EGF-Dependent Activation of ELK1 Contributes to the Induction of CLDND1 Expression Involved in Tight Junction Formation.

Claudin proteins are intercellular adhesion molecules. Increased claudin domain-containing 1 (CLDND1) expression is associated with the malignant transformation of estrogen receptor-negative breast cancer cells with low sensitivity to hormone therapy. Abnormal CLDND1 expression is also implicated in vascular diseases. Previously, we investigated the regulatory mechanism underlying CLDND1 expression and identified a strong enhancer region near the promoter. In silico analysis of the sequence showed high homology to the ETS domain-containing protein-1 (ELK1)-binding sequence which is involved in cell growth, differentiation, angiogenesis, and cancer. Transcriptional ELK1 activation is associated with the mitogen-activated protein kinase (MAPK) signaling cascade originating from the epidermal growth factor receptor (EGFR). Here, we evaluated the effect of gefitinib, an EGFR tyrosine kinase inhibitor, on the suppression of CLDND1 expression using ELK1 overexpression in luciferase reporter and chromatin immunoprecipitation assays. ELK1 was found to be an activator of the enhancer region, and its transient expression increased that of CLDND1 at the mRNA and protein levels. CLDND1 expression was increased following EGF-induced ELK1 phosphorylation. Furthermore, this increase in CLDND1 was significantly suppressed by gefitinib. Therefore, EGF-dependent activation of ELK1 contributes to the induction of CLDND1 expression. These findings open avenues for the development of new anticancer agents targeting CLDND1.

The Effects of Sacran, a Sulfated Polysaccharide, on Gut Microbiota Using Chronic Kidney Disease Model Rats.

The aim of this study was to investigate the beneficial effects of sacran, a sulfated polysaccharide, on renal damage and intestinal microflora, in 5/6 nephrectomy rats as a model for chronic kidney disease (CKD). 5/6 Nephrectomy rats were divided into sacran treated and non-treated groups and examined for lethality after 4 weeks. The 5/6 nephrectomy rats were also divided into three groups: sacran treated, non-treated and AST-120 treated groups, and treated orally in a concentration-dependent manner for 4 weeks. Renal function was estimated by biochemical and histopathological analyses. Metagenomic analysis of feces from each group after 4 weeks was also performed and changes in intestinal microflora were compared. The administration of sacran to CKD rats at ≥19 mg/d increased their survival. In addition, the sacran-treated group improved CKD-related parameters in a concentration-dependent manner, and the inhibitory effect of 40 mg/d of sacran was comparable to that of AST-120. The changes in the intestinal microflora of the sacran treated group were positively correlated with an increase in the number of Lactobacillus species, which are known to be rich in beneficial bacteria, and the increment of this beneficial bacteria was negatively correlated with the concentration of indoxyl sulfate, a uremic toxin, in plasma. These results strongly suggest that the oral administration of sacran could contribute to the stabilization of intestinal microflora in CKD rats and to the reduction of oxidative stress as well as the inhibition of progression of CKD.

Identification of the RORα Transcriptional Network Contributes to the Search for Therapeutic Targets in Atherosclerosis.

The retinoic acid receptor-related orphan receptor α (RORα) is involved in the regulation of several physiological processes, including development, metabolism, and circadian rhythm. RORα-deficient mice display profound atherosclerosis, in which hypoalphalipoproteinemia is reportedly associated with decreased plasma levels of high-density lipoprotein, increased levels of inflammatory cytokines, and ischemia/reperfusion-induced damage. The recent characterization of endogenous ligands (including cholesterol, oxysterols, provitamin D3, and their derivatives), mediators, and initiation complexes associated with the transcriptional regulation of these orphan nuclear receptors has facilitated the development of synthetic ligands. These findings have also highlighted the potential of application of RORα as a therapeutic target for several diseases, including diabetes, dyslipidemia, and atherosclerosis. In this review, the current literature related to the structure and function of RORα, its genetic inter-individual differences, and its potential as a therapeutic target in atherosclerosis is discussed.

Sacran, a sulfated polysaccharide, suppresses the absorption of lipids and modulates the intestinal flora in non-alcoholic steatohepatitis model rats.

AIMS: The objective of this study was to investigate the effects of administering sacran, a sulfated polysaccharide, on liver biology, gut microbiota, oxidative stress, and inflammation on stroke-prone spontaneously hypertensive (SHRSP5/Dmcr) rats that develop fibrotic steatohepatitis with histological similarities to that of non-alcoholic steatohepatitis (NASH). MAIN METHODS: Four groups of 8-week-old SHRSP5/Dmcr rats were fed a high fat-cholesterol (HFC) diet for 4 and 8 weeks and administered either sacran (80 mg/kg/day) or a non-treatment, respectively. Liver function was evaluated by biochemical and histopathological analyses. Hepatic inflammatory markers were measured using mRNA expression. Fecal microbial profiles were determined via 16S rRNA sequencing. A triglyceride (TG) absorption test was administered to the 8-week-old Sprague-Dawley (SD) rats. KEY FINDING: Sacran administration was observed to decrease the extent of oxidative stress and hepatic biochemical parameters in serum and hepatic injury with the levels of transforming growth factor-beta (TGF-ß1) and tumor necrosis factor-alpha (TNF-α), being increased compared to those of the non-treatment group. At the genus level, sacran administration caused a significant decrease in the harmful Prevotella genus, and a significant increase in the useful Blautia genus was observed. Sacran administration also decreased the serum TG increase that was induced by administering corn oil to the SD rats. SIGNIFICANCE: We conclude that sacran administration has the potential to reduce the absorption of lipids into blood and to improve several gut microbiotas, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress and hepatic markers in the systematic circulation on NASH.

Transcription of CLDND1 in human brain endothelial cells is regulated by the myeloid zinc finger 1.

Increased permeability of endothelial cells lining the blood vessels in the brain leads to vascular oedema and, potentially, to stroke. The tight junctions (TJs), primarily responsible for the regulation of vascular permeability, are multi-protein complexes comprising the claudin family of proteins and occludin. Several studies have reported that downregulation of the claudin domain containing 1 (CLDND1) gene enhances vascular permeability, which consequently increases the risk of stroke. However, the transcriptional regulation of CLDND1 has not been studied extensively. Therefore, this study aimed to identify the transcription factors (TFs) regulating CLDND1 expression. A luciferase reporter assay identified a silencer within the first intron of CLDND1, which was identified as a potential binding site of the myeloid zinc finger 1 (MZF1) through in silico and TFBIND software analyses, and confirmed through a reporter assay using the MZF1 expression vector and chromatin immunoprecipitation (ChIP) assays. Moreover, the transient overexpression of MZF1 significantly increased the mRNA and protein expression levels of CLDND1, conversely, which were suppressed through the siRNA-mediated MZF1 knockdown. Furthermore, the permeability of FITC-dextran was observed to be increased on MZF1 knockdown as compared to that of the siGFP control. Our data revealed the underlying mechanism of the transcriptional regulation of CLDND1 by the MZF1. The findings suggest a potential role of MZF1 in TJ formation, which could be studied further and applied to prevent cerebral haemorrhage.

Effects of surface-deacetylated chitin nanofibers on non-alcoholic steatohepatitis model rats and their gut microbiota.

Nonalcoholic steatohepatitis (NASH), a more advanced form of nonalcoholic fatty liver disease (NAFLD), is associated with increased cardiovascular and liver-related mortality. Stroke-prone spontaneously hypertensive rats (SHRSP5/Dmcr) that are fed a high-fat and high-cholesterol diet develop hepatic lesions that are similar to those observed in human NASH pathology. We investigated the hepatic protective and antioxidant effects of surface-deacetylated chitin nanofibers (SDACNFs) that were administered to SHRSP5/Dmcr rats for 8 weeks. The administration of SDACNFs (80 mg/kg/day) resulted in a significant decrease in hepatic injury, oxidative stress, compared with the non-treatment. The SDACNFs also caused a reduction in the population of harmful members of the Morganella and Prevotella genus, and increased the abundance of the Blautia genus, a useful bacterium in gut microbiota. We therefore conclude that SDACNF exerts anti-hepatic and antioxidative effects not only by adsorbing lipid substances but also by reforming the community of intestinal microflora in the intestinal tract.

Orphan Nuclear Receptor RORα Regulates Enzymatic Metabolism of Cerebral 24S-Hydroxycholesterol through CYP39A1 Intronic Response Element Activation.

Oxysterols, important regulators of cholesterol homeostasis in the brain, are affected by neurodegenerative diseases. Early-onset Alzheimer's disease is associated with higher levels of circulating brain-derived 24S-hydroxycholesterol (24S-OHC). Conversion of cholesterol to 24S-OHC is mediated by cholesterol 24S-hydroxylase in the brain, which is the major pathway for oxysterol elimination, followed by oxidation through hepatic first-pass metabolism by CYP39A1. Abnormal CYP39A1 expression results in accumulation of 24S-OHC, influencing neurodegenerative disease-related deterioration; thus, it is important to understand the normal elimination of 24S-OHC and the system regulating CYP39A1, a selective hepatic metabolic enzyme of 24S-OHC. We examined the role of transcriptional regulation by retinoic acid receptor-related orphan receptor α (RORα), a nuclear receptor that responds to oxysterol ligands. In humans, the promoter and first intronic regions of CYP39A1 contain two putative RORα response elements (ROREs). RORα binding and responses of these ROREs were assessed using electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase reporter assays. CYP39A1 was upregulated by RORα overexpression in HEK293 cells, while RORα knockdown by siRNA significantly downregulated CYP39A1 expression in human hepatoma cells. Additionally, CYP39A1 was induced by RORα agonist treatment, suggesting that CYP39A1 expression is activated by RORα nuclear receptors. This may provide a way to increase CYP39A1 activity using RORα agonists, and help halt 24S-OHC accumulation in neurodegenerative illnesses.

Retinoic acid receptor-related orphan receptor α reduces lipid droplets by upregulating neutral cholesterol ester hydrolase 1 in macrophages.

BACKGROUND: Neutral cholesterol ester hydrolase 1 (NCEH1) catalyzes the hydrolysis of cholesterol ester (CE) in macrophages. Genetic ablation of NCEH1 promotes CE-laden macrophages and the development of atherosclerosis in mice. Dysregulation of NCEH1 levels is involved in the pathogenesis of multiple disorders including metabolic diseases and atherosclerosis; however, relatively little is known regarding the mechanisms regulating NCEH1. Retinoic acid receptor-related orphan receptor α (RORα)-deficient mice exhibit several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia and increased susceptibility to atherosclerosis. RESULTS: In this study, inhibition of lipid droplet formation by RORα positively regulated NCEH1 expression in macrophages. In mammals, the NCEH1 promoter region was found to harbor putative RORα response elements (ROREs). Electrophoretic mobility shift, chromatin immunoprecipitation, and luciferase reporter assays showed that RORα binds and responds to ROREs in human NCEH1. Moreover, NCEH1 was upregulated through RORα via a phorbol myristate acetate-dependent mechanism during macrophage differentiation from THP1 cells. siRNA-mediated knockdown of RORα significantly downregulated NCEH1 expression and accumulated lipid droplets in human hepatoma cells. In contrast, NCEH1 expression and removal of lipid droplets were induced by RORα agonist treatments and RORα overexpression in macrophages. CONCLUSION: These data strongly suggested that NCEH1 is a direct RORα target, defining potential new roles for RORα in the inhibition of lipid droplet formation through NCEH1.

Stroke-prone spontaneously hypertensive rats have reduced hydroxysteroid 17-ß dehydrogenase 7 levels for low cholesterol biosynthesis.

Reduced serum cholesterol content was recently reported to be one of the factors responsible for cerebral haemorrhage. Stroke-prone spontaneously hypertensive rats (SHRSP) are known to have lower serum cholesterol content than normotensive Wistar-Kyoto rats (WKY). We previously reported that lower levels of mevalonate pyrophosphate decarboxylase (MPD) and squalene epoxidase (SQE), which are associated with cholesterol biosynthesis in the liver, are involved in the low serum cholesterol content in SHRSP. Here, we investigate the levels of sterol 14-demethylase (CYP51), methylsterol monooxygenase (SC4MOL), and hydroxysteroid 17-ß dehydrogenase 7 (HSD17B7), which contribute to the cholesterol synthesis pathway in the conversion of lanosterol to zymosterol, in SHRSP and WKY. The HSD17B7 mRNA levels in the liver of SHRSP were markedly lower than those in WKY, whereas no significant differences were observed in CYP51 and SC4MOL levels in the two types of rats. The relative levels of protein, heteronuclear RNA, and mRNA of HSD17B7 were also significantly lower in SHRSP than in WKY. The degradation rates of HSD17B7 were the same in SHRSP and WKY. The protein levels of HSD17B7 were not significantly reduced in tissues other than the liver, including the brain, lung, heart, spleen, kidney, and testis, in SHRSP. Moreover, HSD17B7 activity was significantly lower in SHRSP than in WKY. Thus, our results indicated that low protein levels and activity of HSD17B7 are responsible for the reduced cholesterol content in SHRSP, indicating that HSD17B7, along with MPD and SQE, is involved in the decreased cholesterol synthesis in the liver of SHRSP.

Design and Evaluation of An Extended-Release Olmesartan Tablet Using Chitosan/Cyclodextrin Composites.

Sustained-release olmesartan tablets (OLM) were prepared by the simple, direct compression of composites of anionic sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD) and cationic spray-dried chitosan (SD-CS), and were evaluated for use as a sustained release preparation for the treatment of hypertension. An investigation of the interaction between OLM and SBE-ß-CD by the solubility method indicated that the phase diagram of the OLM/SBE-ß-CD system was the AL type, indicating the formation of a 1:1 inclusion complex. The release of OLM from tablets composed of the SD-CS/SBE-ß-CD composite was slow in media at both pH 1.2 and at 6.8. The in vitro slow release characteristics of the SD-CS/SBE-ß-CD composite were reflected in the in vivo absorption of the drug after normal rats were given an oral administration of the preparation. Furthermore, the SD-CS/SBE-ß-CD composite continuously increased the antihypertensive effect of OLM in hypertensive rats, compared with that of the drug itself. These results suggest that a simple mixing of SD-CS and SBE-ß-CD can be potentially useful for the controlled release of a drug for the continuous treatments of hypertension.

Levels of tight junction protein CLDND1 are regulated by microRNA-124 in the cerebellum of stroke-prone spontaneously hypertensive rats.

The claudin family shows organ- and tissue-specific expression of individual members. Deficiency or aberrant expression of distinct claudins has been reported to be associated with severe pathophysiological consequences. Claudin domain-containing 1 (CLDND1), also known as claudin-25, shows homology to this family of proteins. Furthermore, serum CLDND1-derived peptide antibody levels are elevated in patients with cerebral infarction, as compared with healthy controls. We previously reported that, in the adult murine brain, CLDND1 is abundantly expressed in the cerebellum in common sites of intracerebral hemorrhage, and CLDND1 levels are transiently decreased after hemorrhagic insult. However, regulation of CLDND1 expression levels in cerebrovascular disease is poorly studied, and most regulatory microRNAs remain to be defined. We assessed its expression level, according to the presence of early signs of cerebrovascular disease, in the brain of stroke-prone spontaneously hypertensive rats (SHRSPs) and investigated the microRNA regulation of Cldnd1 mRNA. We investigated the post-transcriptional regulation of Cldnd1 by examining the subcellular distribution of its mRNA and evaluating its translational regulation by microRNA in human brain endothelial cells (HBECs) and in the brain of SHRSPs. Using bioinformatics, we identified a conserved microRNA-124 (miR-124)-binding site in the 3'-untranslated region of Cldnd1 and demonstrated that miR-124 regulates the translation of Cldnd1 mRNA reporters in a sequence-specific manner in luciferase assays. HBECs transfected with an miR-124 mimic showed decreased levels of CLDND1 mRNA in reverse transcription quantitative PCR. miR-124 levels were markedly lower in SHRSP than in Wister Kyoto rat brains, whereas Cldnd1 mRNA and protein levels were significantly higher. In SHRSP brains, Cldnd1 mRNA levels increased with a decrease in miR-124. Therefore, by interacting with Cldnd1 mRNA, miR-124 influences CLDNL1 levels in the brain, thus playing a role in the development of cerebrovascular disease in SHRSPs.

Claudin domain containing 1 contributing to endothelial cell adhesion decreases in presence of cerebellar hemorrhage.

The claudin family comprises four-pass transmembrane proteins involved in the formation of tight junctions (TJs). Relatively recently, claudin domain containing (CLDND) 1, also known as claudin-25, was identified as a novel member of the claudin family. In the present study, we revealed that in the adult murine brain, CLDND1 is abundant in the cerebellum among common sites of intracerebral hemorrhage. Thus, the dynamics of CLDND1 after cerebellar hemorrhage were examined. Both CLDND1 mRNA and protein levels transiently decreased at 24 hr after hemorrhagic insult. For immunostaining, an anti-CLDND1 antibody that recognizes the specific epitope in the extracellular first loop was prepared. Dual immunohistochemical staining with CD31 using coronal cryosections of intact murine cerebellum tissue revealed that CLDND1 is expressed on endothelial cells. We therefore performed an in vitro permeability test using a human brain endothelial cell (HBEC) line to reveal whether CLDND1 contributes to cell adhesion like other claudins. CLDND1 was expressed on HBECs as well as in murine cerebellum tissue, and a strong signal was observed at TJs. RNA interference against CLDND1 decreased both the mRNA and protein levels without cytotoxicity. The permeability to small molecules, but not to large ones, across confluent HBECs increased on CLDND1 knockdown compared with mock-treated cells. These results suggest that the transient decrease of CLDND1 after cerebellar hemorrhage is responsible for low-molecular-weight selective vascular hyperpermeability. © 2017 Wiley Periodicals, Inc.

The retinoic acid receptor-related orphan receptor α positively regulates tight junction protein claudin domain-containing 1 mRNA expression in human brain endothelial cells.

Members of the claudin family play important roles in the formation of tight junctions (TJs) in several tissues. Claudin domain containing 1 (CLDND1) is homologous to this family and localizes to TJs and the cytoplasm when exogenously expressed in cultured epithelial cell lines. Furthermore, serum antibody levels of CLDND1-derived peptides are elevated in patients with cerebral infection, cardiovascular disease or diabetes mellitus as compared to healthy controls. However, CLDND1 transcriptional regulation remains poorly analyzed and most regional transcription factor binding sites remain to be defined. Notably, the CLDND1 promoter contains a putative response element for retinoic acid receptor-related orphan receptor α (RORα), which is involved in the above-mentioned disorders. In this study, we found that Cldnd1 and Rora mRNA levels are correlated in rat tissues and that RORα overexpression in human brain endothelial cells enhanced CLDND1 transcript expression. In addition, siRNA-mediated knockdown of RORα significantly decreased CLDND1 transcription. An electrophoresis mobility shift assay indicated that RORα binds to the identified response element in a sequence-specific manner. Furthermore, luciferase reporter assays confirmed that RORα interacts with the CLDND1 promoter to enhance transcription. Taken together, our findings strongly suggest that CLDND1 is a direct RORα target.

High Expression Levels of NADPH Oxidase 3 in the Cerebrum of Ten-Week-Old Stroke-Prone Spontaneously Hypertensive Rats.

We previously demonstrated that the high levels of oxidative stress in the brains of ten-week-old stroke-prone hypertensive rats (SHRSP) were attributable to intrinsic, not extrinsic factors (Biol. Pharm. Bull., 33, 2010, Michihara et al.). The aim of the present study was to determine whether increases in the enzymes producing reactive oxygen species (ROS), reductions in the enzymes and proteins removing ROS, or increases in an enzyme and transporter removing antioxidants promoted oxidative stress in the SHRSP cerebrum. No significant decreases were observed in the mRNA levels of enzymes that remove ROS between SHRSP and normotensive Wistar Kyoto rats. The activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) and the protein and mRNA levels of NOX3, an enzyme that produces ROS, were significantly increased in the SHRSP cerebrum. These results suggested that the high expression levels of NOX3 increased oxidative stress in the SHRSP cerebrum.

Lower Squalene Epoxidase and Higher Scavenger Receptor Class B Type 1 Protein Levels Are Involved in Reduced Serum Cholesterol Levels in Stroke-Prone Spontaneously Hypertensive Rats.

A lower serum cholesterol level was recently shown to be one of the causes of stroke in an epidemiological study. Spontaneously hypertensive rats stroke-prone (SHRSP) have lower serum cholesterol levels than normotensive Wistar-Kyoto rats (WKY). To elucidate the mechanisms responsible for the lower serum cholesterol levels in SHRSP, we determined whether the amounts of cholesterol biosynthetic enzymes or the receptor and transporter involved in cholesterol uptake and efflux in the liver were altered in SHRSP. When the mRNA levels of seven cholesterol biosynthetic enzymes were measured using real-time polymerase chain reaction (PCR), farnesyl pyrophosphate synthase and squalene epoxidase (SQE) levels in the liver of SHRSP were significantly lower than those in WKY. SQE protein levels were significantly reduced in tissues other than the brain of SHRSP. No significant differences were observed in low-density lipoprotein (LDL) receptor (uptake of serum LDL-cholesterol) or ATP-binding cassette transporter A1 (efflux of cholesterol from the liver/formation of high-density lipoprotein (HDL)) protein levels in the liver and testis between SHRSP and WKY, whereas scavenger receptor class B type 1 (SRB1: uptake of serum HDL-cholesterol) protein levels were higher in the livers of SHRSP. These results indicated that the lower protein levels of SQE and higher protein levels of SRB1 in the liver were involved in the reduced serum cholesterol levels in SHRSP.

Phosphoenolpyruvate Carboxykinase, a Key Enzyme That Controls Blood Glucose, Is a Target of Retinoic Acid Receptor-Related Orphan Receptor α.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes a committed and rate-limiting step in hepatic gluconeogenesis, and its activity is tightly regulated to maintain blood glucose levels within normal limits. PEPCK activity is primarily regulated through hormonal control of gene transcription. Transcription is additionally regulated via a cAMP response unit, which includes a cAMP response element and four binding sites for CCAAT/enhancer-binding protein (C/EBP). Notably, the cAMP response unit also contains a putative response element for retinoic acid receptor-related orphan receptor α (RORα). In this paper, we characterize the effect of the RORα response element on cAMP-induced transcription. Electrophoresis mobility shift assay indicates that RORα binds this response element in a sequence-specific manner. Furthermore, luciferase reporter assays indicate that RORα interacts with C/EBP at the PEPCK promoter to synergistically enhance transcription. We also found that cAMP-induced transcription depends in part on RORα and its response element. In addition, we show that suppression of RORα by siRNA significantly decreased PEPCK transcription. Finally, we found that a RORα antagonist inhibits hepatic gluconeogenesis in an in vitro glucose production assay. Taken together, the data strongly suggest that PEPCK is a direct RORα target. These results define possible new roles for RORα in hepatic gluconeogenesis.

Involvement of microRNA214 and transcriptional regulation in reductions in mevalonate pyrophosphate decarboxylase mRNA levels in stroke-prone spontaneously hypertensive rat livers.

Hypocholesterolemia has been epidemiologically identified as one of the causes of stroke (cerebral hemorrhage). We previously reported that lower protein levels of mevalonate pyrophosphate decarboxylase (MPD), which is responsible for reducing serum cholesterol levels in stroke-prone spontaneously hypertensive rats (SHRSP), in the liver were caused by a reduction in mRNA levels. However, the mechanism responsible for reducing MPD expression levels in the SHRSP liver remains unclear. Thus, we compared microRNA (miR)-214 combined with the 3'-untranslated region of MPD mRNA and heterogeneous nuclear RNA (hnRNA) between SHRSP and normotensive Wistar Kyoto rats (WKY). miR-214 levels in the liver were markedly higher in SHRSP than in WKY, whereas hnRNA levels were significantly lower. These results indicate that the upregulation of miR-214 and downregulation of MPD transcription in the liver both play a role in the development of hypocholesterolemia in SHRSP.

Constitutive expression of a COOH-terminal leucine mutant of lysosome-associated membrane protein-1 causes its exclusive localization in low density intracellular vesicles.

Lysosome-associated membrane protein-1 (LAMP-1) is a type I transmembrane protein with a short cytoplasmic tail that possesses a lysosome-targeting signal of GYQTI(382)-COOH. Wild-type (WT)-LAMP-1 was exclusively localized in high density lysosomes, and efficiency of LAMP-1's transport to lysosomes depends on its COOH-terminal amino acid residue. Among many different COOH-terminal amino acid substitution mutants of LAMP-1, a leucine-substituted mutant (I382L) displays the most efficient targeting to late endosomes and lysosomes [Akasaki et al. (2010) J. Biochem. 148: , 669-679]. In this study, we generated two human hepatoma cell lines (HepG2 cell lines) that stably express WT-LAMP-1 and I382L, and compared their intracellular distributions. The subcellular fractionation study using Percoll density gradient centrifugation revealed that WT-LAMP-1 had preferential localization in the high density secondary lysosomes where endogenous human LAMP-1 was enriched. In contrast, a major portion of I382L was located in a low density fraction. The low density fraction also contained approximately 80% of endogenous human LAMP-1 and significant amounts of endogenous ß-glucuronidase and LAMP-2, which probably represents occurrence of low density lysosomes in the I382L-expressing cells. Double immunofluorescence microscopic analyses distinguished I382L-containing intracellular vesicles from endogenous LAMP-1-containing lysosomes and early endosomes. Altogether, constitutive expression of I382L causes its aberrant intracellular localization and generation of low density lysosomes, indicating that the COOH-terminal isoleucine is critical for normal localization of LAMP-1 in the dense lysosomes.

Antioxidant and renoprotective activity of chitosan in nephrectomized rats.

The effect of chitosan on oxidative stress and chronic renal failure was investigated using 5/6 nephrectomized rats. The ingestion of chitosan over a 4-week period resulted in a significant decrease in total body weight, glucose, serum creatinine and indoxyl sulfate levels (P=0.0011, P=0.0006, P=0.0012, and P=0.0005, respectively), compared with the non-treated nephrectomized group. The ingestion of chitosan also resulted in a lowered ratio of oxidized to reduced albumin (P=0.003) and an increase in biological antioxidant potential (P=0.023). Interestingly, the oxidized albumin ratio was correlated with serum indoxyl sulfate levels in vivo. These results suggest that the ingestion of chitosan results in a significant reduction in the levels of pro-oxidants, such as uremic toxins, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation.

Comparison of receptors and enzymes regulating cholesterol levels in liver between SHR/NDmcr-cp rats and normotensive Wistar Kyoto rats at ten weeks of age.

The spontaneously hypertensive rat (SHR)/NDmcr-cp (SHR-cp), which is a metabolic syndrome model rat, was reported to show hypercholesteremia, as compared with lean littermates. The serum total cholesterol level in SHR-cp at 18 weeks of age is higher than that of normotensive Wistar Kyoto rat (WKY), but that in SHR-cp at 10 weeks of age is the same. The objective of this study is to clarify whether there are differences in the system regulating serum cholesterol levels between SHR-cp and WKY at 10 weeks of age. Total serum cholesterol levels, and cholesterol levels of high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) were similar in the two strains. However, the cholesterol levels in the liver of SHR-cp were lower than those of WKY. Next, mRNA levels of receptors (scavenger receptor class B type 1 [SRB1], LDL receptor [LDLR]) involved in uptake from serum to liver or enzymes of cholesterol catabolism (CYP7A1 and CYP8B1) and biosynthesis (mevalonate pyrophosphate decarboxylases [MPD]) in liver were compared between SHR-cp and WKY. High levels of MPD and LDLR and low levels of SRB1 were shown in SHR-cp, as compared with WKY. CYP7A1 and CYP8B1 levels were similar between SHR-cp and WKY. These results suggest that the serum cholesterol level in SHR-cp by the balance or regulation between the rise in cholesterol uptake and reduction in cholesterol biosynthesis in the liver is the same as that in WKY.