Three series of novel sulfonylurea podophyllotoxin derivatives were designed, synthesized, and evaluated for in vitro cytotoxicity against four tumor cell lines (A-549, DU-145, KB and KBvin). Compounds 14c (IC50: 1.41–1.76 μM) and 14e (IC50: 1.72–2.01 μM) showed superior cytotoxic activity compared with etoposide (IC50: 2.03– >20μM), a clinically available anticancer drug. Significantly, most of the compounds exhibited comparable cytotoxicity against the drug-resistant tumor cell line KBvin, while etoposide lost activity completely. Preliminary structure-activity relationship (SAR) correlations indicated that the 4′-O-methyl functionality in podophyllotoxin analogues may be essential to maintain cytotoxic activity, while an arylsulfonylurea side chain at podophyllotoxin’s 4β position can significantly improve cytotoxic activity.
podophyllotoxin; sulfonylurea; synthesis; cytotoxic activity
Previous studies investigating the association between GDF5 rs143383 polymorphism and knee osteoarthritis (OA) have suggested stronger associations in Asians than Caucasians, but limitations on the amount of available data have meant that a definitive assessment has not been possible. Given the availability of more recent data, the aim of this meta-analysis was to determine the overall association between GDF5 rs143383 polymorphism and knee OA and whether the association varies by ethnicity.
Searches of Medline, Embase, and ISI Web of Science were conducted up to July 2013. Summary odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to estimate the strength of association between the GDF5 polymorphism and knee OA risk.
A total of 20 studies with 23,995 individuals were included. There were weak but significant associations present between the GDF5 polymorphism and knee OA at the allele level (C vs. T: OR =0.85, 95% CI = 0.80-0.90) and genotype level (CC vs. TT: OR = 0.73; CT vs. TT: OR = 0.84; CC/CT vs. TT: OR = 0.81; CC vs. CT/TT: OR = 0.81) in the overall population. In the subgroup analysis by ethnicity, we observed a strong significant association (OR = 0.60 to 0.80, all P <0.05) in Asian population and weaker associations (OR =0.78 to 0.87, all P <0.05) in Caucasian population; however marked heterogeneity was detected in all models except for CC vs. TT (I2 = 12.9%) and CC vs. CT + TT (I2 = 0.0%) in Asians.
These results strongly suggest that the C allele and CC genotype of the GDF5 gene are protective for knee OA susceptibility across different populations.
GDF5; Polymorphism; Knee; Osteoarthritis; Meta-analysis
The aim of the present study was to investigate the effects of plasmid-mediated RNA interference targeting of cyclooxygenase-2 (COX-2) on the biological behaviors of SKOV3 human ovarian cancer cells and to analyze the function of COX-2 in carcinogenesis and development of ovarian cancer. A COX-2 small hairpin (sh)RNA sequence was designed and synthesized and pGPU6-COX-2-shRNA plasmids were constructed. The recombinant vector plasmids were stably transfected into SKOV3 cells. The mRNA and protein expression of COX-2 was subsequently analyzed by quantitative polymerase chain reaction and western blot analysis, respectively. MTT and colony formation assays were used to detect the cellular proliferation ability and flow cytometry was performed to detect phase changes in the cell cycle. Finally, a Transwell assay was used to detect cell invasion. The SKOV3 cells, transfected with recombinant vector plasmids, and control cells, were injected into nude mice and the tumor emergence time, volume and weight were measured. The impact of COX-2 gene silencing on the growth of xenograft tumors in nude mice was analyzed. Following transfection of the pGPU6-COX-2-shRNA plasmid, in vitro analyses indicated that the shRNA efficiently suppressed the mRNA and protein expression of COX-2. COX-2 gene silencing significantly inhibited the proliferation and invasion ability of SKOV3 cells, leading to cell cycle arrest in G1. The tumor formation time in the interference group was significantly prolonged, and the tumor volume and weight were significantly decreased, as compared with the control group. Plasmid-mediated shRNA was shown to effectively silence COX-2 expression in SKOV3 ovarian cancer cells. It was identified that COX-2 functioned in regulating proliferation, cell cycle and invasion of ovarian cancer cells. These findings provided a theoretical basis for determining the function of COX-2 in the development of ovarian cancer and suggested that COX-2 may be an effective target for gene therapy and clinical applications.
ovarian cancer; cyclooxygenase-2; RNA interference; proliferation; invasion; gene therapy
In ruminants, lower ruminal pH causes massive disruption of ruminal epithelial structure during periods of feeding high-concentrate diets. However, the influence of excessive organic fatty acids in the lumen of hindgut on the epithelial structure is unclear. In this study, twelve mid-lactating goats were randomly assigned to either a HC diet group (65% concentrate of dry matter; n = 6) or a LC diet group (35% concentrate of dry matter; n = 6) for 10 weeks. The colonic epithelial structure was detected by HE staining and transmission electron microscopy (TEM), and the apoptotic status of epithelial cells was estimated by TUNEL method and caspase activities.
HC goats showed higher level of free lipopolysaccharide (LPS) in rumen fluid (p < 0.01) but not in colonic digesta (p > 0.05), and higher total volatile fatty acid (VFA) concentrations in rumen fluid (p < 0.05) and in colonic digesta (p < 0.01), and higher content of starch in colonic digesta (p < 0.05) compared to LC goats. HC goats demonstrated profound alterations in the colonic epithelial structure and tight junctions (TJ), apparently due to damage of the epithelium with widened TJs space and nuclear breakdown and mitochondrial swelling. HC goats showed higher level of apoptosis in the colonic epithelium with higher proportion of TUNEL-positive apoptotic cells and increases of caspase-3 and −3/7 activities, as well as the lower ratio of bcl-2/bax mRNA expression in the colonic mucosa (p < 0.05). However, β-defense mRNA was significantly down-regulated in the colonic mucosa of HC goats compared to LC (p < 0.05). HC goats showed higher level of TJ proteins including claudin-1 and claudin-4 in the colonic mucosa than LC (p < 0.05). Neither free LPS content in the colonic digesta nor NF-κ B protein expression in tissues showed significant difference between HC and LC goats (p > 0.05).
Our results reveal that long-term feeding HC diet to lactating goats causes severe damages to the colonic mucosa barrier associated with activating cells apoptosis.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-014-0235-2) contains supplementary material, which is available to authorized users.
LPS; Apoptosis; Tight junctions; Colonic mucosa; HC diet; Goat
The human forkhead box A1 (FOXA1) and A2 (FOXA2) transcription factors have been found to control estrogen and androgen signaling through co-regulating target genes with sex hormone receptors. Here we used an integrative strategy to examine the hypothesis that genetic variants at FOXA1/2 binding elements may be associated with sexual dimorphism of hepatocellular carcinoma (HCC) risk. Firstly we extracted chromatin immunoprecipitation-sequencing (ChIP-seq) data of FOXA1, FOXA2 and estrogen receptor 1(ERα) from ENCODE database to obtain dual target regions of FOXA/ERα, and further intersected these regions with genes’ promoters. Then we used MATCH program to predict FOXA binding elements, in which genetic variants were retrieved by dbSNP database (NCBI, build 134). A total of 15 candidate variants were identified in this stage. Secondly we performed a case-control study with 1,081 HCC patients and 2,008 matched controls and found a significant association of SERPINA6-rs1998056 with female HCC risk under common genetic models (e.g. GG versus CC: OR = 2.03, 95% CI = 1.26–3.27, P = 0.004). Moreover, results from our real-time quantitative polymerase chain reaction (qPCR) using 72 normal liver tissues adjacent to the tumors showed that SERPINA6 expression was significantly different among different genotypes of this variant (GG versus CC: P = 0.032; Group test: P = 0.060). In summary, our study suggested that SERPINA6-rs1998056 regulated by FOXA/ERα might be associated with female HCC risk.
To clarify the role of visual feedback in the generation of corrective movements after inaccurate primary saccades, we used a visually-triggered saccade task in which we varied how long the target was visible. The target was on for only 100 ms (OFF100ms), on until the start of the primary saccade (OFFonset) or on for 2 s (ON). We found that the tolerance for the post-saccadic error was small (− 2%) with a visual signal (ON) but greater (−6%) without visual feedback (OFF100ms). Saccades with an error of −10%, however, were likely to be followed by corrective saccades regardless of whether or not visual feedback was present. Corrective saccades were generally generated earlier when visual error information was available; their latency was related to the size of the error. The LATER (Linear Approach to Threshold with Ergodic Rate) model analysis also showed a comparable small population of short latency corrective saccades irrespective of the target visibility. Finally, we found, in the absence of visual feedback, the accuracy of corrective saccades across subjects was related to the latency of the primary saccade. Our findings provide new insights into the mechanisms underlying the programming of corrective saccades: 1) the preparation of corrective saccades begins along with the preparation of the primary saccades, 2) the accuracy of corrective saccades depends on the reaction time of the primary saccades and 3) if visual feedback is available after the initiation of the primary saccade, the prepared correction can be updated.
Primary saccade; Corrective saccade; Visual feedback; LATER model; Forward control
To determine the degree to which hyperglycemia predicts the development of frailty and/or lower extremity mobility limitations.
Secondary data analysis of longitudinal data collected in a prospective cohort study.
We examined 329 women from the Women’s Health and Aging Studies II aged 70–79 years at baseline who had all variables needed for analysis.
Hemoglobin A1c [HbA1c] at baseline was the independent variable and categorized as: <5.5%, 5.5 to 5.9%, 6.0–6.4%, 6.5–7.9%, ≥8%. The incidence of frailty and lower extremity mobility limitations (based on self-reported walking difficulty, walking speed, and short performance physical battery [SPPB] score) was determined (follow-up≈9 years). Frailty was assessed using the Cardiovascular Health Study criteria. Covariates included demographics, body mass index, interleukin-6, and clinical history of comorbidities. Statistical analyses included Kaplan-Meier survival curves and Cox regression models adjusting for key covariates.
In time-to-event analyses, HbA1c category was associated with incidence of walking difficulty (p=0.049) and low physical performance (p=0.001); association with incidence of frailty and low walking speed had a trend towards significance (both p=0.10). In demographics-adjusted regression models, HbA1c≥8% (versus<5.5%) was associated with an approximately three-times increased risk of incident frailty and three-to-five times increased risk of lower extremity mobility limitations (all p<0.05). In fully adjusted models, HbA1c≥8% (versus<5.5%) was associated with incident frailty (hazard ratio[HR]=3.33, 95% confidence interval=1.24–8.93), walking difficulty (HR=3.47,1.26–9.55), low walking speed (HR=2.82,1.19–6.71), and low physical performance (HR=3.60,1.52–8.53).
Hyperglycemia is associated with the development of frailty and lower extremity mobility limitations in older women; future studies should identify mediators of these relationships.
Hyperglycemia; Elderly; Frailty; Mobility; Disability
To determine whether hyperglycemia is related to prevalent frailty status in older women.
Secondary data analysis of baseline data of a prospective cohort study.
Five hundred forty-three women aged 70 to 79.
Research used baseline data from 543 participants in the Women’s Health and Aging Studies I and II aged 70 to 79 who had all variables needed for analyses. The dependent variable was baseline frailty status (not frail, prefrail, frail), measured using an empirically derived model defining frailty according to weight loss, slow walking speed, weakness, exhaustion, and low activity (1–2 characteristics Present = prefrail, ≥3 = frail). Covariates included body mass index (BMI), interleukin-6 (IL-6), age, race, and several chronic diseases. Analyses included descriptive methods and multinomial logistic regression to adjust for key covariates.
A hemoglobin A1c (HbA1c) level of 6.5% or greater in older women was significantly associated with higher likelihood of prefrail and frail status (normal HbA1c <6.0% was reference). The association between HbA1C levels of 6.0% to 6.5% and frailty status was not different from that of normal HbA1c, but HbA1c levels of 6.5% to 6.9% had nearly twice the likelihood of frailty (odds ratio (OR) = 1.96, 95% confidence interval (CI) = 1.47–2.59) as normal HbA1c. A HbA1c level of 9.0% or greater was also strongly associated (OR = 2.57, 95% CI = 1.99,3.32). Significant associations were also seen between baseline prefrail and frail status and low (18.5–20.0 kg/m2) and high (.30.0 kg/m2) body mass index (BMI), interleukin-6, and all chronic diseases evaluated, but controlling for these covariates only minimally attenuated the independent association between HbA1c and frailty status.
Hyperglycemia is associated with greater prevalence of prefrail and frail status; BMI, inflammation, and comorbidities do not explain the association. Longitudinal research and study of alternative pathways are needed.
hyperglycemia; frailty; older women
Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.
Males develop kidney stones far more frequently than females with a ratio of 2–3:1, suggesting that androgen receptor (AR) signaling might play a key role in the development of nephrolithiasis. Using the cre-loxP system to selectively knock out AR in glyoxylate-induced calcium oxalate (CaOx) crystal mouse models, we found that the mice lacking hepatic AR had less oxalate biosynthesis, which might lead to lower CaOx crystal formation, and that the mice lacking kidney proximal or distal epithelial AR also had lower CaOx crystal formation. We found that AR could directly up-regulate hepatic glycolate oxidase and kidney epithelial NADPH oxidase subunit p22-PHOX at the transcriptional level. This up-regulation might then increase oxalate biosynthesis and oxidative stress that resulted in induction of kidney tubular injury. Targeting AR with the AR degradation enhancer ASC-J9 led to suppression of CaOx crystal formation via modulation of oxalate biosynthesis and oxidative stress in both in vitro and in vivo studies. Taken together, these results established the roles of AR in CaOx crystal formation.
Selenium (Se), an essential trace element for human health, mainly exerts its biological function via selenoproteins. Among the 25 selenoproteins identified in human, selenoprotein P (SelP) is the only one that contains multiple selenocysteines (Sec) in the sequence, and has been suggested to function as a Se transporter. Upon feeding a selenium-deficient diet, mice lacking SelP develop severe neurological dysfunction and exhibit widespread brainstem neurodegeneration, indicating an important role of SelP in normal brain function. To further elucidate the function of SelP in the brain, SelP was screened by the yeast two-hybrid system from a human fetal brain cDNA library for interactive proteins. Our results demonstrated that SelP interacts with tubulin, alpha 1a (TUBA1A). The interaction between SelP and tubulin was verified by fluorescence resonance energy transfer (FRET) and co-immunoprecipitation (co-IP) assays. We further found that SelP interacts with the C-terminus of tubulin by its His-rich domain, as demonstrated by FRET and Isothermal Titration Calorimetry (ITC) assays. The implications of the interaction between SelP and tubulin in the brain and in Alzheimer’s disease are discussed.
selenoprotein P (SelP); tubulin; protein-protein interaction; yeast two-hybrid system; fluorescence resonance energy transfer (fret)
The transcriptional co-activator YAP plays an important role in organ size control and tumorigenesis. However, how Yap gene expression is regulated remains unknown. This study shows that the Ets family member GABP binds to the Yap promoter and activates YAP transcription. The depletion of GABP downregulates YAP, resulting in a G1/S cell cycle block and increased cell death, both of which are substantially rescued by reconstituting YAP. GABP can be inactivated by oxidative mechanisms, and acetaminophen-induced GSH depletion inhibits GABP transcriptional activity and depletes YAP. In contrast, activating YAP by deleting Mst1/Mst2 strongly protects acetaminophen-induced liver injury. Similar to its effects on YAP, the Hippo signaling inhibits GABP transcriptional activity through several mechanisms. In human liver cancers, enhanced YAP expression is correlated with increased nuclear expression of GABP. Therefore, we conclude that GABP is an activator of Yap gene expression and a potential therapeutic target for cancers driven by YAP.
GABP; Hippo pathway; YAP; Glutathione depletion; Liver injury; liver cancer
Using next-generation sequencing technology alone, we have successfully generated and assembled a draft sequence of the giant panda genome. The assembled contigs (2.25 gigabases (Gb)) cover approximately 94% of the whole genome, and the remaining gaps (0.05 Gb) seem to contain carnivore-specific repeats and tandem repeats. Comparisons with the dog and human showed that the panda genome has a lower divergence rate. The assessment of panda genes potentially underlying some of its unique traits indicated that its bamboo diet might be more dependent on its gut microbiome than its own genetic composition. We also identified more than 2.7 million heterozygous single nucleotide polymorphisms in the diploid genome. Our data and analyses provide a foundation for promoting mammalian genetic research, and demonstrate the feasibility for using next-generation sequencing technologies for accurate, cost-effective and rapid de novo assembly of large eukaryotic genomes.
We previously reported that calycosin, a natural phytoestrogen structurally similar to estrogen, successfully triggered apoptosis of estrogen receptor (ER)-positive breast cancer cell line, MCF-7. To better understand the antitumor activities of calycosin against breast cancer, besides MCF-7 cells, another ER-positive cell line T-47D was analyzed here, with ER-negative cell lines (MDA-231, MDA-435) as control. Notably, calycosin led to inhibited cell proliferation and apoptosis only in ER-positive cells, particularly in MCF-7 cells, whereas no such effect was observed in ER-negative cells. Then we investigated whether regulation of ERβ, a subtype of ER, contributed to calycosin-induced apoptosis in breast cancer cells. The results showed that incubation of calycosin resulted in enhanced expression ERβ in MCF-7 and T-47D cells, rather than MDA-231 and MDA-435 cells. Moreover, with the upregulation of ERβ, successive changes in downstream signaling pathways were found, including inactivation of insulin-like growth factor 1 receptor (IGF-1R), then stimulation of p38 MAPK and suppression of the serine/threonine kinase (Akt), and finally poly(ADP-ribose) polymerase 1 (PARP-1) cleavage. However, the other two members of the mitogen-activated protein kinase (MAPK) family, extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK), were not consequently regulated by downregulated IGF-1R, indicating ERK 1/2 and JNK pathways were not necessary to allow proliferation inhibition by calycosin. Taken together, our results indicate that calycosin tends to inhibit growth and induce apoptosis in ER-positive breast cancer cells, which is mediated by ERβ-induced inhibition of IGF-1R, along with the selective regulation of MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways.
To evaluate the sensitivity, specificity, and predictive value of 7 different referral criteria used for the plusoptiX photoscreener on the same cohort of children.
Retrospective chart review of patients presenting to a pediatric ophthalmology clinic who underwent plusoptiX photoscreening as part of a comprehensive examination. We applied multiple referral criteria from previously published studies as well as the manufacturer’s criteria in order to calculate specificity, sensitivity, and predictive value differences between the various referral criteria. We compared all criteria to the results of a pediatric ophthalmology examination based upon the 2003 American Association for Pediatric Ophthalmology and Strabismus (AAPOS) criteria, as well as the newly accepted revision of the AAPOS referral criteria.
109 children were examined with a thorough pediatric ophthalmic exam and with the plusoptiX photoscreener. Of these, 58 (53%) were confirmed to demonstrate amblyopia risk factors, according to 2003 AAPOS criteria. The plusoptiX referral criteria were adjusted to match 7 different published plusoptiX referral paradigms so that the differing referral paradigms could be analyzed for sensitivity and specificity. When comparing the differing plusoptiX referral paradigms to 2003 AAPOS criteria, the sensitivity/specificity of the 7 different paradigms were respectively: Matta/Silbert 98%/80%, Arthur (2) 67%/96%, Arnold 81%/96%, Arthur 81%/92%, PediaVision 80%/94%, plusoptiX 98%/41%, AAPOS 74%/86%. When comparing the 7 differing referral paradigms to the newly approved (2013) AAPOS criteria, the sensitivity/specificity were respectively: Matta/Silbert 98%/68%, Arthur (2) 73%/92%, Arnold 92%/90%, Arthur 86%/85%, PediaVision 90%/92%, plusoptiX 98%/35%, AAPOS 87%/87%.
There are multiple referral criteria available for the plusoptiX photoscreener. Screening programs need to evaluate their own requirements with respect to desired sensitivity and specificity and decide on the most appropriate referral criteria for their program. The “Arnold” criteria is the best at maximizing sensitivity and specificity utilizing the 2003 “AAPOS” criteria and the “Arnold” and “PediaVision” were best at maximizing sensitivity and specificity for the newly accepted AAPOS referral criteria. Screening programs will need to decide the level of sensitivity and specificity that they wish to obtain, but for most screening programs the “Arnold” criteria may be preferred.
Amblyopia; pediatric; strabismus screening; vision screening; vision screening/diagnosis
Chromatin remodeling has been newly established as an important cancer genome characterization and recent exome and whole-genome sequencing studies of hepatocellular carcinoma (HCC) showed that recurrent inactivating mutations in SWI/SNF subunits involved in the molecular basis of hepatocarcinogenesis. To test the hypothesis that genetic variants in the key subunits of SWI/SNF complexes may contribute to HCC susceptibility, we systematically assessed associations of genetic variants in SWI/SNF complexes with HCC risk using a two-staged case-control study in Chinese population. A set of 24 single nucleotide polymorphisms (SNPs) in SWI/SNF complexes were examined in stage 1 with 502 HCC patients and 487 controls and three promising SNPs (SMARCA4 rs11879293, rs2072382 and SMARCB1 rs2267032) were further genotyped in stage 2 comprising 501 cases and 545 controls for validation. SMARCA4 rs11879293 presented consistently significant associations with the risk of HCC at both stages, with an OR of 0.73 (95% CI: 0.62–0.87) using additive model in combined analysis. Moreover, the decreased risk of HCC associated with SMARCA4 rs11879293 AG/AA was more evident among HBsAg positive individuals (OR = 0.47, 95% CI: 0.27–0.80) in combined analysis. The study highlighted the potential role of the SWI/SNF complexes in conferring susceptibility to HCC, especially modified HCC risk by HBV infection.
To evaluate the relationship between amblyopia and accommodative ability.
The open-field Grand Seiko binocular autorefractor has become the gold standard for automated measurement of static accommodation. We measured the accommodation amplitudes in 52 children ages 3 to 14 years employing the Grand Seiko auto refractor. Children wore their glasses for this test, which was prescribed based on a cycloplegic refraction performed by one pediatric ophthalmologist. No readings could be obtained for 9 eyes (5 patients).
Normal accommodation with correction utilizing full accommodative effort at 1/3 meter is 3D assuming no accommodative lag, and would generate a reading of −3.00D from the Grand Seiko auto refractor. Lack of any accommodative should give a reading of 0.00D. Accommodative gradually declined as the acuity worsened.
Our results suggest that amblyopic eyes do not accommodate as well as non-amblyopic eyes. Because accommodation amplitude is not subjective it may be a more sensitive indicator of regression of amblyopia than visual acuity. The Grand Seiko autorefractor could prove to be a useful tool to monitor the progress of patients with amblyopia.
Accommodation; amblyopia; amblyopia treatment; pharmacology
We compare the plusoptiX S04 and A09 photoscreeners on a single cohort of children.
One hundred and thirteen children were evaluated on both the plusoptiX S04 and A09 photoscreener. A lay screener performed all of the testing prior to a pediatric ophthalmology examination. The order was alternated so that the S04 was performed first on one patient then second on the next to minimize fatigue bias.
Utilizing our modified criteria previously published, the plusoptiX S04 was found to have a sensitivity of 85%, specificity of 94%, false positive rate of 15%, and false negative rate of 6% in detecting American Association for Pediatric Ophthalmology and Strabismus–defined amblyopia factors. The plusoptiX A09 was found to have a sensitivity of 92%, specificity of 88%, false positive rate of 11%, and false negative rate of 8%.
The plusoptiX S04 and A09 photoscreeners perform similarly when used by a lay screener to evaluate the same population of children.
Pediatric; screening and infant vision development; strabismus; strabismus screening; vision screening; vision screening/diagnosis
Previous studies have shown that acute increases in oxidative stress induced by the addition of hydrogen peroxide (H2O2) can increase endothelial nitric oxide synthase (eNOS) catalytic activity via an increase in the phosphorylation of eNOS at serine 1177. However, it is unclear how increased H2O2 affects nitric oxide (NO) signaling when endothelial cells are exposed to biomechanical forces. Thus, the purpose of this study was to evaluate the acute effects of H2O2 on NO signaling in the presence or absence of laminar shear stress. We found that acute sustained increases in cellular H2O2 levels in bovine aortic endothelial cells did not alter basal NO generation but the NO produced in response to shear stress was significantly increased. This amplification in NO signaling was found to correlate with an H2O2-induced increase in eNOS localized to the plasma membrane and an increase in total caveolin-1 protein levels. We further demonstrated that overexpressing caveolin-1 increased eNOS localized to the plasma membrane again without altering total eNOS protein levels. We also found that caveolin-1 overexpression increased NO generation in response to shear stress but only in the presence of H2O2. Conversely, depleting caveolin-1 with an siRNA decreased eNOS localized to the plasma membrane and abolished the enhanced NO generation. Finally, we found that expressing a caveolin-1 binding-site deletion mutant of eNOS in COS-7 cells decreased its plasma membrane localization and resulted in attenuated NO production in response to calcium activation. In conclusion, we have identified a new role for caveolin-1 in enhancing eNOS trafficking to the plasma membrane that seems to be involved in priming eNOS for flow-mediated activation under conditions of oxidative stress. To our knowledge, this is the first report that H2O2 modulates eNOS activity by altering its subcellular location and that caveolin-1 can play a stimulatory role in NO signaling.
Oxidative stress; Protein trafficking; Gene expression; Free radicals
Selenium, an essential trace element for human health, mainly exerts its biological function through selenoproteins. Selenoprotein M (SelM) is one of the highly expressed selenoproteins in the brain, but its biological effect and molecular mechanism remain unclear. Thus, the interactive protein of SelM was investigated in this paper to guide further study. In order to avoid protein translational stop, the selenocysteine-encoding UGA inside the open reading frame of SelM was site-directly changed to the cysteine-encoding UGC to generate the SelM′ mutant. Meanwhile, its N terminal transmembrane signal peptide was also cut off. This truncated SelM′ was used to screen a human fetal brain cDNA library by the yeast two-hybrid system. A new interactive protein of SelM′ was found to be galectin-1 (Gal-1). This protein-protein interaction was further verified by the results of fluorescence resonance energy transfer techniques, glutathione S-transferase pull-down and co-immunoprecipitation assays. As Gal-1 plays important roles in preventing neurodegeneration and promoting neuroprotection in the brain, the interaction between SelM′ and Gal-1 displays a new direction for studying the biological function of SelM in the human brain.
selenoprotein M (SelM); galectin-1 (Gal-1); protein-protein interaction; yeast two-hybridization; fluorescence resonance energy transfer (FRET); co-immunoprecipitation; Glutathione S-transferase pull-down (GST pull-down)
♦ Background: The aim of the present study was to investigate the effect of oral pioglitazone (PIO) on lipid metabolism, insulin resistance, inflammation, and adipokine metabolism in continuous ambulatory peritoneal dialysis (CAPD) patients.
♦ Methods: In this randomized crossover trial, 36 CAPD patients with serum triglyceride levels above 1.8 mmol/L were randomly assigned to receive either oral PIO 15 mg once daily or no PIO for 12 weeks. Then, after a 4-week washout, the patients were switched to the alternative regimen. The primary endpoint was change in serum triglycerides during the PIO regimen compared with no PIO. Secondary endpoints included changes in other lipid levels, homeostatic model assessment of insulin resistance (HOMA-IR), adipocytokines, and C-reactive protein (CRP).
♦ Results: All 36 CAPD patients (age: 64 ± 11 years; 33% men; 27.8% with diabetes mellitus) completed the study. Comparing patients after PIO and no PIO therapy, we found no significant differences in mean serum triglycerides (3.83 ± 1.49 mmol/L vs 3.51 ± 1.98 mmol/L, p = 0.2). However, mean high-density lipoprotein (0.94 ± 0.22 mmol/L vs 1.00 ± 0.21 mmol/L, p = 0.004) and median total adiponectin [10.34 μg/mL (range: 2.59 - 34.48 μg/mL) vs 30.44 μg/mL (3.47 - 93.41 μg/mL), p < 0.001] increased significantly. Median HOMA-IR [7.51 (1.39 - 45.23) vs 5.38 (0.97 - 14.95), p = 0.006], mean fasting blood glucose (7.31 ± 2.57 mmol/L vs 6.60 ± 2.45 mmol/L, p = 0.01), median CRP [8.78 mg/L (0.18 - 53 mg/L) vs 3.50 mg/L (0.17 - 26.30 mg/L), p = 0.005], and mean resistin (32.70 ± 17.17 ng/mL vs 28.79 ± 11.83 ng/mL, p = 0.02) all declined. The PIO was well tolerated, with only one adverse event: lower-extremity edema in a patient with low residual renal function.
♦ Conclusions: Blood triglycerides were not altered after 12 weeks of PIO 15 mg once daily in CAPD patients, but parameters of dysmetabolism were markedly improved, including insulin resistance, inflammation, and adipokine balance, suggesting that PIO could be of value for this high-risk patient group. Larger, more definitive studies are needed to confirm these findings.
Pioglitazone; lipid dysmetabolism; insulin resistance; inflammation; adipocytokines
The Hippo signaling pathway has emerged as a critical regulator for organ size control. The serine/threonine protein kinases Mst1 and Mst2, mammalian homologs of the Hippo kinase from Drosophila, play the central roles in the Hippo pathway controlling the cell proliferation, differentiation, and apoptosis during development. Mst1/2 can be activated by cellular stressors and the activation of Mst1/2 might enforce a feedback stimulation system to regulate oxidant levels through several mechanisms, in which regulation of cellular redox state might represent a tumor suppressor function of Mst1/2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in multiple organs, although considerable diversification in the pathway composition and regulation is observed in some of them. Generally, loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by the reduction or elimination of YAP. The Hippo pathway integrates with other signaling pathways e.g. Wnt and Notch pathways and coordinates with them to impact on the tumor pathogenesis and development. Furthermore, Mst1/2 kinases also act as an important regulator in immune cell activation, adhesion, migration, growth, and apoptosis. This review will focus on the recent updates on those aspects for the roles of Mst1/2 kinases.
The Hippo pathway; Mst1; Mst2; Reactive oxygen species; YAP; Cancer; Immune diseases
The development of pulmonary hypertension is a common accompaniment of congenital heart disease (CHD) with increased pulmonary blood flow. Our recent evidence suggests that asymmetric dimethylarginine (ADMA)-induced mitochondrial dysfunction causes endothelial nitric oxide synthase (eNOS) uncoupling secondary to a proteasome-dependent degradation of GTP cyclohydrolase I (GCH1) that results in a decrease in the NOS co-factor, tetrahydrobiopterin (BH4). Decreases in NO signaling are thought to be an early hallmark of endothelial dysfunction. As L-carnitine plays an important role in maintaining mitochondrial function in this study we examined the protective mechanisms and the therapeutic potential of L-carnitine on NO signaling in pulmonary arterial endothelial cells (PAEC) and in a lamb model of CHD and increased pulmonary blood flow (Shunt). Acetyl L-carnitine (ALC) attenuated the ADMA-mediated proteasomal degradation of GCH1. This preservation was associated with a decrease in the association of GCH1 with the Hsp70 and the C-terminus of Hsp70-interacting protein (CHIP) and a decrease in its ubiquitination. This in turn prevented the decrease in BH4 levels induced by ADMA and preserved NO signaling. Treatment of Shunt lambs with L-carnitine also reduced GCH1/CHIP interactions, attenuated the ubiquitination and degradation of GCH1, and increased BH4 levels compared to vehicle treated Shunt lambs. The increases in BH4 were associated with decreased NOS uncoupling and enhanced NO generation. Thus, we conclude that L-carnitine may have a therapeutic potential in the treatment of pulmonary hypertension in children with CHD with increased pulmonary blood flow.
mitochondrial dysfunction; BH4; Hsp90; Hsp70; CHIP; ubiquitination
C-Jun has been proved as playing an important role in the pathogenesis of tumors, as a main component of Activator protein 1 and c-Jun gene polymorphisms are associated with colorectal cancer. However, the relationship between the c-Jun gene polymorphism and the susceptibility to systemic lupus erythematosus (SLE) has not been known. Our purpose is to evaluate whether the c-Jun gene polymorphism (SNP rs3748814) is associated with susceptibility to SLE in a Chinese population. In this study, we enrolled 502 SLE patients and 652 healthy controls. The c-Jun polymorphism (rs3748814) was specified from genomic DNA using the TaqMan genotyping assay on a 7300 real-time reverse transcription polymerase chain reaction system. We found that the frequency of the A/G genotype in SLE patients was lower than in healthy controls, whereas the frequency of the G/G genotype was significantly higher in SLE patients than in healthy controls (A/G vs. G/G p=8.670e-08, odds ratio [OR]=0.290, 95% confidence interval [CI]=0.184–0.456). In addition, the frequency of allele A in the patients group was significantly lower than in the control group (A vs. G p=5.221e-09, OR=0.308, 95% CI=0.212–0.466). The distribution of genotype and allele frequency in SLE patients with lupus nephritis (LN) compared with SLE patients without LN was not statistically significant (A/G vs. G/G p=0.744, OR=1.157, 95% CI=0.481–2.785; A vs. G p=0.748, OR=1.152, 95% CI=0.486–2.734; A/A+A/G vs. G/G p=0.744, OR=1.157, 95% CI=0.481–2.785). Furthermore, we did not find any significant association between other clinical features and genotypes. Our findings suggest that the c-Jun polymorphism (rs3748814) may be significantly associated with the susceptibility to SLE in a Chinese population.
Selenoprotein R (SelR) plays an important role in maintaining intracellular redox balance by reducing the R-form of methionine sulfoxide to methionine. As SelR is highly expressed in brain and closely related to Alzheimer′s disease (AD), its biological functions in human brain become a research focus. In this paper, the selenocysteine-coding TGA of SelR gene was mutated to cysteine-coding TGC and used to screen the human fetal brain cDNA library with a yeast two-hybrid system. Our results demonstrated that SelR interacts with clusterin (Clu), a chaperone protein. This protein interaction was further verified by fluorescence resonance energy transfer (FRET), coimmunoprecipitation (co-IP), and pull-down assays. The interacting domain of Clu was determined by co-IP to be a dynamic, molten globule structure spanning amino acids 315 to 381 with an amphipathic-helix. The interacting domain of SelR was investigated by gene manipulation, ligand replacement, protein over-expression, and enzyme activity measurement to be a tetrahedral complex consisting of a zinc ion binding with four Cys residues. Study on the mutual effect of SelR and Clu showed synergic property between the two proteins. Cell transfection with SelR gene increased the expression of Clu, while cell transfection with Clu promoted the enzyme activity of SelR. Co-overexpression of SelR and Clu in N2aSW cells, an AD model cell line, significantly decreased the level of intracellular reactive oxygen species. Furthermore, FRET and co-IP assays demonstrated that Clu interacted with β-amyloid peptide, a pathological protein of AD, which suggested a potential effect of SelR and Aβ with the aid of Clu. The interaction between SelR and Clu provides a novel avenue for further study on the mechanism of SelR in AD prevention.