retinal barrier (BRB). METHODS. Integration plot and retinal uptake index (RUI) were used to investigate the in vivo [ 3 H]L-ornithine transport across the BRB. In vitro transport studies of [ 3 H]L-ornithine were performed with TR-iBRB2 cells and RPE-J cells, the model cells of the inner and outer BRB, respectively. Immunohistochemistry was performed on cationic amino acid transporter 1 (CAT1/SLC7A1). RESULTS. The apparent influx permeability clearance of [ 3 H]L-ornithine was found to be 18. 7 lL/(minÁg retina), and the RUI of [ 3 H]L-ornithine was reduced by L-ornithine and L-arginine, suggesting the blood-to-retina transport of L-ornithine at the BRB. [ 3 H]L-Ornithine uptake by TR-iBRB2 cells showed a time-, temperature-and concentration-dependence with a MichaelisMenten constant (K m ) of 33.2 lM and a nonsaturable uptake rate (K d ) of 2.18 lL/(minÁmg protein). The uptake was Na þ -independent, and was inhibited by L-ornithine, L-arginine, and L-lysine, suggesting the involvement of CAT1 in L-ornithine transport at the inner BRB. Immunohistochemistry revealed the luminal and abluminal localization of CAT1 at the inner BRB, and at the basal localization at the outer BRB. Retinal pigment epithelium-J cells showed that the basal-to-cell (B-to-C) uptake of [ 3 H]L-ornithine was greater than that of the apical-tocell (A-to-C) uptake, and the B-to-C transport was inhibited by unlabeled L-ornithine, suggesting the involvement of CAT1 in the blood-to-cell transport of L-ornithine across the basal membrane at the outer BRB. CONCLUSIONS. These suggest the involvement of CAT1 in L-ornithine transport at the luminal and abluminal sides of the inner BRB and the basal side of the outer BRB

Development of a Novel Output Value for Quantitative Assessment in Methylated DNA Immunoprecipitation-CpG Island Microarray Analysis

In DNA methylation microarray analysis, quantitative assessment of intermediate methylation levels in samples with various global methylation levels is still difficult. Here, specifically for methylated DNA immunoprecipitation-CpG island (CGI) microarray analysis, we developed a new output value. The signal log ratio reflected the global methylation levels, but had only moderate linear correlation (r = 0.72) with the fraction of DNA molecules immunoprecipitated. By multiplying the signal log ratio using a coefficient obtained from the probability value that took account of signals in neighbouring probes, its linearity was markedly improved (r = 0.94). The new output value, Me value, reflected the global methylation level, had a strong correlation also with the fraction of methylated CpG sites obtained by bisulphite sequencing (r = 0.88), and had an accuracy of 71.8 and 83.8% in detecting completely methylated and unmethylated CGIs. Analysis of gastric cancer cell lines using the Me value showed that methylation of CGIs in promoters and gene bodies was associated with low and high, respectively, gene expression. The degree of demethylation of promoter CGIs after 5-aza-2'-deoxycytidine treatment had no association with that of induction of gene expression. The Me value was considered to be useful for analysis of intermediate methylation levels of CGIs

Attenuation of prostaglandin E2 elimination across the mouse blood-brain barrier in lipopolysaccharide-induced inflammation and additive inhibitory effect of cefmetazole

<p>Abstract</p> <p>Background</p> <p>Peripheral administration of lipopolysaccharide (LPS) induces inflammation and increases cerebral prostaglandin E₂(PGE₂) concentration. PGE₂is eliminated from brain across the blood-brain barrier (BBB) in mice, and this process is inhibited by intracerebral or intravenous pre-administration of anti-inflammatory drugs and antibiotics such as cefmetazole and cefazolin that inhibit multidrug resistance-associated protein 4 (Mrp4/Abcc4)-mediated PGE₂transport. The purpose of this study was to examine the effect of LPS-induced inflammation on PGE₂elimination from brain, and whether antibiotics further inhibit PGE₂elimination in LPS-treated mice.</p> <p>Methods</p> <p>[³H]PGE₂elimination across the BBB of intraperitoneally LPS-treated mice was assessed by the brain efflux index (BEI) method. Transporter protein amounts in brain capillaries were quantified by liquid chromatography-tandem mass spectrometry.</p> <p>Results</p> <p>The apparent elimination rate of [³H]PGE₂from brain was lower by 87%, in LPS-treated mice compared with saline-treated mice. The Mrp4 protein amount was unchanged in brain capillaries of LPS-treated mice compared with saline-treated mice, while the protein amounts of organic anion transporter 3 (Oat3/Slc22a8) and organic anion transporting polypeptide 1a4 (Oatp1a4/Slco1a4) were decreased by 26% and 39%, respectively. Either intracerebral or intravenous pre-administration of cefmetazole further inhibited PGE₂elimination in LPS-treated mice. However, intracerebral or intravenous pre-administration of cefazolin had little effect on PGE₂elimination in LPS-treated mice, or in LPS-untreated mice given Oat3 and Oatp1a4 inhibitors. These results indicate that peripheral administration of cefmetazole inhibits PGE₂elimination across the BBB in LPS-treated mice.</p> <p>Conclusion</p> <p>PGE₂elimination across the BBB is attenuated in an LPS-induced mouse model of inflammation. Peripheral administration of cefmetazole further inhibits PGE₂elimination in LPS-treated mice.</p

Lactobacillus helveticus SBT2171 Attenuates Experimental Autoimmune Encephalomyelitis in Mice

We recently reported that Lactobacillus helveticus SBT2171 (LH2171) inhibited the proliferation and inflammatory cytokine production of primary immune cells in vitro, and alleviated collagen-induced arthritis (CIA) in mice, a model of human rheumatoid arthritis (RA). In this study, we newly investigated whether LH2171 could relieve the severity of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), which is an autoimmune disease, but develop the symptoms by different mechanisms from RA. In MS and EAE, main cause of the disease is the abnormality in CD4+ T cell immunity, whereas in RA and CIA, is that in antibody-mediated immunity. The intraperitoneal administration of LH2171 significantly decreased the incidence and clinical score of EAE in mice. LH2171 also reduced the numbers of pathogenic immune cells, especially Th17 cells, in the spinal cord at the peak stage of disease severity. Interestingly, before the onset of EAE, LH2171 administration remarkably decreased the ratio of Th17 cells to CD4+ T cells in the inguinal lymph nodes (LNs), where pathogenic immune cells are activated to infiltrate the central nervous system, including the spinal cord. Furthermore, the expression of interleukin (IL)-6, an inflammatory cytokine essential for Th17 differentiation, decreased in the LNs of LH2171-administered mice. Moreover, LH2171 significantly inhibited IL-6 production in vitro from both DC2.4 and RAW264.7 cells, model cell lines of antigen-presenting cells. These findings suggest that LH2171 might down-regulate IL-6 production and the subsequent Th17 differentiation and spinal cord infiltration, consequently alleviating EAE symptoms

Topoisomerase II beta targets DNA crossovers formed between distant homologous sites to induce chromatin opening

Type II DNA topoisomerases (topo II) flip the spatial positions of two DNA duplexes, called G- and T- segments, by a cleavage-passage-resealing mechanism. In living cells, these DNA segments can be derived from distant sites on the same chromosome. Due to lack of proper methodology, however, no direct evidence has been described so far. The beta isoform of topo II (topo II beta) is essential for transcriptional regulation of genes expressed in the final stage of neuronal differentiation. Here we devise a genome-wide mapping technique (eTIP-seq) for topo II beta target sites that can measure the genomic distance between G- and T-segments. It revealed that the enzyme operates in two distinctive modes, termed proximal strand passage (PSP) and distal strand passage (DSP). PSP sites are concentrated around transcription start sites, whereas DSP sites are heavily clustered in small number of hotspots. While PSP represent the conventional topo II targets that remove local torsional stresses, DSP sites have not been described previously. Most remarkably, DSP is driven by the pairing between homologous sequences or repeats located in a large distance. A model-building approach suggested that topo II beta acts on crossovers to unknot the intertwined DSP sites, leading to chromatin decondensation

Author Correction:C151 in KEAP1 is the main cysteine sensor for the cyanoenone class of NRF2 activators, irrespective of molecular size or shape

Correction to: Scientific Reportshttps://doi.org/10.1038/s41598-018-26269-9, published online 23 May 2018 This Article contains an error in Figure 3.As a result of an error during the preparation of the figures for this Article, the western blots shown in Figure 3A and 3B contained an additional lane for the protein Tubulin. This is because an additional sample was loaded in the last lane of the gel to prevent potential stretching of the gel in this lane during electrophoresis if left empty. It was subsequently left uncropped from the tubulin blot shown in the published figure. The corrected Figure 3 and its accompanying legend appear below. C151 in KEAP1 is the primary sensor for MCE-23 and MCE-1 in MEF cells. Western blot analyses of total cell lysates of KEAP1-knockout MEF cells rescued with either wild-type (WT), single cysteine mutant C151S, double cysteine mutant C273W/C288E or triple cysteine mutant C151S/C273W/C288E of mouse N-terminally tagged HA-KEAP1. Cells (3 × 105 per well), growing in 6-well plates, were exposed to vehicle (0.1% DMSO) (A,B), MCE-23 (A) or MCE-1 (B) for 3 h, after which the cells were lysed. Immunoblotting was performed on cell lysates using antibodies raised against NRF2, HA and α-tubulin.</p