Mechanism of hydroxyl radical scavenging by rebamipide: identification of mono-hydroxylated rebamipide as a major reaction product

Rebamipide, an antiulcer agent, is known as a potent hydroxyl radical (_•OH) scavenger. In the present study, we further characterized the scavenging effect of rebamipide against _•OH generated by ultraviolet (UV) irradiation of hydrogen peroxide (H₂O₂), and identified the reaction products to elucidate the mechanism of the reaction. Scavenging effect of rebamipide was accessed by ESR using DMPO as a _•OH-trapping agent after UVB exposure (305 nm) to H₂O₂ for 1 min in the presence of rebamipide. The signal intensity of _•OH adduct of DMPO (DMPO-OH) was markedly reduced by rebamipide in a concentration-dependent fashion as well as by dimethyl sulfoxide and glutathione as reference radical scavengers. Their second order rate constant values were 5.62 × 10₁₀, 8.16 × 10₉ and 1.65 × 10₁₀ M_-1 s_-1, respectively. As the rebamipide absorption spectrum disappeared during the reaction, a new spectrum grew due to generation of rather specific reaction product. The reaction product was characterized by LC-MS/MS and NMR measurements. Finally, a hydroxylated rebamipide at the 3-position of the 2(1H)-quinolinone nucleus was newly identified as the major product exclusively formed in the reaction between rebamipide and the _•OH generated by UVB/H₂O₂. Specific formation of this product explained the molecular characteristics of rebamipide as a potential _•OH scavenger.     

Lipid-lowering efficacy of 3,4-di(OH)-phenylpropionic L-leucine in high-cholesterol fed rats

A preliminary study revealed that 3,4-di(OH)-hydrocinnamate (HC), a polyphenolic compound, lowered the plasma lipids in high-cholesterol fed rats. Accordingly, this study was designed to test the lipid-lowering efficacy of a synthetic derivative, 3,4-di(OH)-phenylpropionic (L-leucine) amide (PPLA), in rats fed a high-cholesterol (1%, wt/wt) diet. As such, HC or PPLA was given as supplement to a high-cholesterol diet for 6 weeks at a dose of 0.137 mmol/100 g diet. The supplementation of HC and PPLA significantly lowered the plasma and hepatic cholesterol and triglyceride levels compared to the control group. The activities of hepatic HMG-CoA reductase (164 +/- 9.12 and 124.74 +/- 17.09 pmol/min/mg protein vs. 245.41 +/- 13.01 pmol/min/mg protein, p < 0.05) and ACAT (411.49 +/- 11.48 and 334.35 +/- 17.68 pmol/min/mg protein vs. 490.41 +/- 16.69 pmol/min/mg protein, p < 0.05) were significantly lower in the HC- and PPLA-supplemented groups than in the control group. However, PPLA was more effective in inhibiting the enzyme activities than HC. The excretion of neutral sterol was significantly higher in HC- and PPLA-supplemented groups than in the control group. Therefore, these results indicate that PPLA, a leucine-attached version of HC, exhibited a similar significant hypocholesterolemic effect to HC in rats fed a high-cholesterol diet.     

Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants

The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P2 and PI(3,4,5)P3. A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein.     

NADPH-dependent metabolism of 17beta-estradiol and estrone to polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms

The endogenous estrogens, 17beta-estradiol (E(2)) and estrone (E(1)), undergo extensive metabolism in animals and humans, and a large number of their hydroxylated and keto metabolites have been identified in biological samples. The formation of most of the oxidative estrogen metabolites is catalyzed by cytochrome P450 (CYP) enzymes. Precise knowledge of the CYP-mediated formation of these metabolites, particularly those with unique biological activities (e.g., 4-hydroxy-E(2), 16alpha-hydroxy-E(1), 15alpha-hydroxy-E(2), 16-epiestriol, and 2-methoxyestradiol) in human liver and extrahepatic target tissues and cells, would add significantly to our understanding of the diverse biological functions that are associated with endogenous estrogens. In this article, we review recent results on the NADPH-dependent metabolism of endogenous estrogens to polar (hydroxylated and keto) metabolites as well as to nonpolar metabolites by human tissues and recombinant human CYP isoforms. The available data show that a large number of polar and nonpolar metabolites of E(2) and E(1) are formed by human tissues, and a variety of human CYP isoforms are involved in the NADPH-dependent formation of polar as well as nonpolar estrogen metabolites. These enzymes have varying degrees of catalytic activity and distinct regioselectivity.     

The effect of VDR gene polymorphisms and vitamin D level on blood pressure,risk of preeclampsia,gestational age,and body mass index

We investigated the influence of vitamin D receptor (VDR) polymorphisms and vitamin D level on the blood pressure and the risk of preeclampsia. In a case-control study, 200 pregnant women, including 100 individuals with preeclampsia along with 100 healthy pregnant women, were studied for VDR FokI, TaqI, and BmsI polymorphisms and serum 25 (OH)-D level using polymerase chain reaction-restriction fragment length polymorphism method and commercial kit, respectively. The mean level of 25 (OH)-D in preeclamptic patients was significantly lower (16.6 ± 4.2 ng/mL, P < 0.001) compared with controls (19.6 ± 3.8 ng/mL). Among all women, a significantly higher systolic blood pressure and before-pregnancy body mass index and also lower gestational age were observed in the presence of 25 (OH)-D level < 20 ng/mL compared with the 20 to 30 ng/mL. A significantly higher frequency of VDR FokI C allele in preeclamptic patients (83%) than controls (74%) was associated with a 1.72-fold increased risk of preeclampsia. In all the studied individuals, the systolic and diastolic blood pressures were significantly higher in the presence of the FokI CC genotype compared with the TC and TT+TC genotypes. Neither VDR Taq1 nor VDR BmsI was associated with the risk of preeclampsia. The haplotype FokI C, TaqI C and BmsI A (CCA) compared with haplotype CTG increased the risk of preeclampsia by 1.4-fold (P = 0.33). Our study suggests an association between VDR FokI polymorphism and an insufficient serum level of 25 (OH)-D with the risk of preeclampsia and also the influence of insufficient 25 (OH)-D level and VDR FokI polymorphism on maternal factors, including blood pressure.     

Increased WD-repeat containing protein 1 in interstitial fluid from ovarian carcinomas shown by comparative proteomic analysis of malignant and healthy gynecological tissue

We aimed to identify differentially expressed proteins in interstitial fluid from ovarian cancer employing multiple fractioning and high resolution mass spectrometry-based proteomic analysis, and asked whether specific proteins that may serve as biomarker candidates or therapeutic targets could be identified. High throughput proteomics was conducted on immunodepleted and fractioned interstitial fluid from pooled samples of ovarian carcinomas, using endometrial carcinomas and healthy ovarian tissue as controls. Differential analysis revealed the up-regulation of extracellular proteasomes in tumor interstitial fluid compared to the healthy control. Moreover, a number of differentially expressed proteins in interstitial fluid from ovarian carcinomas compared with control tissues were identified. Detection of proteasome 20S related proteins in TIF compared to IF from healthy tissue indicates that the 20S proteasome can have a role in the tumor microenvironment. Six selected proteins, CEACAM5, FREM2, MUC5AC, TFF3, PYCARD and WDR1, were independently validated in individual tumor lysates from ovarian carcinomas by multiple reaction monitoring initiated detection and sequence analysis, Western blot and/or selected reaction monitoring. Quantification of specific proteins revealed substantial heterogeneity between individual samples. Nevertheless, WD repeat-containing protein 1 was confirmed as being significantly overexpressed in interstitial fluid from ovarian carcinomas compared to healthy ovarian tissue by Orbitrap analysis of individual native interstitial fluid from ovarian and endometrial carcinomas and healthy ovarian tissue. We suggest that this protein should be explored as a therapeutic target in ovarian carcinomas. This article is part of a Special Issue entitled: An Updated Secretome.     

The role of N-acetylcysteine in protecting synovial fluid biomolecules against radiolytically-mediated oxidative damage: A high field proton NMR study

High field proton (¹H) NMR spectroscopy has been employed to evaluate the abilities of the antioxidant thiol drug N-acetylcysteine and exogenous cysteine to protect metabolites present in intact inflammatory synovial fluid samples against oxidative damage arising from gamma-radiolysis (5.00 kGy) in the presence of atmospheric O₂. Although oxidation of urate to allantoin by radiolytically-generated *OH radical was readily circumventable by pre-treatment of synovial fluids with N-acetylcystine (1.00 or 3.00 × 10^-3 mol · dm^-3) or cysteine (1.00, 2.00 or 5.00 × 10^-3 mol · dm^-3), both thiols offered only a limited protective capacity with respect to hyaluronate depolymerisation and the production of formate from carbohydrates in general. Radiolytic products generated from the added thiols (predominantly their corresponding disulphides) were simultaneously detectable in ¹H Hahn spin-echo spectra of gamma-irradiated synovial fluids, permitting a quantitative evaluation of the radioprotective capacity of these agents. It is concluded that the multicomponent analytical ability of high field ¹H NMR spectroscopy provides much useful molecular information regarding mechanisms associated with the radioprotectant actions of thiols in intact biofluids.     

肥胖儿童血清25- 羟维生素D 水平的研究

目的:探讨肥胖儿童血清25-(OH)D的水平,为临床评估维生素D营养状况与儿童肥胖症关系的研究提供参考。方法:将2009年5月至10月收治的儿童依据体重指数分成3组,抽取空腹静脉血检测血清中25-(OH)D水平,并进行组间比较。结果:通过分析25-(OH)D水平,肥胖组婴儿指标显著低于正常儿和瘦婴儿,统计学分析差异有显著性意义。结论:肥胖婴儿较正常儿和瘦婴儿25-(OH)D水平低,更易患佝偻病。提示防治佝偻病,对肥胖婴儿尤应重视,更要补充维生素D。     

Effect of 1,25(OH)(2)D(3) on rat peritoneal mesothelial cells treated with high glucose plus lipopolysaccharide

1,25(OH)₂D₃, the active metabolite of vitamin D₃, its activity is not limited to mineral and skeletal homeostasis. In recent years, there has been increasing evidence pointing to the role of its activity in the regulation of cell proliferation, cell differentiation and immunomodulation. Here we report lipopolysaccharide (LPS), a glycolipid that is produced and secreted by gram-negative bacteria during peritonitis, plus high glucose (HG) can significantly inhibit mesothelial cell viability while induce more apoptosis in rat peritoneal mesothelial cells (RPMC). Pretreatment with 1,25(OH)₂D₃ can reverse the above effect in a concentration dependent manner. HG plus LPS can down-regulate the levels of both mRNA and protein of VDR, and up-regulate the expression of TGF-β1 and TNF-α in RPMC, which can also be effectively reversed by pretreatment with 1,25(OH)₂D₃. The above results suggest that HG plus LPS may induce changes in RPMC’s viability and apoptosis, leading to peritoneal injury. 1,25(OH)₂D₃ can reverse the inhibition of cell viability, the increase of apoptotic rate and induction of fibrosis related cytokine TGF-β1 and TNF-α by HG plus LPS in RPMC, thus protect peritoneal membrane.     

Association between ultraviolet radiation,skin sun sensitivity and risk of pancreatic cancer

Background: Ecological studies showing an inverse association between pancreatic cancer incidence and mortality and levels of ultraviolet radiation (UVR), suggest that higher levels of sun exposure may reduce risks of pancreatic cancer but there has been only one individual-level study that examined this issue. We aimed to examine the association between pancreatic cancer and markers of exposure to solar UVR, namely skin type, treatment of skin lesions, ambient UVR and time outdoors on work days. Methods: We used data from an Australian case-control study. Location at birth, residential location during adulthood, outdoors work, history of skin lesion treatment and sensitivity of the skin to the sun were obtained by questionnaire. We limited the analyses to Caucasians who answered the questionnaire about UVR (controls = 589/711 recruited; cases = 496/705 recruited). We used NASA's Total Ozone Mapping Spectrometer to estimate ambient UVR. Results: Being born in or living in areas of higher ambient UVR (compared to lower ambient UVR) was associated with about 30–40% lower risk of pancreatic cancer. People with fair skin colour had 47% lower risk of pancreatic cancer than those with dark skin colour (95% CI 0.37–0.75). There was some suggestion of increased risk with increased average number of hours spent outside at work. Conclusions: This study suggests that people with light skin colour or those born or living in areas of high ambient UVR have lower risk of pancreatic cancer. Our analysis supports an association between UVR and pancreatic cancer, possibly mediated through production of vitamin D.