A Visual Discrimination of Existing States of Virus Capsid Protein by a Giant Molybdate Cluster.

We report a unique phenomenon, the opposite color response of a giant polyoxometalate, (NH4)42[Mo132O372(CHCOO)30] (H2O)72 ([Mo132]), to the existing states of human papillomavirus (HPV) major capsid protein, L1-pentamer (L1-p), and virus-like particles (VLPs). The color responses originate from the different assembly forms between [Mo132] and the capsid protein. The latter were inspected and separated by using CsCl gradient centrifugation, and validated in detail by sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE), dynamic light scattering (DLS), and transmission electron microscopy (TEM) imaging. Furthermore, the intrinsic mechanisms were investigated in-depth by using XPS-based semi-quantitative analysis and well-designed peptides, revealing the critical points of L1 that determine the charge-transfer ratio between Mo(V) to Mo(VI), and consequently, the levels of [Mo132] hypochromic in different assemblies. Such a unique phenomenon is significant as it supplies a colorimetry approach to distinguish the existing states of the HPV capsid protein and would be significant in the quality assay of the HPV vaccine and existing states of other viruses in the future.

Proteomics analysis reveals diabetic kidney as a ketogenic organ in type 2 diabetes.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease. To date, the molecular mechanisms of DN remain largely unclear. The present study aimed to identify and characterize novel proteins involved in the development of DN by a proteomic approach. Proteomic analysis revealed that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase 2 (HMGCS2), the key enzyme in ketogenesis, was increased fourfold in the kidneys of type 2 diabetic db/db mice. Consistently, the activity of HMGCS2 in kidneys and 24-h urinary excretion of the ketone body ß-hydroxybutyrate (ß-HB) were significantly increased in db/db mice. Immunohistochemistry, immunofluorescence, and real-time PCR studies further demonstrated that HMGCS2 was highly expressed in renal glomeruli of db/db mice, with weak expression in the kidneys of control mice. Because filtered ketone bodies are mainly reabsorbed in the proximal tubules, we used RPTC cells, a rat proximal tubule cell line, to examine the effect of the increased level of ketone bodies. Treating cultured RPTC cells with 1 mM ß-HB significantly induced transforming growth factor-ß1 expression, with a marked increase in collagen I expression. ß-HB treatment also resulted in a marked increase in vimentin protein expression and a significant reduction in E-cadherin protein levels, suggesting an enhanced epithelial-to-mesenchymal transition in RPTCs. Collectively, these findings demonstrate that diabetic kidneys exhibit excess ketogenic activity resulting from increased HMGCS2 expression. Enhanced ketone body production in the diabetic kidney may represent a novel mechanism involved in the pathogenesis of DN.

Enhanced pressor response to acute Ang II infusion in mice lacking membrane-associated prostaglandin E2 synthase-1.

AIM: To examine the contribution of vascular membrane-associated prostaglandin E2 synthase-1 (mPGES-1) to acute blood pressure homeostasis. METHODS: Angiotensin II (AngII, 75 pmol·kg⁻¹·min⁻¹) was continuously infused via the jugular vein into wild-type and mPGES-1(-/-) mice for 30 min, and blood pressure was measured by carotid arterial catheterization. RT-PCR and immunohistochemistry were performed to detect the expression and localization of mPGES-1 in the mouse arterial vessels. Mesenteric arteries were dissected from mice of both genotypes to study vessel tension and measure vascular PGE2 levels. RESULTS: Wild-type and mPGES-1(-/-) mice showed similar blood pressure levels at baseline, and the acute intravenous infusion of AngII caused a greater increase in mean arterial pressure in the mPGES-1(-/-) group, with a similar diuretic and natriuretic response in both groups. mPGES-1 was constitutively expressed in the aortic and mesenteric arteries and vascular smooth muscle cells of wild-type mice. Strong staining was detected in the smooth muscle layer of arterial vessels. Ex vivo treatment of mesenteric arteries with AngII produced more vasodilatory PGE2 in wild-type than in mPGES-1(-/-) mice. In vitro tension assays further revealed that the mesenteric arteries of mPGES-1(-/-) mice exhibited a greater vasopressor response to AngII than those arteries of wild-type mice. CONCLUSION: Vascular mPGES-1 acts as an important tonic vasodilator, contributing to acute blood pressure regulation.

Level Of Natriuretic Peptide Determines Outcome In Atrial Fibrillation.

Background: Natriuretic peptide (NP) is high in atrial fibrillation (AF) and may decrease after cardioversion to sinus rhythm and the levels of atrial NP (ANP) and brain NP (BNP) in different types of AF and whether ANP and BNP have predictive values for relapsed AF have not been determined. Purpose: We aimed to examine the levels of ANP and BNP in AF to determine their roles in different types of AF, including a predictive value in relapsed AF. Methods and Results: ANP and BNP were measured in 100 consecutive patients with AF and without heart dysfunction at baseline and in 20 controls. All patients had higher levels than controls (p<0.01). After cardioversion treatment with antiarrhythmic therapy, 40 patients failed to cardioversion successfully and still showed AF, whereas 60 patients were successful. ANP and BNP levels decreased significantly after cardioversion (163.55±54.27pg/ml vs. 200.20±55.63 pg/ml; 124.15±43.00 pg/ml vs. 161.99±48.04 pg/ml, for ANP and BNP respectively, both p<0.0001). 18 of the 60 successfully cardioverted patients had AF recurred within 24 hours, who were then excluded from 500-day follow-up and the remaining 42 patients were enrolled. During 500-day follow-up period, AF relapsed in 16 patients. Comparing with the 42 patients, the 16 patients showed higher concentrations of ANP (187.72±32.79 pg/ml vs. 138.42±30.65 pg/ml, p<0.0001). Besides, both ANP and BNP were significantly higher in the relapsed patients than those remained SR during follow-up (153.38±29.61pg/ml vs. 129.21±27.98pg/ml for ANP, p=0.01 and 147.41±25.95pg/ml vs. 121.87±20.53pg/ml for BNP, p=0.001). The area under the receiver-operating characteristic curve was 0.799 for BNP and 0.706 for ANP in predicting a relapse of AF. Using the BNP optimized cut-off level of 138 pg/ml, relapsed AF can be predicted with relatively acceptable accuracy. CONCLUSIONS: ANP and BNP decrease significantly after cardioversion in patients with AF, and both can be useful predictors of relapsed AF.

Induction of MIF expression by oxidized LDL via activation of NF-kappaB in vascular smooth muscle cells.

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine playing important roles in atherosclerosis. MIF gene deficiency and neutralizing antibodies against MIF have been reported to exert anti-atherosclerotic effects in various animal models. However, the mechanism by which MIF is induced in atherosclerotic lesions remains unclear. In the present studies, we cloned a 540bp full-length rabbit MIF cDNA by screening a rabbit uterine library. The cDNA contains a 348bp open-reading frame which encodes a deduced 115-amino acid polypeptide with approximately 90% similarity to human and mouse homologs. Constitutive MIF mRNA expression was detected in most rabbit tissues including aortas. The expression of MIF obviously abounded in vascular smooth muscle cells (VSMCs) of the atherosclerotic plaques. In cultured VSMCs, MIF expression was significantly induced by a pro-atherogenic factor, oxidized low-density lipoprotein (oxLDL). Promoter analysis showed there were two NF-kappaB binding sites in the MIF proximal promoter region. Deletion or mutation of the two sites abolished oxLDL-enhanced MIF promoter activity. Moreover, the induction of MIF by oxLDL can be blocked by IkappaB-alpha overexpression. Taken together, our results revealed that MIF expression can be induced by oxLDL in VSMCs via a NF-kappaB dependent manner, which may contribute to the pathogenesis of atherosclerosis.

[Activation of liver X receptor regulates fatty acid synthase expression in diabetic liver].

OBJECTIVE: To investigate the effects of liver X receptor (LXR) on the expression of fatty acid synthase (FAS) in diabetic liver. METHODS: Sixteen-week-old male db/db mice with C57BL/6 background were administered via gavaging of T0901317 (TO), a LXR synthetic agonist, at the dose of 3 mg x kg(-1) x d(-1) or dimethyl sulfide (DMSO), a vehicle alone for 7 days. Then the mice were killed with their livers taken out to undergo immunohistochemistry to observe the distribution of FAS protein. Human hepatocellular liver carcinoma cell of the line HepG2 were cultured with TO (10 micromol/L) or DMSO for 24 hours. Another HepG2 cells were transfected with mouse FAS promoter-luciferase reporter recombinants with or without pcDNA3.1, LXR expression vector, or an active sterol regulatory element binding protein-1c (SREBP-1c) expression vector for 12 hours. Real-time PCR and Western blotting were used to detect the levels of mRNA and protein of FAS and SREBP-1c respectively. Luciferase reporter assay was utilized to examine the activity of mouse FAS promoter. RESULTS: FAS was abundantly expressed in the mouse livers, especially in the cytoplasm of liver cells. The FAS mRNA levels of the livers of the db/db mice was about 5.5 times as high as that of the db/m mice (P < 0.01). The FAS protein levels in the livers of db/db and db/m mice treated with TO were 1.7 and 3.5 times higher than those of the control mice (both P < 0.05). The SREBP-1 mRNA levels in the liver of the db/m and db/db mice treated with TO were 2.4 and 2.1 times higher compared with the control mice (P < 0.05, P < 0.01). Luciferase test showed that the FAS promoter activity of the HepG2 cells treated with TO was 1.5 times that of the control cells (P < 0.01). The FAS promoter activities of the HepG2 cells transfected with LXR and SREBP-1c were 1.9 and 1.6 times those of the control cells (botn P < 0.01). CONCLUSION: LXRE directly or indirect (via SREBP-lc) upregulates the expression of FAS gene in the diabetic liver. LXR may mediate the lipid accumulation in liver of diabetes.

Expression profiling of hepatic genes associated with lipid metabolism in nephrotic rats.

Hyperlipidemia is one of the major features of nephrotic syndrome (NS). Although many factors have been implicated in the pathogenesis of NS-related dyslipidemia, the underlying mechanisms remain largely uncharacterized. The present study was designed to examine the gene profile associated with lipid metabolism in the livers of nephrotic rats. NS was created in male Sprague-Dawley rats (n = 6) receiving sequential intraperitoneal injections of puromycin aminonucleoside. Analysis by Affymetrix assay, quantitative RT-PCR, and Northern and Western blotting revealed 21 genes associated with cholesterol and fatty acid metabolism. Eight genes involved in cholesterol metabolism, Apo A-I, Acly, Acat, Mpd, Fdps, Ss, Lss, and Nsdhl, were significantly upregulated under NS. Four genes involved in fatty acid biosynthesis, Acc, FAS, ELOVL 2, and ELOVL6, and three critical for triglyceride biosynthesis, Gpam, Agpat 3, and Dgat 1, were significantly upregulated, whereas two genes involved in fatty acid oxidation, Dci and MCAD, were downregulated. Expression of several genes in sterol-regulatory element-binding protein (SREBP)-1 activation was also aberrantly altered in nephrotic livers. The expression and transcriptional activity of SREBP-1 but not SREBP-2 were increased in nephrotic rats as assessed by real-time PCR, immunoblotting, and gel shift assays. The upregulation of hepatic genes involved in cholesterol biosynthesis may play an important role in the pathogenesis of hypercholesterolemia, whereas upregulation of genes participating in hepatic fatty acid and triglyceride biosynthesis and downregulation of genes involved in hepatic fatty acid oxidation may contribute to hypertriglyceridemia in nephrotic rats. Activation of SREBP-1 transcription factor may represent an underlying molecular mechanism of hyperlipidemia in NS.

Expression of mouse membrane-associated prostaglandin E2 synthase-2 (mPGES-2) along the urogenital tract.

Prostaglandin E(2) (PGE(2)) is the most common prostanoid and has a variety of bioactivities including a crucial role in urogenital function. Multiple enzymes are involved in its biosynthesis. Among 3 PGE(2) terminal synthetic enzymes, membrane-associated PGE(2) synthase-2 (mPGES-2) is the most recently identified, and its role remains uncharacterized. In previous studies, membrane-associated PGE(2) synthase-1 (mPGES-1) and cytosolic PGE(2) synthase (cPGES) were reported to be expressed along the urogenital tracts. Here we report the genomic structure and tissue distribution of mPGES-2 in the urogenital system. Analysis of several bioinformatic databases demonstrated that mouse mPGES-2 spans 7 kb and consists of 7 exons. The mPGES-2 promoter contains multiple Sp1 sites and a GC box without a TATA box motif. Real-time quantitative PCR revealed that constitutive mPGES-2 mRNA was most abundant in the heart, brain, kidney and small intestine. In the urogenital system, mPGES-2 was highly expressed in the renal cortex, followed by the renal medulla and ovary, with lower levels in the ureter, bladder and uterus. Immunohistochemistry studies indicated that mPGES-2 was ubiquitously expressed along the nephron, with much lower levels in the glomeruli. In the ureter and bladder, mPGES-2 was mainly localized to the urothelium. In the reproductive system, mPGES-2 was restricted to the epithelial cells of the testis, epididymis, vas deferens and seminal vesicle in males, and oocytes, stroma cells and corpus luteum of the ovary and epithelial cells of the oviduct and uterus in females. This expression pattern is consistent with an important role for mPGES-2-mediated PGE(2) in urogenital function.

[Liver X receptors mediate cholesterol efflux in mouse glomerular mesangial cells].

OBJECTIVE: To examine the role of liver X receptors (LXRs) in lipid metabolism in cultured mouse mesangial cells. METHODS: To determine whether LXRalpha and LXRbeta are expressed in the kidney, RT-PCR and western blot assay were utilized. Cultured mesangial cells were treated with either vehicle or LXR agonist TO901317(10 micromol/L) for 24 hours. Real-time PCR analysis was used to detect ABCA1 and ABCG1 expressions. Cells were also transfected with a human ABCA1 promoter driven luciferase reporter plasmid and then stimulated with or without TO901317 for 24 hours. In order to determine the effect of TO901317 on protein expression of ABCA1, LXRalpha adenovirus was used to overexpress LXRalpha in the cultured cells. Finally, [3H] cholesterol efflux assay was performed to evaluate the efflux of cholesterol upon TO901317 stimulation. RESULTS: Both LXRalpha and LXRbeta were expressed in the kidney, freshly isolated glomeruli and mesangial cells. After treatment with TO901317, both ABCA1 and ABCG1 expressions were induced. Moreover, ABCA1 protein level was increased after the cells were simultaneously treated with LXRalpha-adenovirus and TO901317. The cholesterol efflux was also significantly enhanced after TO901317 treatment. CONCLUSION: LXRalpha and LXRbeta were functionally expressed in mouse mesangial cells. Activation of LXRs enhanced cholesterol efflux possibly through upregulating ABCA1 and ABCG1 expressions in mesangial cells. Therefore, LXR agonist might ameliorate lipid accumulation and reduce related cell injury in mesangial cells.