Recombinant tissue-type plasminogen activator transiently enhances blood-brain barrier permeability during cerebral ischemia through vascular endothelial growth factor-mediated endothelial endocytosis in mice.

Recombinant tissue-type plasminogen activator (rt-PA) modulates cerebrovascular permeability and exacerbates brain injury in ischemic stroke, but its mechanisms remain unclear. We studied the involvement of vascular endothelial growth factor (VEGF)-mediated endocytosis in the increase of blood-brain barrier (BBB) permeability potentiated by rt-PA after ischemic stroke. The rt-PA treatment at 4 hours after middle cerebral artery occlusion induced a transient increase in BBB permeability after ischemic stroke in mice, which was suppressed by antagonists of either low-density lipoprotein receptor families (LDLRs) or VEGF receptor-2 (VEGFR-2). In immortalized bEnd.3 endothelial cells, rt-PA treatment upregulated VEGF expression and VEGFR-2 phosphorylation under ischemic conditions in an LDLR-dependent manner. In addition, rt-PA treatment increased endocytosis and transcellular transport in bEnd.3 monolayers under ischemic conditions, which were suppressed by the inhibition of LDLRs, VEGF, or VEGFR-2. The rt-PA treatment also increased the endocytosis of endothelial cells in the ischemic brain region after stroke in mice. These findings indicate that rt-PA increased BBB permeability via induction of VEGF, which at least partially mediates subsequent increase in endothelial endocytosis. Therefore, inhibition of VEGF induction may have beneficial effects after thrombolytic therapy with rt-PA treatment after stroke.

Characterization of a new cold-adapted lipase from Pseudomonas sp. TK-3.

A psychrotrophic Pseudomonas sp. TK-3 was isolated from dirty and cool stream water in Toyama, Japan from which we cloned and characterized the bacterial lipase LipTK-3. The sequenced DNA fragment contains an open reading frame of 1,428 bp that encoded a protein of 476 amino acids with an estimated molecular mass of 50,132 Da. The lipase showed high sequence similarity to those of subfamily Ι.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 20-25 °C, lower than in most other subfamily Ι.3 lipases. The lipase exhibited about 30 % of maximal activity at 5 °C. The optimal pH value was 8.0. The activity was strongly inhibited by EDTA and was highly dependent on Ca(2+). Tricaprylin and p-nitrophenyl caprylate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipTK-3 also showed high activity towards natural substrates including edible vegetable oils and animal fats. Furthermore, LipTK-3 was very active and stable in the presence of several detergents, metal ions, and organic solvents. This cold-adapted lipase may prove useful for future applications.

Detection of fluoroquinolone-induced tendon disorders using a hospital database in Japan.

PURPOSE: Tendinitis and tendon rupture are well-known side effects of fluoroquinolones (FQs) in Western countries. In Japan, some case reports have been reported; however, the incidence of FQ-induced tendon disorders has not been investigated. The aims of this study were to measure the occurrence of tendon disorders associated with FQs in Japanese patients and to compare the observed risk with that of previous reports. Moreover, the observed risk in FQ-prescribed patients was compared with that in cephalosporin-prescribed patients. METHODS: The Hamamatsu University Hospital database was examined to determine the risk of tendon disorders that occurred in all inpatients and outpatients between the first day of prescription of an oral FQ or cephalosporins to the calculated end date plus 30 days. The risk of tendon disorders, the risk ratio, and their 95% confidence intervals (CIs) were evaluated. RESULTS: From April 1996 to December 2009, FQs were prescribed to 17 147 patients, 14 of whom had tendon disorders (risk: 0.082%, 95%CI: 0.049-0.137). The risk of a tendon disorder in FQ-prescribed patients was significantly higher than that in cephalosporin-prescribed patients (five tendon disorders in 38 517 patients, risk: 0.013%, 95%CI: 0.006-0.030, and p < 0.001). The risk ratio of a tendon disorder in FQ-prescribed patients in relation to cephalosporin-prescribed patients was 6.29 (95%CI: 2.27-17.46). A large discrepancy in the risk of tendon disorders was not observed between our findings and previous reports. CONCLUSION: A hospital database search revealed that the risk of tendon disorders in Japanese patients administered with FQs was higher than in those administered with cephalosporins.

Combination of reactive oxygen species and tissue-type plasminogen activator enhances the induction of gelatinase B in brain endothelial cells.

Tissue-type plasminogen activator (t-PA) increases the risk of intracranial hemorrhage by gelatinase B (matrix metalloprotease-9; MMP-9) production in brain endothelial cells. It was recently reported that the free-radical scavenger edaravone significantly decreases t-PA-mediated MMP-9 production. Therefore, by using cultured brain endothelial cells (b.End3), we investigated whether t-PA-mediated MMP-9 production was enhanced by reactive oxygen species (ROS) and its signaling pathways. Moreover, we also investigated that whether this combined enhancement is reduced by edaravone. The b.End3 cells were exposed to t-PA (10 µg/mL), followed by H2O2 (30 µM); further, the MMP-9 protein level was measured. ROS enhanced MMP-9 production, and ROS plus t-PA significantly increased MMP-9 production more than t-PA or ROS alone. The results showed that H2O2 or t-PA alone caused a significant increase in NF-κB translocation to the nucleus, whereas the combination of t-PA and H2O2 increased the translocation of NF-κB to an even greater extent. Moreover, the combination of t-PA and ROS significantly increased I-κB degradation as well as NF-κB expression. Edaravone completely decreased the ROS plus t-PA-mediated MMP-9 enhancement. In conclusion, ROS enhanced t-PA-mediated MMP-9 production in brain endothelial cells; this MMP-9 production was decreased by the addition of edaravone, which inhibited the NF-κB pathway, specifically by enhancing I-κB degradation.

Tissue-type plasminogen activator (t-PA) induces stromelysin-1 (MMP-3) in endothelial cells through activation of lipoprotein receptor-related protein.

Tissue-type plasminogen activator (t-PA) is approved for treatment of ischemic stroke patients, but it increases the risk of intracranial bleeding (ICB). Previously, we have shown in a mouse stroke model that stromelysin-1 (matrix metalloproteinase-3 [MMP-3]) induced in endothelial cells was critical for ICB induced by t-PA. In the present study, using bEnd.3 cells, a mouse brain-derived endothelial cell line, we showed that MMP-3 was induced by both ischemic stress and t-PA treatment. This induction by t-PA was prevented by inhibition either of low-density lipoprotein receptor-related protein (LRP) or of nuclear factor-kappaB activation. LRP was up-regulated by ischemic stress, both in bEnd.3 cells in vitro and in endothelial cells at the ischemic damage area in the mouse stroke model. Furthermore, inhibition of LRP suppressed both MMP-3 induction in endothelial cells and the increase in ICB by t-PA treatment after stroke. These findings indicate that t-PA deteriorates ICB via MMP-3 induction in endothelial cells, which is regulated through the LRP/nuclear factor-kappaB pathway.

Comparison of enzymatically synthesized inulin, resistant maltodextrin and clofibrate effects on biomarkers of metabolic disease in rats fed a high-fat and high-sucrose (cafeteria) diet.

BACKGROUND: While naturally occurring inulin has anti-hyperlipidemic effects in animals and humans, health effects of synthetic inulin with different degrees of fructose polymerization remain poorly understood. AIM OF THE STUDY: Our study aimed at distinguishing health effects of synthetic inulin with different degrees of fructose polymerization (DP) from those of resistant maltodextrin and clofibrate. METHODS: We examined effects of synthetic inulin on serum and liver lipid profiles and blood biochemical parameters in rats fed a high-fat and high-sucrose (HF, cafeteria) diet when compared to resistant maltodextrin and clofibrate. RESULTS: Treatment with inulin (average DP = 6-8, 16-17 and 23) and resistant maltodextrin for 3 weeks reduced the elevation in liver levels of triacylglycerol and total cholesterol of rats fed the cafeteria diet but not the standard diet. In these groups, inulin (average DP = 16-17) significantly reduced the portal plasma glucose level. Moreover, the levels of portal plasma propionate and circulating serum adiponectin, which were decreased in cafeteria rats, recovered to nearly normal levels after administration of inulin (average DP = 16-17). In addition, the dietary inulin suppressed elevation in levels of portal plasma insulin and circulating serum leptin and induction of acetyl-CoA carboxylase and fatty acid synthase mRNAs in the liver of cafeteria rats, consistent with the reduction of liver lipids. The dietary inulin and clofibrate markedly reduced triacylglycerol levels in serum very low density lipoprotein (VLDL) and liver and epididymal adipose tissue weights of cafeteria rats; the extent of suppression by the dietary inulin was higher than that by clofibrate. No additive or synergistic effect of the dietary inulin and clofibrate was found in decrease in circulating serum VLDL and liver lipid levels. CONCLUSION: These observations indicate that the dietary inulin may prevent the development of metabolic disease such as hyperlipidemia and hyperinsulinemia caused by intake of cafeteria diet, in association with suppression of liver lipogenesis.

Transcriptional regulation of human UGT1A1 gene expression through distal and proximal promoter motifs: implication of defects in the UGT1A1 gene promoter.

Human UDP-glucuronosyltransferase (UGT)1A1 is a critical enzyme responsible for detoxification and metabolism of endogenous and exogenous lipophilic compounds, such as potentially neurotoxic bilirubin and the anticancer drug irinotecan SN-38, via conjugation with glucuronic acid. A 290-bp distal enhancer module, phenobarbital-responsive enhancer module of UGT1A1 (gtPBREM), fully accounts for constitutive androstane receptor (CAR)-, pregnane X receptor (PXR)-, glucocorticoid receptor (GR)-, and aryl hydrocarbon receptor (AhR)-mediated activation of the UGT1A1 gene. This study indicates that hepatocyte nuclear factor 1alpha (HNF1alpha) bound to the proximal promoter motif not only enhances the basal reporter activity of UGT1A1, including the distal (-3570/-3180) and proximal (-165/-1) regions, but also influences the transcriptional regulation of UGT1A1 by CAR, PXR, GR, and AhR to markedly enhance reporter activities. Moreover, we assessed the influence of the TA repeat polymorphism and gtPBREM T-3279G mutation on transcriptional activation of UGT1A1 by CAR, PXR, GR, and AhR. Transcriptional activation of the A(TA)(7)TAA mutant by CAR, the PXR activator rifampicin, the GR activator dexamethasone, and the AhR activator benzo[a]pyrene was more reduced than that of the T-3279G variant, and the activity of the UGT1A1 promoter with both T-3279G and A(TA)(7)TAA mutations was still lower. Thus, UGT1A1 gene promoter variations, including the TA repeat polymorphism and T-3279G gtPBREM, have important clinical implications.

Dietary inulin alleviates hepatic steatosis and xenobiotics-induced liver injury in rats fed a high-fat and high-sucrose diet: association with the suppression of hepatic cytochrome P450 and hepatocyte nuclear factor 4alpha expression.

Inulin enzymatically synthesized from sucrose is a dietary component that completely escapes glucide digestion. Supplementing inulin to a high-fat and high-sucrose diet (HF) ameliorated hypertriglycemia and hepatic steatosis in 8-week-fed rats by suppressing elevated levels of serum triacylglycerols, fatty acids, and glucose, and the accumulation of hepatic triacylglycerols and fatty acids. Inulin intake prevented phenobarbital (PB)- and dexamethasone-induced liver injuries in the HF group. No significant alteration in the baseline expression of CYP2B, CYP2C11, CYP3A, and NADPH-cytochrome P450 (P450) reductase mRNAs and proteins was found. In contrast, baseline and PB-treated expressions of CYP2E1 mRNA were reduced in HF-fed rats. The induction of P450s in response to PB was affected by the nutritional status of the rats; mRNA levels of CYP2B1 and CYP3A1 after PB treatment, as assessed by quantitative real-time polymerase chain reaction analysis were reduced in the inulin-supplemented HF (HF+I) group, compared with those in the HF group. Western blot analysis detected the corresponding changes of CYP2B and CYP3A proteins. These alterations were correlated with changes in hepatic thiobarbituric acid-reactive substances. Furthermore, no significant difference in the expression of nuclear receptors constitutive androstane receptor, pregnane X receptor, and retinoid X receptor alpha and coactivator peroxisome proliferator-activated receptor-gamma coactivator 1alpha proteins was found in the hepatic nucleus between the HF and HF+I groups, but the expression of hepatocyte nuclear factor alpha (HNF4alpha) protein was significantly reduced in the HF+I group. Taken together, these results indicate that inulin intake ameliorates PB-induced liver injury, associated with a decline in lipid accumulation and PB-induced expression of CYP2B and CYP3A, which may be related by a reduction in the nuclear expression of HNF4alpha.

Transcriptional regulation of human UGT1A1 gene expression: activated glucocorticoid receptor enhances constitutive androstane receptor/pregnane X receptor-mediated UDP-glucuronosyltransferase 1A1 regulation with glucocorticoid receptor-interacting protein 1.

UDP-glucuronosyltransferase (UGT) 1A1 glucuronidates endogenous metabolites, such as bilirubin, and exogenous substances, and plays a critical role in their detoxification and excretion. In a previous article, we described the phenobarbital response activity to a 290-base pair (bp) distal enhancer sequence (-3499/-3210) of the human UGT1A1 gene that is activated by the constitutive androstane receptor (CAR). Here, we show that dexamethasone at submicromolar concentrations enhances the pregnane X receptor (PXR) activator-mediated expression of the UGT1A1 gene and protein in HepG2 cells. We investigated the molecular mechanism of UGT1A1 induction by glucocorticoids at submicromolar concentrations and PXR activators and the functional cross-talk between the glucocorticoid receptor (GR) and CAR/PXR. The glucocorticoid-response element (GRE) was characterized by cotransfection experiments, site-directed mutagenesis, and electrophoretic mobility shift assays. Analysis of the human UGT1A1 promoter revealed GREs at -3404/-3389 and -3251/-3236 close to the CAR/PXR response element gtNR1 (-3382/-3367). Furthermore, in an in vitro reporter gene assay, dexamethasone effectively enhanced CAR/PXR-mediated transactivation of the 290-bp distal enhancer module in HepG2 cells and CV-1 cells in the presence of exogenously expressed GR and glucocorticoid receptor-interacting protein 1 (GRIP1). In glutathione S-transferase pull-down experiments, CAR and PXR interacted with GRIP1. Together, these results demonstrate a rational mechanistic basis for UGT1A1 induction by glucocorticoids and PXR activators, showing that activated GR enhances CAR/PXR-mediated UGT1A1 regulation with the transcriptional cofactor GRIP1 and that GR may be involved synergistically in the xenobiotic-responsive regulation of UGT1A1 by CAR/PXR.

The induction of human UDP-glucuronosyltransferase 1A1 mediated through a distal enhancer module by flavonoids and xenobiotics.

We identified the UDP-glucuronosyltransferase (UGT) 1A1 5'-upstream region that confers UGT1A1 induction by various agents, including flavonoids, on a luciferase reporter gene and has the properties of a transcriptional enhancer. Chrysin- and rifampicin-response activities were traced to the same element as a 290-bp distal enhancer module (-3483/-3194), in which the reporter activities were enhanced by activators of nuclear receptors [constitutive androstane receptor (CAR) and pregnane X receptor (PXR)] and transcription factor [aryl hydrocarbon receptor (AhR)]. Utilizing transactivation experiments with the UGT1A1 290-bp reporter gene, we assessed UGT1A1 induction by various flavonoids. 5,7-Dihydroxyflavones with varying substituents in the B-ring and gallocatechin dimers increased the reporter activity in a time- and dose-dependent manner. The treatment of HepG2 cells with the flavonoids for 24 hr elevated the expression of mRNAs and proteins of UGT1A1 and CYP1A1, while the mRNA levels of CYP2B6, CYP3A4, CAR, PXR and AhR was not altered. Chrysin and rifampicin induced the activation of the wild-type reporter gene and T-3263G-mutated gene to a similar extent in HepG2 cells cotransfected with expression vectors of CAR and PXR. Mutation of the AhR core binding region most prominently suppressed the activation of the 290-bp reporter gene by chrysin and baicalein, while mutations of four putative nuclear receptor motifs (DR4 element, PXRE, CARE and DR3 element) partly decreased its activation. Taken together, the results indicate that UGT1A1 was induced in response to flavonoids and xenobiotics through the transactivation of the 290-bp reporter gene, that was a multi-component enhancer containing CAR, PXR and AhR motifs.

Identification of a defect in the UGT1A1 gene promoter and its association with hyperbilirubinemia.

The UDP-glucuronosyltransferase UGT1A1 plays a critical role in the detoxification of potentially neurotoxic bilirubin by conjugating it with glucuronic acid. We identified a polymorphism that results in a T to G substitution at nucleotide number -3263 of the phenobarbital-responsive enhancer module of the UGT1A1 gene, thereby significantly decreasing transcriptional activity as indicated by the luciferase-reporter assay. At least one T-3263G allele was found in 21 of 25 subjects with mild hyperbilirubinemia (Gilbert's syndrome); this frequency (0.58) was significantly higher than that in normobilirubinemic controls (0.17; n = 8 of 27). Homozygous mutations in the TATA element (A[TA](7)TAA) or at nucleotide 211 of exon 1 (G to A substitution) were found in 5 and 2 of the hyperbilirubinemic group, respectively, while 12 of these subjects were double heterozygotes for the T-3263G and G211A mutations. Plasma total bilirubin levels in these double heterozygotes were significantly higher than those in control subjects carrying one or other of these mutations singly, indicating that compound heterozygous mutations may result in more strongly reduced UGT1A1 activity. Our results indicate that homozygosity and compound heterozygosity for mutations in the UGT1A1 gene promoter (T-3263G and A[TA](7)TAA) and/or exon 1 of the gene (G211A) could explain the hyperbilirubinemia seen in the majority of individuals with Gilbert's syndrome.