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1.  FOXP3 is an X-linked breast cancer suppressor gene and an important repressor of the HER-2/ErbB2 oncogene 
Cell  2007;129(7):1275-1286.
The X-linked Foxp3 is a member of the forkhead/winged helix transcription factor family. Germ-line mutations cause lethal autoimmune diseases in males. Serendipitously, we observed that Foxp3sf/+ heterozygous mice developed cancer at a high rate. The majority of the cancers were mammary carcinomas in which the wild-type Foxp3 allele was inactivated and ErbB2 was over-expressed. Foxp3 bound and repressed the ErbB2 promoter. Deletion, functionally significant somatic mutations and down-regulation of the FOXP3 gene were commonly found in human breast cancer samples and correlated significantly with HER-2 over-expression, regardless of the status of HER-2 amplification. In toto, the data demonstrate that FOXP3 is an X-linked breast cancer suppressor gene and an important regulator of the HER-2/ErbB2 oncogene.
PMCID: PMC1974845  PMID: 17570480
2.  A Dinucleotide Deletion in CD24 Confers Protection against Autoimmune Diseases 
PLoS Genetics  2007;3(4):e49.
It is generally believed that susceptibility to both organ-specific and systemic autoimmune diseases is under polygenic control. Although multiple genes have been implicated in each type of autoimmune disease, few are known to have a significant impact on both. Here, we investigated the significance of polymorphisms in the human gene CD24 and the susceptibility to multiple sclerosis (MS) and systemic lupus erythematosus (SLE). We used cases/control studies to determine the association between CD24 polymorphism and the risk of MS and SLE. In addition, we also considered transmission disequilibrium tests using family data from two cohorts consisting of a total of 150 pedigrees of MS families and 187 pedigrees of SLE families. Our analyses revealed that a dinucleotide deletion at position 1527∼1528 (P1527del) from the CD24 mRNA translation start site is associated with a significantly reduced risk (odds ratio = 0.54 with 95% confidence interval = 0.34–0.82) and delayed progression (p = 0.0188) of MS. Among the SLE cohort, we found a similar reduction of risk with the same polymorphism (odds ratio = 0.38, confidence interval = 0.22–0.62). More importantly, using 150 pedigrees of MS families from two independent cohorts and the TRANSMIT software, we found that the P1527del allele was preferentially transmitted to unaffected individuals (p = 0.002). Likewise, an analysis of 187 SLE families revealed the dinucleotide-deleted allele was preferentially transmitted to unaffected individuals (p = 0.002). The mRNA levels for the dinucleotide-deletion allele were 2.5-fold less than that of the wild-type allele. The dinucleotide deletion significantly reduced the stability of CD24 mRNA. Our results demonstrate that a destabilizing dinucleotide deletion in the 3′ UTR of CD24 mRNA conveys significant protection against both MS and SLE.
Author Summary
When an individual's immune system attacks self tissues or organs, he/she develops autoimmune diseases. Although it is well established that multiple genes control susceptibility to autoimmune diseases, most of the genes remain unidentified. In addition, although different autoimmune diseases have a common immunological basis, a very small number of genes have been identified that affect multiple autoimmune diseases. Here we show that a variation in CD24 is a likely genetic factor for the risk and progression of two types of autoimmune diseases, including multiple sclerosis (MS), an organ-specific autoimmune disease affecting the central nervous system, and systemic lupus erythematosus, a systemic autoimmune disease. Our data indicated that if an individual's CD24 gene has a specific two-nucleotide deletion in the noncoding region of CD24 mRNA, his/her risk of developing MS or SLE is reduced by 2- to 3-fold. As a group, MS patients with the two-nucleotide deletion will likely have a slower disease progression. Biochemical analysis indicated that the deletion leads to rapid decay of CD24 mRNA, which should result in reduced synthesis of the CD24 protein. Our data may be useful for the treatment and diagnosis of autoimmune diseases.
PMCID: PMC1847692  PMID: 17411341
3.  Determination of the Substrate Binding Mode to the Active Site Iron of (S)-2-Hydroxypropylphosphonic Acid Epoxidase Using 17O-Enriched Substrates and Substrate Analogues† 
Biochemistry  2007;46(44):12628-12638.
(S)-2-hydroxypropylphosphonic acid epoxidase (HppE) is an O2-dependent, nonheme Fe(II)-containing oxidase that converts (S)-2-hydroxypropylphosphonic acid ((S)-HPP) to the regio-and enantiomerically specific epoxide, fosfomycin. Use of (R)-2-hydroxypropylphosphonic acid ((R)-HPP) yields the 2-keto-adduct rather than the epoxide. Here we report the chemical synthesis of a range of HPP analogs designed to probe the basis for this specificity. In past studies, NO has been used as an O2 surrogate to provide an EPR probe of the Fe(II) environment. These studies suggest that O2 binds to the iron, and substrates bind in a single orientation that strongly perturbs the iron environment. Recently, the X-ray crystal structure showed direct binding of the substrate to the iron, but both monodentate (via the phosphonate) and chelated (via the hydroxyl and phosphonate) orientations were observed. In the current study, hyperfine broadening of the homogeneous S = 3/2 EPR spectrum of the HppE-NO-HPP complex was observed when either the hydroxyl or the phosphonate group of HPP was enriched with 17O (I = 5/2). These results indicate that both functional groups of HPP bind to Fe(II) ion at the same time as NO, suggesting that the chelated substrate binding mode dominates in solution. (R)- and (S)-analog compounds that maintained the core structure of HPP but added bulky terminal groups were turned over to give products analogous to those from (R)- and (S)-HPP, respectively. In contrast, substrate analogs lacking either the phosphonate or hydroxyl group were not turned over. Elongation of the carbon chain between the hydroxyl and phosphonate allowed binding to the iron in a variety of orientations to give keto and diol products at positions determined by the hydroxyl substituent, but no stable epoxide was formed. These studies show the importance of the Fe(II)-substrate chelate structure to active antibiotic formation. This fixed orientation may align the substrate next to the iron-bound activated oxygen species thought to mediate hydrogen atom abstraction from the nearest substrate carbon.
PMCID: PMC2780580  PMID: 17927218
4.  Potent diuretic effects of prednisone in heart failure patients with refractory diuretic resistance 
The Canadian Journal of Cardiology  2007;23(11):865-868.
Refractory congestive heart failure (CHF) with diuretic resistance is life-threatening and predicts a short life expectancy. Glucocorticoids have been proven to have potent diuretic effects in animal studies; however, their efficacy in CHF patients with diuretic resistance is not known.
Thirteen CHF patients with significant volume overload and diuretic resistance who failed to respond to a conventional sequential nephron blockade therapeutic strategy; that is, the coad-ministration of a thiazide (hydrochlorothiazide) and spironolactone, in combination with loop diuretics, were studied. Prednisone (1 mg/kg daily) was then added to standard care, with other medications unchanged, to determine diuretic efficacy in these CHF patients. Variables included body weight, urine volume, serum electrolytes and renal function.
Adding prednisone resulted in striking diuresis with a mean (± SD) body weight reduction of 9.39±3.09 kg. Prednisone significantly decreased serum creatinine by 52.21±48.68 μmol/L and increased glomerular filtration rate by 33.63±15.87 mL/min/1.73 m2 compared with baseline. All patients were discharged from hospital with improved clinical status and renal function, and 11 patients remained alive in the long term. The main side effect of prednisone appeared to be hyperglycemia in diabetic patients.
The present study demonstrated that prednisone can rapidly eliminate volume overload and improve clinical status and renal function in CHF patients with diuretic resistance. Further prospective randomized clinical studies are warranted to confirm its clinical efficacy.
PMCID: PMC2651362  PMID: 17876376
Diuretic resistance; Furosemide; Heart failure; Prednisone; Renal function; Spironolactone
5.  Comparison of Immunogenicity between Codon Optimized HIV-1 Thailand Subtype B gp140 and gp145 Vaccines 
Vaccine  2007;25(26):4949-4959.
HIV-1 pandemic posed an unprecedent challenge to the global health and it is believed that an effective vaccine will be the final solution against HIV-1. HIV-1 envelope is the primary immunogen in developing neutralization antibody based HIV vaccine. To define the suitable Env derived immunogen, we systemically compared the immunogenicity of gp140 and gp145 in a DNA vaccination alone and a prime-boost modalities. 2 DNA vaccines and 2 recombinant Tiantan vaccinia vaccines (rTTV) were constructed for vaccination of female Balb/c mice. Elispot assay was used to read out the T cell immunity and ELISA assay was used to quantify antibody immunity. PLL (poly-L-Leucine)-ELISA assay was used in linear antibody epitope mapping. Mice primed with gp145 tended to elicit more Env-specific T cells responses than those primed with gp140, significant difference was observed in DNA immunization alone. The ultimate T cell responses in prime-boost regimen tends to be determined mainly by the priming efficacy. Linear antibody epitope mapping displayed that sera raised by gp145 priming were vigorously reactive to more peptides than that by gp140. Our data demonstrated HIV-1 Thailand B-derived gp145 may raise higher T-cell responses and broader linear peptide-specific antibody responses than gp140 does. However, it remains to be determined that how these observations are relevant to neutralization antibody activities.
PMCID: PMC1961633  PMID: 17350736
HIV-1; Vaccine; Envelope; Immunogen design
6.  LPS-induced down-regulation of signal regulatory protein α contributes to innate immune activation in macrophages 
The Journal of Experimental Medicine  2007;204(11):2719-2731.
Activation of the mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) cascades after Toll-like receptor (TLR) stimulation contributes to innate immune responses. Signal regulatory protein (SIRP) α, a member of the SIRP family that is abundantly expressed in macrophages, has been implicated in regulating MAPK and NF-κB signaling pathways. In addition, SIRPα can negatively regulate the phagocytosis of host cells by macrophages, indicating an inhibitory role of SIRPα in innate immunity. We provide evidences that SIRPα is an essential endogenous regulator of the innate immune activation upon lipopolysaccharide (LPS) exposure. SIRPα expression was promptly reduced in macrophages after LPS stimulation. The decrease in SIRPα expression levels was required for initiation of LPS-induced innate immune responses because overexpression of SIRPα reduced macrophage responses to LPS. Knockdown of SIRPα caused prolonged activation of MAPKs and NF-κB pathways and augmented production of proinflammatory cytokines and type I interferon (IFN). Mice transferred with SIRPα-depleted macrophages were highly susceptible to endotoxic shock, developing multiple organ failure and exhibiting a remarkable increase in mortality. SIRPα may accomplish this mainly through its association and sequestration of the LPS signal transducer SHP-2. Thus, SIRPα functions as a biologically important modulator of TLR signaling and innate immunity.
PMCID: PMC2118489  PMID: 17954568
7.  FOXP3 is a novel transcriptional repressor for the breast cancer oncogene SKP2  
The Journal of Clinical Investigation  2007;117(12):3765-3773.
S-phase kinase-associated protein 2 (SKP2) is a component of the E3 ubiquitin ligase SKP1-Cul1-Fbox complex. Overexpression of SKP2 results in cell cycle dysregulation and carcinogenesis; however, the genetic lesions that cause this upregulation are poorly understood. We recently demonstrated that forkhead box P3 (FOXP3) is an X-linked breast cancer suppressor and an important repressor of the oncogene ERBB2/HER2. Since FOXP3 suppresses tumor growth regardless of whether the tumors overexpress ERBB2/HER2, additional FOXP3 targets may be involved in its tumor suppressor activity. Here, we show that mammary carcinomas from mice heterozygous for a Foxp3 mutation exhibited increased Skp2 expression. Ectopic expression of FOXP3 in mouse mammary cancer cells repressed SKP2 expression with a corresponding increase in p27 and polyploidy. Conversely, siRNA silencing of the FOXP3 gene in human mammary epithelial cells increased SKP2 expression. We also show that Foxp3 directly interacted with and repressed the Skp2 promoter. Moreover, the analysis of over 200 primary breast cancer samples revealed an inverse correlation between FOXP3 and SKP2 levels. Finally, we demonstrated that downregulation of SKP2 was critical for FOXP3-mediated growth inhibition in breast cancer cells that do not overexpress ERBB2/HER2. Our data provide genetic, biochemical, and functional evidence that FOXP3 is a novel transcriptional repressor for the oncogene SKP2.
PMCID: PMC2075479  PMID: 18008005
8.  Development and evaluation of immunoassay for zeranol in bovine urine*  
A high affinity polyclonal antibody-based enzyme linked immunosorbent assay (ELISA) was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1:5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 μg/ml , and the detection limit was 0.02 μg/ml for the assay. The overall recoveries and the coefficients of variation (CVs) were in the range of 82%~127% and 3.5%~8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol (ranging from 0.2 to 10 μg/ml) were detected by the ELISA and liquid chromatography (LC) method, and good correlations were obtained between the two methods (R 2=0.9643). We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an alternative for the conventional LC method for zeranol in bovine urine.
PMCID: PMC2100163  PMID: 18257125
Zeranol; Enzyme linked immunosorbent assay (ELISA); Bovine urine
9.  Establishment and primary application of a mouse model with hepatitis B virus replication 
AIM: To establish a rapid and convenient animal model with hepatitis B virus (HBV) replication.
METHODS: A naked DNA solution of HBV-replication-competent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen (HBeAg) was detected by enzyme-linked immunosorbent assay (ELISA). Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid (polyIC) or phosphate-buffered saline (PBS).
RESULTS: After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS.
CONCLUSION: A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.
PMCID: PMC4171321  PMID: 17879401
Animal model; Gene expression; Hepatitis B Virus; Hydrodynamic transfection; Polyinosinic-polytidylin acid; Virus replication
10.  DNA Microarray-Based Identification of Serogroups and Virulence Gene Patterns of Escherichia coli Isolates Associated with Porcine Postweaning Diarrhea and Edema Disease▿ †  
Applied and Environmental Microbiology  2007;73(12):4082-4088.
Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 103 CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.
PMCID: PMC1932722  PMID: 17449692
11.  Diverse genome structures of Salmonella paratyphi C 
BMC Genomics  2007;8:290.
Salmonella paratyphi C, like S. typhi, is adapted to humans and causes typhoid fever. Previously we reported different genome structures between two strains of S. paratyphi C, which suggests that S. paratyphi C might have a plastic genome (large DNA segments being organized in different orders or orientations on the genome). As many but not all host-adapted Salmonella pathogens have large genomic insertions as well as the supposedly resultant genomic rearrangements, bacterial genome plasticity presents an extraordinary evolutionary phenomenon. Events contributing to genomic plasticity, especially large insertions, may be associated with the formation of particular Salmonella pathogens.
We constructed a high resolution genome map in S. paratyphi C strain RKS4594 and located four insertions totaling 176 kb (including the 90 kb SPI7) and seven deletions totaling 165 kb relative to S. typhimurium LT2. Two rearrangements were revealed, including an inversion of 1602 kb covering the ter region and the translocation of the 43 kb I-CeuI F fragment. The 23 wild type strains analyzed in this study exhibited diverse genome structures, mostly as a result of recombination between rrn genes. In at least two cases, the rearrangements involved recombination between genomic sites other than the rrn genes, possibly homologous genes in prophages. Two strains had a 20 kb deletion between rrlA and rrlB, which is a highly conservative region and no deletion has been reported in this region in any other Salmonella lineages.
S. paratyphi C has diverse genome structures among different isolates, possibly as a result of large genomic insertions, e.g., SPI7. Although the Salmonella typhoid agents may not be more closely related among them than each of them to other Salmonella lineages, they may have evolved in similar ways, i.e., acquiring typhoid-associated genes followed by genome structure rearrangements. Comparison of multiple Salmonella typhoid agents at both single sequenced genome and population levels will facilitate the studies on the evolutionary process of typhoid pathogenesis, especially the identification of typhoid-associated genes.
PMCID: PMC2000905  PMID: 17718928
12.  Developmental expression of three small GTPases in the mouse eye 
Molecular Vision  2007;13:1144-1153.
The small GTPases function as "molecular switches" by binding and releasing GTP to mediate downstream signaling effects. The Rho-family of GTPases is central in modulating cell differentiation and cytoskeletal changes. Since eye development requires comprehensive morphogenetic movements and extensive cellular differentiation, we hypothesize that different small GTPases may play important roles during morphogenesis of eye development. To explore this possibility, we examined the expression patterns of three major Rho-GTPases: RhoA, Rac1, and Cdc42 in embryonic, postnatal (one day after birth), and adult (two-month old) mouse eye.
Various ocular tissues were collected from embryonic, postnatal, and adult C57BL/6 mice. Western blots were conducted using total proteins extracted from cornea, retina, lens epithelial cells, and lens fiber cells of the adult mice or different fractions of rat lenses. Immunohistochemistry (IHC) was performed with 6 μm thick sections cut through the eye ball region of 11.5 pc, 14.5 pc, 17.5 pc, postnatal, and adult mice. Parallel controls were run using the rabbit preimmune and GTPase-specific antibodies blocked with saturating levels of corresponding peptide antigen.
In the embryonic mouse eye, RhoA and Cdc42 expressions were initially detectable in all three compartments at 11.5 pc. However, Rac1 became easily detectable in these compartments at 14.5 pc. Increased levels of RhoA, Rac1, and Cdc42 were detected in the three compartments at 17.5 pc and the strongest signals for RhoA, Rac1, and Cdc42 were observed in the primary lens fiber cells at 17.5 pc. In the postnatal mouse eye, the three small GTPases were significantly expressed in both endothelial and epithelial cells of mouse cornea, epithelial cells of the ocular lens, photoreceptors, horizontal/amacrine/Muller's cells, and some ganglian cells of the retina. Much lower level of expression was observed in the corneal stroma fibroblasts, lens fiber cells, and the inner and outer plexiform layers of the mouse retina. In the adult mouse eye, all three Rho-GTPases were expressed in corneal epithelial cells and retina. However, only RhoA protein was detected in corneal endothelial cells and Rac1 protein detected in the ocular lens.
The strong expression of the three small GTPases in the cornea, lens, and retina of mouse eye at embryonic 17.5 pc and postnatal stage suggests their important functions for the morphogenesis of the different compartments of the mouse eye. Particularly, high levels of expression of RhoA, Rac1, and Cdc42 in embryonic lens fiber cells suggest their involvement in differentiation of primary lens fiber cells. In the adult mouse eye, all three Rho-GTPases seem to be involved in differentiation of corneal epithelial cells and retina, however, RhoA alone may be required for endothelial cell differentiation and Rac1 likely plays an important role in supporting continuous lens growth and maintenance of lens transparency.
PMCID: PMC2779149  PMID: 17653061
13.  BRAF V600E Maintains Proliferation, Transformation, and Tumorigenicity of BRAF-Mutant Papillary Thyroid Cancer Cells 
Although the BRAF V600E mutant can initiate the formation of papillary thyroid cancer (PTC), it is unclear whether it is required to maintain cell proliferation, transformation, and tumor growth of BRAF mutation-harboring PTC.
The aim of the study was to investigate whether BRAF V600E is required for the proliferation, transformation, and tumorigenicity of BRAF mutation-harboring PTC cells.
We addressed this issue using BRAF small interference RNA (siRNA) to transfect stably several BRAF mutation-harboring PTC cell lines, isolated clones with stable suppression of BRAF, and assessed their ability to proliferate, transform, and grow xenograft tumors in nude mice.
PTC cell proliferation and transformation were suppressed in specific BRAF siRNA clones, but not in control scrambled siRNA clones. Specifically, taking the advantage of stable BRAF knockdown, we were able to show continued suppression of PTC cell proliferation and transformation, or anchorage-independent colony formation in soft agar, after long-term culture. Moreover, we also demonstrated that in vivo tumorigenicity and growth of tumors from the specific BRAF siRNA cell clones in nude mice were suppressed compared with control clones.
BRAF V600E is not only an initiator of PTC as demonstrated previously but is also a maintainer of proliferation, transformation, and tumorigenicity of PTC cells harboring BRAF mutation, and growth of tumors derived from such cells continues to depend on BRAF V600E. These results provide further support for potentially effective therapy targeted at BRAF for BRAF mutation-harboring PTC.
PMCID: PMC4152621  PMID: 17374713
14.  Calcineurin Promotes Hypoxia-inducible Factor 1α Expression by Dephosphorylating RACK1 and Blocking RACK1 Dimerization* 
The Journal of biological chemistry  2007;282(51):37064-37073.
Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of transcriptional responses to changes in O2 concentration. HIF-1 is a heterodimer of HIF-1α and HIF-1β subunits. O2-dependent degradation of the HIF-1α subunit is mediated by prolyl hydroxylase, von Hippel-Lindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O2-independent degradation of HIF-1α is regulated by the competition of RACK1 and HSP90 for binding to HIF-1α. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1α. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1α. Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin. The phosphatase activity of calcineurin is required for its regulation of HIF-1α. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1α degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1α each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF-1α. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by calcineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1α degradation. These results indicate that intracellular calcium levels can regulate HIF-1α expression by modulating calcineurin activity and RACK1 dimerization.
PMCID: PMC3754800  PMID: 17965024
15.  Involvement of Xeroderma Pigmentosum Group A (XPA) in Progeria Arising from Defective Maturation of Prelamin A 
Cellular accumulation of DNA damage has been widely implicated in cellular senescence, aging, and premature aging. In Hutchinson-Gilford progeria syndrome (HGPS) and restrictive dermopathy (RD), premature aging is linked to accumulation of DNA double-strand breaks (DSBs) which results in genome instability. However, how DSBs accumulate in cells despite the presence of intact DNA repair proteins remains unknown. Here we report that the recruitment of DSB repair factors Rad50 and Rad51 to the DSB sites, as marked by γ-H2AX, was impaired in human HGPS and Zmpste24-deficient cells. Consistently, the progeria-associated DSBs appeared to be unrepairable although DSBs induced by camptothecin were efficiently removed in the progeroid cells. We also found that these progeroid cells exhibited nuclear foci of XPA, a unique nucleotide excision repair protein. Strikingly, these XPA foci colocalized with the DSB sites in the progeroid cells. This XPA-DSB association was further confirmed, and found to be mediated by DNA, using a modified chromatin immunoprecipitation assay and co-immunoprecipitation. RNAi knockdown of XPA in HGPS cells partially restored DSB repair as evidenced by Western blot analysis, immunofluorescence and comet assays. We propose that the uncharacteristic localization of XPA to or near DSBs inhibits DSB repair, thereby contributing to the premature aging phenotypes observed in progeria arising from genetic defects in prelamin A maturation.
PMCID: PMC3116236  PMID: 17848622
XPA; Hutchinson-Gilford progeria syndrome; lamin A; Zmpste24; DNA double strand breaks and repair; premature aging; aging; DNA damage
16.  Emergent cardiopulmonary bypass in canines with penetrating cardiac wounds caused by gunshot 
Emergency Medicine Journal : EMJ  2007;24(11):764-768.
Most patients with penetrating cardiac wounds die within minutes of injury from uncontrolled haemorrhage and acute cardiac dysfunction. Thus, sustaining sufficient circulation rapidly is crucial to saving lives. Emergent cardiopulmonary bypass (CPB) is a potential intervention to maintain circulation after penetrating cardiac wounds from a gunshot.
Canines were wounded with a bullet and randomly split into one of three treatment groups. Animals in group 1 (Gp1) were treated with conventional methods. Animals in group 2 (Gp2) received emergent CPB for 180 min and autologous blood transfusion. Animals in group 3 (Gp3) received emergent CPB for 30 min followed by surgical repair. Animal survival, haemodynamics and blood chemistry were measured, and lung water content was evaluated at the end of the experiment.
The right ventricle was the most severely wounded cardiac chamber. In Gp1, mean arterial pressure and central venous pressure were dramatically decreased 8 min after injury, and all animals died within 18 min. In Gp2 and Gp3, mean arterial pressure ranged from 60–90 mm Hg during CPB. 60 min after terminating CPB in Gp2, mean arterial pressure and heart rate were decreased compared to Gp3. In Gp3, most animals maintained haemodynamic stability. 60 min after CPB, free haemoglobin in circulating blood was elevated compared to pre‐trauma levels. Pulmonary water content was significantly higher in Gp2 and Gp3 than in Gp1.
Emergent CPB in the field can maintain haemodynamic stability and supply vital organs with sufficient blood flow, but surgery following CPB is essential to rescue patients with penetrating cardiac wounds.
PMCID: PMC2658320  PMID: 17954829
17.  Molecular-Targeted Antitumor Agents 15: Neolamellarins from the Marine Sponge Dendrilla nigra Inhibit Hypoxia-Inducible Factor-1 (HIF-1) Activation and Secreted Vascular Endothelial Growth Factor (VEGF) Production in Breast Tumor Cells 
Journal of natural products  2007;70(11):1741-1745.
The transcription factor Hypoxia-Inducible Factor 1 (HIF-1) has emerged as a major antitumor molecular target. Inhibition of HIF-1 activation has been shown to suppress the growth, survival, and metastatic spread of hypoxic tumors. The NCI Open Repository of marine invertebrates and algae lipid extracts was evaluated for HIF-1 inhibitory activity in a T47D human breast tumor cell-based reporter assay. Bioassay-guided chromatographic separation of the active extract from the sponge Dendrilla nigra produced four new lamellarin-like phenolic pyrroles, which most closely resemble the structure of the known D. cactos compound lamellarin O. However, unlike lamellarins, the structures of neolamellarin A (1), neolamellarin B (2), 5-hydroxyneolamellarin B (3), and 7-hydroxyneolamellarin A (4) lack the carboxyl moiety at position C-2 of the substituted pyrrole ring and have a significantly different pattern of oxidation. Compound 4 was found to inhibit hypoxia-induced HIF-1 activation (IC50 1.9 μM) in T47D cells. Hypoxic induction of vascular endothelial growth factor (VEGF), a potent angiogenic factor and HIF-1 target gene, was also inhibited by 4 at the secreted protein level.
PMCID: PMC2914556  PMID: 17958397
18.  Comparison of classical and non‐classical cardiovascular risk factors influencing the patency of native arteriovenous fistulas after percutaneous transluminal angioplasty therapy among haemodialysis patients 
Postgraduate Medical Journal  2007;83(982):547-551.
To evaluate the classical and non‐classical cardiovascular risk factors that effect patency of native arteriovenous fistulas (AVF) in end stage renal disease (ESRD) patients who are undergoing regular haemodialysis treatment and have a percutaneous transluminal angioplasty (PTA) procedure.
All PTAs performed between 1 October 2002 and 30 September 2004 were identified from case notes and the computerised database and follow up to 31 March 2005. The definition of patency of AVF after PTA was including primary or secondary patencies. Risks were analysed to assess the influence on survival following PTAs of age, sex, serum cholesterol, serum triglyceride, diabetes, use of aspirin, current smoking and hypertension, serum albumin, serum calcium–phosphate product, intact parathyroid hormone (I‐PTH), and urea reduction ratio (URR).
The patency rate of AVFs of all interventions was 65% at 6 months. Factors with poor patencies of AVFs after PTA procedures were higher serum calcium–phosphate product (p = 0.033), higher URR (p<0.001), lower serum albumin (p<0.001), non‐hypertension (p = 0.010) and “non‐smoker + ex‐smoker group” (p = 0.033). The hypertensive patients and current smokers had lower patency failure after PTAs (p<0.01 and p<0.05, respectively).
Unfavourable cumulative patency rates are observed in haemodialysis patients with higher URR, higher serum calcium–phosphate product and hypoalbuminaemia (lower serum albumin before the PTA procedure). Hypertension and current smoking were associated with better patency rates of AVF after PTA.
PMCID: PMC2600117  PMID: 17675549
19.  Hydroxytyrosol protects retinal pigment epithelial cells from acrolein-induced oxidative stress and mitochondrial dysfunction 
Journal of neurochemistry  2007;103(6):2690-2700.
Hydroxytyrosol (HTS) is a natural polyphenol abundant in olive oil. Increasing evidence indicates HTS has beneficial effect on human health for preventing various diseases. In the present study, we investigated the protective effects of HTS on acrolein-induced toxicity in human retinal pigment epithelial cell line, ARPE-19, a cellular model of smoking- and age-related macular degeneration. Acrolein, a major component of the gas phase cigarette smoke and also a product of lipid peroxidation in vivo, at 75 µmol/L for 24 h caused significant loss of cell viability, oxidative damage (increase in oxidant generation and oxidative damage to proteins and DNA, decrease in antioxidants and antioxidant enzymes, and also inactivation of the Keap1/Nrf2 pathway), and mitochon-drial dysfunction (decrease in membrane potential, activities of mitochondrial complexes, viable mitochondria, oxygen consumption, and factors for mitochondrial biogenesis, and increase in calcium). Pre-treatment with HTS dose dependently and also time dependently protected the ARPE-19 cells from acrolein-induced oxidative damage and mitochondrial dysfunction. A short-term pre-treatment with HTS (48 h) required >75 µmol/L for showing protection while a long-term pre-treatment (7 days) showed protective effect from 5 µmol/L on. The protective effect of HTS in this model was as potent as that of established mitochondria-targeting antioxidant nutrients. These results suggest that HTS is also a mitochondrial-targeting antioxidant nutrient and that dietary administration of HTS may be an effective measure in reducing and or preventing cigarette smoke-induced or age-related retinal pigment epithelial degeneration, such as age-associated macular degeneration.
PMCID: PMC2889218  PMID: 20938484
DNA damage; hydroxytyrosol; macular degeneration; mitochondrial membrane potential; nuclear factor-E2-related factor 2; protein carbonyl
20.  Irritable bowel syndrome and negative appendectomy: a prospective multivariable investigation 
Gut  2007;56(5):655-660.
To examine prospectively whether irritable bowel syndrome (IBS) or other variables—that is, psychiatric profiles, health‐related quality of life (HRQoL) and clinical features—are associated with negative appendectomy (NA).
Longitudinal study.
Inpatient and emergency service in a university‐affiliated teaching hospital.
430 consecutive patients underwent emergent surgery for suspected appendicitis.
Main outcome measures
Rome‐II IBS questionnaire; the Hospital Anxiety and Depression Scale; the Short‐Form 36 survey; the clinical, pathological and CT findings.
The NA group (n = 68, 15.8%) was younger, with female predominance, higher prevalence of Rome‐II IBS, higher anxiety/depression scores and lower levels of HRQoL than the positive appendectomy group. The patients with NA tended to have atypical presentations (absence of migration pain/fever/muscle guarding), lower white cell count and percentage of polymorphonuclear cells (PMNC) and lower rate of CT scan usage than the positive group. After multiple logistic regression, IBS (OR 2.17; 95% CI 1.14 to 4.24), degree of anxiety (OR 1.12; 95% CI 1.02 to 1.49), absence of migrating pain (OR 3.43; 95% CI 1.90 to 5.95)/muscle guarding (OR 3.72; 95% CI 2.07 to 6.70), a lower PMNC percentage (<75%; OR 3.05; 95% CI 1.69 to 5.51) and no CT scan usage (OR 2.32; 95% CI 1.27 to 4.26) were found to be the independent factors in predicting NA.
Both patient (IBS, anxiety, atypical presentation) and physician (low CT scan usage) factors are the independent determinants predicting NA. Physicians should be cautious before operating on or referring patients with IBS for appendectomy. CT scan should be considered in patients with suspected appendicitis, particularly in those with IBS and atypical clinical presentations.
PMCID: PMC1942162  PMID: 17440183
21.  Management of patients with implantable cardioverter defibrillators at emergency departments 
Emergency Medicine Journal : EMJ  2007;24(2):106-109.
With rapid improvements in technology and accumulation of clinical evidence, the implantable cardioverter defibrillator (ICD) has become a standard treatment for either primary or secondary prevention of sudden cardiac death. However, no analysis based on the perspective of emergency department has been reported, and managing patients with ICD remains a challenge to the emergency department doctors.
This study reviewed the emergency department visits of patients who received ICD implantation in a single university hospital from 1995 to 2004. The baseline demographic and laboratory data were compared between groups with the non‐parametric method of the Mann–Whitney U test for continuous data and the χ2 test for categorical data; p<0.05 was considered significant.
81 patients (56 men and 25 women) were included in this study. 43% of patients had at least one emergency department visit during the follow‐up period, and a total of 86 emergency department visits were recorded. The most frequent aetiology of emergency department visits was ICD discharge (37 episodes; 43.1%) and the most frequent presenting symptom was electric shock sensation (25 episodes; 29.1%). Only 11 (12.8%) emergency department visits were because of non‐cardiac aetiologies. Patients with emergency department visits had significant lower left ventricular ejection fraction (mean (SD) 41.5 (19.8) v 55.2 (18.4) ejection fraction units; p = 0.005) and more use of warfarin (8.6% v 0%; p<0.05). Although most emergency department visits were device or arrhythmia related, the acute coronary syndrome and congestive heart failure still accounted for 27.9% of hospital returns in combination.
Defibrillator discharge, acute coronary syndrome and heart failure constitute most aetiologies of emergency department visits of patients with ICD. The risk factors include lower left ventricular ejection fraction and use of warfarin.
PMCID: PMC2658183  PMID: 17251615
22.  Micro-magnetic resonance imaging of avian embryos 
Journal of Anatomy  2007;211(6):798-809.
Chick embryos are useful models for probing developmental mechanisms including those involved in organogenesis. In addition to classic embryological manipulations, it is possible to test the function of molecules and genes while the embryo remains within the egg. Here we define conditions for imaging chick embryo anatomy and for visualising living quail embryos. We focus on the developing limb and describe how different tissues can be imaged using micro-magnetic resonance imaging and this information then synthesised, using a three-dimensional visualisation package, into detailed anatomy. We illustrate the potential for micro-magnetic resonance imaging to analyse phenotypic changes following chick limb manipulation. The work with the living quail embryos lays the foundations for using micro-magnetic resonance imaging as an experimental tool to follow the consequences of such manipulations over time.
PMCID: PMC2375841  PMID: 18045352
anatomy; avian; chick; embryo; limb development; using micro-magnetic resonance imaging; magnetic resonance imaging; quail
23.  Effect of Pioglitazone on Progression of Subclinical Atherosclerosis in Non-Diabetic Premenopausal Hispanic Women with Prior Gestational Diabetes 
Atherosclerosis  2007;199(1):207-214.
The Pioglitazone in the Prevention of Diabetes (PIPOD) study was a single arm 3-year open-label pioglitazone treatment to determine the effects of pioglitazone in women with prior gestational diabetes mellitus (GDM) who had completed the Troglitazone in the Prevention of Diabetes (TRIPOD) study. Here we report the results on progression of subclinical atherosclerosis, measured by carotid intima-media thickness (CIMT) in non-diabetic women. Data were analyzed to compare CIMT progression rates during pioglitazone treatment to rates that had been observed during either placebo or troglitazone treatment in the TRIPOD study. Sixty-one women met the entry criteria with mean age of 40 years. In the 30 women who came to PIPOD from the placebo arm of TRIPOD, the CIMT rate was 69% lower during pioglitazone treatment than it had been during placebo (0.0031 vs. 0.0100 mm/yr, p=0.006). In the 31 women who came to PIPOD from the troglitazone arm of TRIPOD, CIMT rate was 38% lower during pioglitazone than it had been during troglitazone, a difference that was not statistically significant (0.0037 vs. 0.0060 mm/year; p=0.26). Adjustment for differences in baseline characteristics and potential on-trial confounders did not alter the conclusion but did increase the CIMT rates differences slightly. We conclude that treatment with pioglitazone slowed CIMT progression in women who had been on placebo in the TRIPOD study and maintained a relatively low rate of progression in women who had been on troglitazone. Pioglitazone slows progression of subclinical atherosclerosis in young Hispanic women at increased risk for type 2 diabetes.
PMCID: PMC2493568  PMID: 18054942
Pioglitazone; intima-media thickness; premenopausal; gestational diabetes; atherosclerosis
24.  Brachial artery vasoreactivity is associated with cross-sectional and longitudinal anatomical measures of atherosclerosis in postmenopausal women with coronary artery disease 
Atherosclerosis  2007;196(2):674-681.
The diagnostic and prognostic importance of brachial artery flow-mediated dilation (BFMD) for cardiovascular disease (CVD) is not certain and associations between BFMD and recognized measures of atherosclerosis have not been well established.
We investigated cross-sectional and longitudinal correlations between repeated measures of BFMD and quantitative coronary artery angiographic (QCA) measurements of average percent diameter stenosis, number of lesions and minimum luminal diameter (MLD), and ultrasonographic measurement of carotid artery intima-media thickness (CIMT) in an ethnically diverse cohort of postmenopausal women (n = 132) with coronary artery disease (CAD). Subjects were participants in a 3-year randomized, placebo-controlled clinical trial, testing the efficacy of hormone therapy on atherosclerosis progression. Associations between BFMD and QCA measures, and between BFMD and CIMT were examined using measurements from the same study visit.
BFMD was significantly inversely correlated with coronary artery stenosis at baseline (β = −1.21% [S.E.(β) = 0.38], p = 0.002). BFMD levels significantly predicted rate of change in CIMT over the trial period (β = −0.76 μm/year [S.E.(β) = 0.29], p = 0.008).
Physiological and anatomical measures of atherosclerosis are correlated among postmenopausal women with CAD, which provides some validation of BFMD as a measure of atherosclerosis in high-risk populations.
PMCID: PMC2693963  PMID: 17803999
Brachial artery flow-mediated dilation; Endothelial function; Cardiovascular disease; Atherosclerosis; Coronary artery; Angiography; Post-menopausal women; Longitudinal
25.  Neuronal effects of 4-t-Butylcatechol: A model for catechol-containing antioxidants 
Toxicology and applied pharmacology  2007;228(2):247-255.
Many herbal medicines and dietary supplements sold as aids to improve memory or treat neurodegenerative diseases or have other favorable effects on the CNS contain a catechol or similar 1,2-dihydroxy aromatic moiety in their structure. As an approach to isolate and examine the neuroprotective properties of catechols, a simple catechol 4-t-butylcatechol (TBC) has been used as a model. In this study, we investigated the effects of TBC on lipopolysaccharide (LPS)-activated microglial-induced neurotoxicity by using the in vitro model of coculture murine microglial-like cell line HAPI with the neuronal-like human neuroblastoma cell line SH-SY5Y. We also examined the effects of TBC on 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. TBC at concentration from 0.1-10 μM had no toxic effect on HAPI cells and SH-SY5Y cells, and it inhibited LPS (100 ng/ml)-induced increases of superoxide, intracellular ROS, gp91Phox, iNOS and a decrease of HO-1 in HAPI cells. Under coculture condition, TBC significantly reduced LPS-activated microglia-induced dopaminergic SH-SY5Y cells death. Moreover, TBC (0.1-10 μM) inhibited 6-OHDA-induced increases of intracellular ROS, iNOS, nNOS, and a decrease of mitochondria membrane potential, and cell death in SH-SY5Y cells. However, the neurotoxic effects of TBC (100 μM) on SH-SY5Y cells were also observed including the decrease in mitochondria membrane potential and the increase in COX-2 expression and cell death. TBC-induced SH-SY5Y cell death was attenuated by pretreatment with NS-398, a selective COX-2 inhibitor. In conclusion, this study suggests that TBC might possess protective effects on inflammation- and oxidative stress-related neurodegenerative disorders. However, the high concentration of TBC might be toxic, increasing COX-2 expression.
PMCID: PMC2486429  PMID: 18190940
4-t-Butylcatechol; antioxidant; neurons; microglia; oxidative stress; inflammation

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