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1.  Effect of luteinizing hormone-induced prohibitin and matrix metalloproteinases on ovarian epithelial tumor cell proliferation 
Recent studies have suggested that elevated gonadotropins contribute to ovarian epithelial tumor (OET) cell proliferation. However, the cellular effects of luteinizing hormone, a member of gonadotropins, on OET proliferation are controversial. Our previous work showed that luteinizing hormone has no effect on cell proliferation, but the molecular mechanism of such finding remains to be clarified. Considering that the cell growth in various types of tumors has been associated with regulations of prohibitin and matrix metalloproteinases, we aim to investigate a possible regulatory role of luteinizing hormone on prohibitin and matrix metalloproteinases to determine the roles of these molecules in OET proliferation. We found that LH stimulation resulted in a dose-dependent expression of prohibitin and MMPs and time-dependent phosphorylations of ERK and AKT. Blocking MAPK or PI3K/AKT signaling could attenuate LH-induced prohibitin and MMPs expression. Additionally, the depletion of prohibitin reduced the level of MMPs expression, and increased prohibitin expression abolished the positive effect of LH-induced MMP-9 on cellular growth. Therefore, we conclude that LH is able to up-regulate both prohibitin and MMP-9 in OET cells without the cellular growth effect due to opposing biologic functions for cell proliferation between these two molecules. The opposing cellular growth function between prohibitin and MMP-9 is a novel finding. Regulation of either molecule may be useful for future targeted therapy for ovarian epithelial cancers.
PMCID: PMC4300715  PMID: 25628924
Prohibitin; MMP-2; MMP-9; LH; proliferation
2.  Core structure of the U6 snRNP at 1.7 Å resolution 
The spliceosome is a dynamic assembly of five small nuclear ribonucleoproteins (snRNPs) that removes introns from eukaryotic pre-mRNA. U6 is the most conserved of the spliceosomal snRNAs and participates directly in catalysis. Here, we report the crystal structure of the Saccharomyces cerevisiae U6 snRNP core, containing most of U6 snRNA and all four RRM domains of the Prp24 protein. It reveals a unique interlocked RNP architecture that sequesters the 5′ splice site-binding bases of U6 snRNA. RRMs 1, 2 and 4 of Prp24 form an electropositive groove that binds double-stranded RNA and may nucleate annealing of U4 and U6 snRNAs. Substitutions in Prp24 that suppress a mutation in U6 localize to direct RNA-protein contacts. Our results provide the most complete view to date of a multi-RRM protein bound to RNA, and reveal striking co-evolution of protein and RNA structure.
PMCID: PMC4141773  PMID: 24837192
3.  Five-year epidemiological survey of valvular heart disease: changes in morbidity, etiological spectrum and management in a cardiovascular center of Southern China 
Journal of Thoracic Disease  2014;6(12):1724-1730.
The objective of the present study is to analyze the epidemiological profile of patients with abnormal valvular structure and function and highlight the etiological spectrum and management of valvular heart disease (VHD) in a single cardiovascular center of Southern China in five years.
The retrospective study included 19,428 consecutive patients (9,441 men and 9,987 women with a mean age of 52.03±20.50 years) with abnormal valvular structure and function who were screened by transthoracic echocardiography (TTE) or transesophageal echocardiography (TEE) at the in-patient department of Guangdong General Hospital from January 2009 to December 2013. Data on baseline characteristics, potential etiology, treatment strategies and discharge outcomes were collected from electronic medical records.
There were 13,549 (69.7%) patients with relatively definite etiology for VHD. VHD was rheumatic in 7,197 (37.0%) patients, congenital in 2,697 (13.9%), degenerative in 2,241 (11.5%), ischemic in 2,460 (12.7%). The prevalence decreased significantly in rheumatic VHD from 2009 to 2013 (from 42.8% to 32.8%, P<0.001), but increased markedly in congenital VHD (from 9.0% to 12.3%, P<0.001), ischemic VHD (from 9.2% to 11.3%, P=0.003) and degenerative VHD (from 8.8% to 14.5%, P<0.001). Meantime, the prevalence of ischemic VHD increased after the age of 45, similar to that of degenerative VHD. From 2009 to 2013, the proportion of patients with VHD undergoing open cardiac valvular surgery decreased (from 49.5% to 44.3%, P<0.001) and that of patients treated with general medication increased (from 49.2% to 54.1%, P<0.001). However, there was markedly increment in video-assisted thoracoscopic surgery (VATS) from 2009 to 2013 (from 0.3% to 4.4%, P<0.001). Increasing tendencies were showed in aortic mechanical valve replacement (from 32.1% to 34.5%, P=0.001) and double mechanical valve replacement (from 20.9% to 22.3%, P=0.035), especially in mitral valvuloplasty (from 8.5% to 15.7%, P<0.001). However, the proportion of patients undergoing bioprosthetic valve replacement decreased from 2009 to 2013 (from 26.3% to 15.5%, P<0.001).
Despite a significant shift from rheumatic towards degenerative etiology from 2009 to 2013, rheumatic VHD remains the leading etiology in Southern China, with a significant increase in the prevalence of ischemic, congenital and degenerative VHD. General medication and cardiac valvular surgery remain the main treatment options. The proportion of VATS increased markedly from 2009 to 2013, and mechanical valve replacement and mitral valvuloplasty showed an increasing tendency.
PMCID: PMC4283290  PMID: 25589965
Epidemiological; valvular heart disease (VHD); etiology; cardiac valvular surgery
4.  Nafamostat mesilate attenuates neuronal damage in a rat model of transient focal cerebral ischemia through thrombin inhibition 
Scientific Reports  2014;4:5531.
Evidence suggests that thrombin, a blood coagulation serine protease, mediates neuronal injury in experimental cerebral ischemia. Here, we test the hypothesis that nafamostat mesilate, a serine protease inhibitor, may ameliorate ischemia-induced neuronal damage through thrombin inhibition after ischemic stroke. Focal ischemia was induced in adult Sprague-Dawley rats by occlusion of the middle cerebral artery for 2 hours followed by 22 hours of reperfusion. The administration of nafamostat mesilate during ischemia and reperfusion reduced the brain infarct volume, edema volume and neurological deficit. Thrombin expression and activity in the ipsilateral striatum were increased after ischemia, whereas the administration of nafamostat mesilate significantly inhibited thrombin expression and activity. Immunostaining showed that the majority of thrombin was expressed in neurons. TUNEL staining showed that nafamostat mesilate reduced the number of dying cells during ischemia. A rat behavioral test showed that nafamostat mesilate treatment significantly improved the learning ability of ischemic rats. These results suggest that nafamostat mesilate may have a potential therapeutic role for neuroprotection against focal cerebral ischemia through thrombin inhibition.
PMCID: PMC4078306  PMID: 24985053
5.  Integrin-mediated type II TGF-β receptor tyrosine dephosphorylation controls SMAD-dependent profibrotic signaling 
The Journal of Clinical Investigation  2014;124(8):3295-3310.
Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-β mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-β receptor (TβRII), which in turn promotes a TβRI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within TβRII is considered the principal regulatory mechanism of TβRII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate TβRII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the TβRII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated TβRII tail tyrosine residues, resulting in inhibition of TβR-dependent fibrotic signaling. The collagen-binding receptor integrin α1β1 was required for recruitment of TCPTP to the TβRII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of TβRII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin α1β1 and TβRII that is essential for TβRII-mediated SMAD activation and fibrotic signaling pathways.
PMCID: PMC4109532  PMID: 24983314
6.  Telmisartan protects central neurons against nutrient deprivation-induced apoptosis in vitro through activation of PPARγ and the Akt/GSK-3β pathway 
Acta Pharmacologica Sinica  2014;35(6):727-737.
To determine whether angiotensin II receptor blockers (ARBs) could protect central neurons against nutrient deprivation-induced apoptosis in vitro and to elucidate the underlying mechanisms.
Primary rat cerebellar granule cells (CGCs) underwent B27 (a serum substitute) deprivation for 24 h to induce neurotoxicity, and cell viability was analyzed using LDH assay and WST-1 assay. DNA laddering assay and TUNEL assay were used to detect cell apoptosis. The expression of caspase-3 and Bcl-2, and the phosphorylation of Akt and GSK-3β were detected using Western blot analysis. AT1a mRNA expression was determined using RT-PCR analysis.
B27 deprivation significantly increased the apoptosis of CGCs, as demonstrated by LDH release, DNA laddering, caspase-3 activation and positive TUNEL staining. Pretreatment with 10 μmol/L ARBs (telmisartan, candesartan or losartan) partially blocked B27 deprivation-induced apoptosis of CGCs with telmisartan being the most effective one. B27 deprivation markedly increased the expression of AT1a receptor in CGCs, inhibited Akt and GSK-3β activation, decreased Bcl-2 level, and activated caspase-3, which were reversed by pretreatment with 1 μmol/L telmisartan. In addition, pretreatment with 10 μmol/L PPARγ agonist pioglitazone was more effective in protecting CGCs against B27 deprivation-induced apoptosis, whereas pretreatment with 20 μmol/L PPARγ antagonist GW9662 abolished all the effects of telmisartan in CGCs deprived of B27.
ARBs, in particular telmisartan, can protect the nutrient deprivation-induced apoptosis of CGCs in vitro through activation of PPARγ and the Akt/GSK-3β pathway.
PMCID: PMC4086392  PMID: 24793312
cerebellar granule cell; nutrient deprivation; apoptosis; angiotensin II receptor blocker; telmisartan; AT1a receptor; Akt; GSK-3β; PPARγ; neurotoxicity
7.  Root Interactions in a Maize/Soybean Intercropping System Control Soybean Soil-Borne Disease, Red Crown Rot 
PLoS ONE  2014;9(5):e95031.
Within-field multiple crop species intercropping is well documented and used for disease control, but the underlying mechanisms are still unclear. As roots are the primary organ for perceiving signals in the soil from neighboring plants, root behavior may play an important role in soil-borne disease control.
Principal Findings
In two years of field experiments, maize/soybean intercropping suppressed the occurrence of soybean red crown rot, a severe soil-borne disease caused by Cylindrocladium parasiticum (C. parasiticum). The suppressive effects decreased with increasing distance between intercropped plants under both low P and high P supply, suggesting that root interactions play a significant role independent of nutrient status. Further detailed quantitative studies revealed that the diversity and intensity of root interactions altered the expression of important soybean PR genes, as well as, the activity of corresponding enzymes in both P treatments. Furthermore, 5 phenolic acids were detected in root exudates of maize/soybean intercropped plants. Among these phenolic acids, cinnamic acid was released in significantly greater concentrations when intercropped maize with soybean compared to either crop grown in monoculture, and this spike in cinnamic acid was found dramatically constrain C. parasiticum growth in vitro.
To the best of our knowledge, this study is the first report to demonstrate that intercropping with maize can promote resistance in soybean to red crown rot in a root-dependent manner. This supports the point that intercropping may be an efficient ecological strategy to control soil-borne plant disease and should be incorporated in sustainable agricultural management practices.
PMCID: PMC4014482  PMID: 24810161
8.  SPX1 is an important component in the phosphorus signalling network of common bean regulating root growth and phosphorus homeostasis 
Journal of Experimental Botany  2014;65(12):3299-3310.
PvSPX1 was found to be a positive regulator in the P signalling network of common bean, and is downstream of PvPHR1.
Proteins containing the SPX domain are believed to play vital roles in the phosphorus (P) signalling network in plants. However, the functions of SPX proteins in legumes remain largely unknown. In this study, three SPX members, PvSPX1–PvSPX3 were cloned from common bean (Phaseolus vulgaris L.). It was found that the transcripts of all three PvSPX members were significantly enhanced in both bean leaves and roots by phosphate (Pi) starvation. Among them, the expression of nuclear localized PvSPX1 showed more sensitive and rapid responses to Pi starvation. Consistently, only overexpression of PvSPX1 resulted in increased root P concentration and modified morphology of transgenic bean hairy roots, such as inhibited root growth and an enlarged root hair zone. It was further demonstrated that PvSPX1 transcripts were up-regulated by overexpressing PvPHR1, and overexpressing PvSPX1 led to increased transcripts of 10 Pi starvation-responsive genes in transgenic bean hairy roots. Taken together, it is suggested that PvSPX1 is a positive regulator in the P signalling network of common bean, and is downstream of PvPHR1.
PMCID: PMC4071846  PMID: 24790114
Bean; hairy roots; phosphate starvation; phosphorus homeostasis; root growth; SPX domain.
9.  Acetylcholinesterase function in apoptotic retina pigment epithelial cells induced by H2O2 
To investigate the acetylcholinesterase (AChE) expression involved in retina pigment epithelial (RPE) apoptosis induced by higher concentrations H2O2.
The human retinal pigment epithelium cell line ARPE-19 was from ATCC (Rockville, MD). Cultured ARPE-19 cells were treated with H2O2 at 0, 250, 500, 1 000, 2 000µmol/L and cell viability was measured with MTT assay. AChE expression and DNA fragments were analyzed by immunocytochemistry, TUNEL and PARP-1 Western blotting.
Immunofluorescence detected AChE exist in the normal human retinal tissue. When H2O2 >500µmol/L, AChE expression showed an increase after 2h, and this concentration was selected for the present study. RPE cell was induced with 1 000µmol/L H2O2 for 2h, compared to the control group, cell activity decline detected by MTT, AChE and PARP-1 protein expression was significantly increased detected by Western blotting. AChE immunofluorescence staining was positive in RPE cell after H2O2 incubate 2h. In addition, pretreatment with 100µmol/L epigallocatechin gallate (EGCG), cell viability increased from 31.20%±3.90% to 70.23%±12.96%.
AChE is weakly expressed in normal human RPE cells. Stimulation with H2O2 caused the stable increase of AChE expression in RPE cells, which may indicate that AChE may be an important role in AMD.
PMCID: PMC3874514  PMID: 24392323
acetylcholinesterase; retina pigment epithelial cells; oxidative stress; age-related macular degeneration
10.  Structural and Morphological Evolution of Lead Dendrites during Electrochemical Migration 
Scientific Reports  2013;3:3227.
The electrochemical deposition and dissolution of lead on gold electrodes immersed in an aqueous solution of lead nitrate were studied in situ using a biasing liquid cell by transmission electron microscopy (TEM). We investigate in real time the growth mechanisms of lead dendrites as deposited on the electrodes under an applied potential. TEM images reveal that lead dendrites are developed by the fast protrusion of lead branches in the electrolyte and tip splitting. And, the fast growing tip of the dendritic branch is composed of polycrystalline nanograins and it develops into a single crystalline branch eventually. This study demonstrated unique electrochemical growth of single crystal dendrites through nucleation, aggregation, alignment and attachment of randomly oriented small grains. Additionally, we found the lead concentration in the electrolyte drastically influences the morphology of dendritic formation.
PMCID: PMC3828566  PMID: 24233151
11.  Identification and expression analysis of the Glycine max CYP707A gene family in response to drought and salt stresses 
Annals of Botany  2012;110(3):743-756.
Background and Aims
Abscisic acid (ABA) plays crucial roles in plants' responses to abiotic stresses. ABA 8′-hydroxylation controlled by CYP707A genes has been well studied in Arabidopsis and rice, but not in legumes. The aims of the present study were to identify and functionally analyse the soybean CYP707A gene family, and to explore their expression patterns under dehydration and salt stresses.
A complementation experiment was employed to verify the function of soybean CYP707A1a in ABA catabolism. Genomic and cDNA sequences of other soybean CYP707A genes were isolated from the Phytozome database based on soybean CYP707A1a. The structure and phylogenetic relationship of this gene family was further analysed. The expression patterns of soybean CYP707A genes under dehydration and salt stress were analysed via quantitative real-time PCR.
Key Results
Over-expression of GmCYP707A1a in the atcyp707a2 T-DNA insertion mutant decreased its sensitivity to ABA, indicating that GmCYP707A1a indeed functions as an ABA 8′-hydroxylase in higher plants. The soybean genome contains ten CYP707A genes. Gene structure and phylogenetic analysis showed high conservation of ten GmCYP707A genes to the other CYP707A genes from monocots and dicots. Seed imbibition induced expression of A1a, A1b, A2a, A2b, A2c, A3a and A5 in embryo, and expression of A1a, A1b, A2a and A2b in cotyledon. Dehydration induced expression of A1a, A1b, A2b, A2c, A3a, A3b, A4a, A4b and A5 both in roots and in leaves, whereas rehydration stimulated transcription of A2a, A2b, A3b, A4a and A5 in roots, and only A3b and A5 in leaves. Expression of all soybean CYP707A genes was induced either by short- or by long-term salt stress.
The first biological evidence is provided that GmCYP7071a encodes an ABA 8′-hydroxylase through transgenic studies. Ten soybean GmCYP707A genes were identified, most of them expressed in multiple soybean tissues, and were induced by imbibition, dehydration and salinity.
PMCID: PMC3400457  PMID: 22751653
Abscisic acid; ABA catabolism; Glycine max; CYP707A gene family; drought; salt stress
12.  Neurogenic and Myogenic Properties of Pan-Colonic Motor Patterns and Their Spatiotemporal Organization in Rats 
PLoS ONE  2013;8(4):e60474.
Background and Aims
Better understanding of intrinsic control mechanisms of colonic motility will lead to better treatment options for colonic dysmotility. The aim was to investigate neurogenic and myogenic control mechanisms underlying pan-colonic motor patterns.
Analysis of in vitro video recordings of whole rat colon motility was used to explore motor patterns and their spatiotemporal organizations and to identify mechanisms of neurogenic and myogenic control using pharmacological tools.
Study of the pan-colonic spatiotemporal organization of motor patterns revealed: fluid-induced or spontaneous rhythmic propulsive long distance contractions (LDCs, 0.4–1.5/min, involving the whole colon), rhythmic propulsive motor complexes (RPMCs) (0.8–2.5/min, dominant in distal colon), ripples (10–14/min, dominant in proximal colon), segmentation and retrograde contractions (0.1–0.8/min, prominent in distal and mid colon). Spontaneous rhythmic LDCs were the dominant pattern, blocked by tetrodotoxin, lidocaine or blockers of cholinergic, nitrergic or serotonergic pathways. Change from propulsion to segmentation and distal retrograde contractions was most prominent after blocking 5-HT3 receptors. In the presence of all neural blockers, bethanechol consistently evoked rhythmic LDC-like propulsive contractions in the same frequency range as the LDCs, indicating the existence of myogenic mechanisms of initiation and propulsion.
Neurogenic and myogenic control systems orchestrate distinct and variable motor patterns at different regions of the pan-colon. Cholinergic, nitrergic and serotonergic pathways are essential for rhythmic LDCs to develop. Rhythmic motor patterns in presence of neural blockade indicate the involvement of myogenic control systems and suggest a role for the networks of interstitial cells of Cajal as pacemakers.
PMCID: PMC3618275  PMID: 23577116
13.  Disruption of AtWNK8 Enhances Tolerance of Arabidopsis to Salt and Osmotic Stresses via Modulating Proline Content and Activities of Catalase and Peroxidase 
With no lysine kinases (WNKs) play important roles in plant growth and development. However, its role in salt and osmotic stress tolerance is unclear. Here, we report that AtWNK8 is mainly expressed in primary root, hypocotyl, stamen and pistil and is induced by NaCl and sorbitol treatment. Compared to the wild-type, the T-DNA knock-out wnk8 mutant was more tolerant to severe salinity and osmotic stresses, as indicated by 27% and 198% more fresh weight in the NaCl and sorbitol treatment, respectively. The wnk8 mutant also accumulated 1.43-fold more proline than the wild-type in the sorbitol treatment. Under NaCl and sorbitol stresses, catalase (CAT) activity in wnk8 mutant was 1.92- and 3.7-times of that in Col-0, respectively. Similarly, under salt and osmotic stress conditions, peroxidase (POD) activities in wnk8 mutant were 1.81- and 1.58-times of that in Col-0, respectively. Taken together, we revealed that maintaining higher CAT and POD activities might be one of the reasons that the disruption of AtWNK8 enhances the tolerance to salt stress, and accumulating more proline and higher activities of CAT and POD might result in the higher tolerance of WNK8 to osmotic stress.
PMCID: PMC3645675  PMID: 23535337
AtWNK8; Arabidopsis; salt stress; osmotic stress; proline; catalase; peroxidase
14.  Trans-subclavian approach for radiofrequency ablation of premature ventricular contractions originating from subtricuspid annulus: a case report 
Catheter ablation has been established as a curative treatment strategy for ventricular arrhythmias. The standard procedure of most ventricular arrhythmias originating from the right ventricle is performed via the femoral vein. However, a femoral vein access may not achieve a successful ablation in some patients.
Case presentation
We reported a case of a 29-year old patient with symptomatic premature ventricular contractions was referred for catheter ablation. Radiofrequency energy application at the earliest endocardial ventricular activation site via the right femoral vein could not eliminate the premature ventricular contractions. Epicardial mapping could not obtain an earlier ventricular activation when compared to the endocardial mapping, and at the earliest epicardial site could not provide an identical pace mapping. Finally, we redeployed the ablation catheter via the right subclavian vein by a long sheath. During mapping of the subvalvular area of the right ventricle, a site with a good pace mapping and early ventricular activation was found, and premature ventricular contractions were eliminated successfully.
Ventricular arrhythmias originating from the subtricuspid annulus may be successfully abolished via a trans-subclavian approach and a long sheath. Although access via the right subclavian vein for mapping and ablation is an effective alternative, it is not a routine approach.
PMCID: PMC3582537  PMID: 23419096
Premature ventricular contractions; Tricuspid annulus; Radiofrequency catheter ablation
15.  Genome-wide identification of soybean microRNAs and their targets reveals their organ-specificity and responses to phosphate starvation 
BMC Genomics  2013;14:66.
Phosphorus (P) plays important roles in plant growth and development. MicroRNAs involved in P signaling have been identified in Arabidopsis and rice, but P-responsive microRNAs and their targets in soybean leaves and roots are poorly understood.
Using high-throughput sequencing-by-synthesis (SBS) technology, we sequenced four small RNA libraries from leaves and roots grown under phosphate (Pi)-sufficient (+Pi) and Pi-depleted (-Pi) conditions, respectively, and one RNA degradome library from Pi-depleted roots at the genome-wide level. Each library generated ∼21.45−28.63 million short sequences, resulting in ∼20.56−27.08 million clean reads. From those sequences, a total of 126 miRNAs, with 154 gene targets were computationally predicted. This included 92 new miRNA candidates with 20-23 nucleotides that were perfectly matched to the Glycine max genome 1.0, 70 of which belong to 21 miRNA families and the remaining 22 miRNA unassigned into any existing miRNA family in miRBase 18.0. Under both +Pi and -Pi conditions, 112 of 126 total miRNAs (89%) were expressed in both leaves and roots. Under +Pi conditions, 12 leaf- and 2 root-specific miRNAs were detected; while under -Pi conditions, 10 leaf- and 4 root-specific miRNAs were identified. Collectively, 25 miRNAs were induced and 11 miRNAs were repressed by Pi starvation in soybean. Then, stem-loop real-time PCR confirmed expression of four selected P-responsive miRNAs, and RLM-5’ RACE confirmed that a PHO2 and GmPT5, a kelch-domain containing protein, and a Myb transcription factor, respectively are targets of miR399, miR2111, and miR159e-3p. Finally, P-responsive cis-elements in the promoter regions of soybean miRNA genes were analyzed at the genome-wide scale.
Leaf- and root-specific miRNAs, and P-responsive miRNAs in soybean were identified genome-wide. A total of 154 target genes of miRNAs were predicted via degradome sequencing and computational analyses. The targets of miR399, miR2111, and miR159e-3p were confirmed. Taken together, our study implies the important roles of miRNAs in P signaling and provides clues for deciphering the functions for microRNA/target modules in soybean.
PMCID: PMC3673897  PMID: 23368765
MicroRNA; Soybean; Phosphorus; Root; Leaf; Genome; Degradome; RLM-5’ RACE; Deep sequencing
16.  Identification of soybean purple acid phosphatase genes and their expression responses to phosphorus availability and symbiosis 
Annals of Botany  2011;109(1):275-285.
Background and Aims
Purple acid phosphatases (PAPs) are members of the metallo-phosphoesterase family and have been known to play important roles in phosphorus (P) acquisition and recycling in plants. Low P availability is a major constraint to growth and production of soybean, Glycine max. Comparative studies on structure, transcription regulation and responses to phosphate (Pi) deprivation of the soybean PAP gene family should facilitate further insights into the potential physiological roles of GmPAPs.
BLAST searches were performed to identify soybean PAP genes at the phytozome website. Bioinformatic analyses were carried out to investigate their gene structure, conserve motifs and phylogenetic relationships. Hydroponics and sand-culture experiments were carried out to obtain the plant materials. Quantitative real-time PCR was employed to analyse the expression patterns of PAP genes in response to P deficiency and symbiosis.
Key Results
In total, 35 PAP genes were identified from soybean genomes, which can be classified into three distinct groups including six subgroups in the phylogenetic tree. The expression pattern analysis showed flowers possessed the largest number of tissue-specific GmPAP genes under normal P conditions. The expression of 23 GmPAPs was induced or enhanced by Pi starvation in different tissues. Among them, nine GmPAP genes were highly expressed in the Pi-deprived nodules, whereas only two GmPAP genes showed significantly increased expression in the arbuscular mycorrhizal roots under low-P conditions.
Most GmPAP genes are probably involved in P acquisition and recycling in plants. Also we provide the first evidence that some members of the GmPAP gene family are possibly involved in the response of plants to symbiosis with rhizobia or arbuscular mycorrhizal fungi under P-limited conditions.
PMCID: PMC3241574  PMID: 21948626
Soybean; Glycine max; purple acid phosphatase; expression analysis; phylogenetic analysis; phosphorus nutrition; symbiosis
17.  WIN55,212-2 protects oligodendrocyte precursor cells in stroke penumbra following permanent focal cerebral ischemia in rats 
Acta Pharmacologica Sinica  2012;34(1):119-128.
To explore whether the synthetic cannabinoid receptor agonist WIN55,212-2 could protect oligodendrocyte precursor cells (OPCs) in stroke penumbra, thereby providing neuroprotection following permanent focal cerebral ischemia in rats.
Adult male SD rats were subjected to permanent middle cerebral artery occlusion (p-MCAO). The animals were administered WIN55,212-2 at 2 h, and sacrificed at 24 h after the ischemic insult. The infarct volumes and brain swelling were assessed. The expression of cannabinoid receptor type 1 (CB1) in the stroke penumbra was examined using Western blot assay. The pathological changes and proliferation of neural glial antigen 2-positive OPCs (NG2+ cells) in the stroke penumbra were studied using immunohistochemistry staining.
p-MCAO significantly increased the expression of CB1 within the stroke penumbra with the highest level appearing at 2 h following the ischemic insult. Administration of WIN55,212-2 (9 mg/kg, iv) significantly attenuated the brain swelling, and reduced the infarct volume as well as the number of tau-immunoreactive NG2+ cells (tau-1+/NG2+ cells) in the stroke penumbra. Moreover, WIN55,212-2 significantly promoted the proliferation of NG2+ cells in the stroke penumbra and in the ipsilateral subventricular zone at 24 h following the ischemic insult. Administration of the selective CB1 antagonist rimonabant (1 mg/kg, iv) partially blocked the effects caused by WIN55,212-2.
Tau-1 is expressed in NG2+ cells following permanent focal cerebral ischemic injury. Treatment with WIN55,212-2 reduces the number of tau-1+/NG2+ cells and promotes NG2+ cell proliferation in the stroke penumbra, which are mediated partially via CB1 and may contribute to its neuroprotective effects.
PMCID: PMC4086494  PMID: 23202804
stroke; permanent focal cerebral ischemia; penumbra; oligodendrocyte precursor cells; neural glial antigen 2 (NG2); tau-1; cannabinoid receptor type 1 (CB1); WIN55,212-2; rimonabant
18.  Novel cancerization marker, TP53, and its role in distinguishing normal tissue adjacent to cancerous tissue from normal tissue adjacent to benign tissue 
The histopathological and molecular heterogeneity of normal tissue adjacent to cancerous tissue (NTAC) and normal tissue adjacent to benign tissue (NTAB), and the availability of limited specimens make deciphering the mechanisms of carcinogenesis challenging. Our goal was to identify histogenetic biomarkers that could be reliably used to define a transforming fingerprint using RNA in situ hybridization.
We evaluated 15 tumor-related RNA in situ hybridization biomarkers using tumor microarray and samples of seven tumor-adjacent normal tissues from 314 patients. Biomarkers were determined using comprehensive statistical methods (significance of support vector machine-based artificial intelligence and area under curve scoring of classification distribution).
TP53 was found to be a most reliable index (P <10-7; area under curve >87%) for distinguishing NTAC from NTAB, according to the results of a significance panel (BCL10, BECN1, BRCA2, FITH, PTCH11 and TP53).
The genetic alterations in TP53 between NTAC and NTAB may provide new insight into the field of cancerization and tumor transformation.
PMCID: PMC3544683  PMID: 23170979
Cancerization; Genetic biomarkers; Normal tissue adjacent to benign; Normal tissue adjacent to cancer; Tissue microarray
19.  Functional Characterization of 14 Pht1 Family Genes in Yeast and Their Expressions in Response to Nutrient Starvation in Soybean 
PLoS ONE  2012;7(10):e47726.
Phosphorus (P) is essential for plant growth and development. Phosphate (Pi) transporter genes in the Pht1 family play important roles in Pi uptake and translocation in plants. Although Pht1 family genes have been well studied in model plants, little is known about their functions in soybean, an important legume crop worldwide.
Principal Findings
We identified and isolated a complete set of 14 Pi transporter genes (GmPT1-14) in the soybean genome and categorized them into two subfamilies based on phylogenetic analysis. Then, an experiment to elucidate Pi transport activity of the GmPTs was carried out using a yeast mutant defective in high-affinity Pi transport. Results showed that 12 of the 14 GmPTs were able to complement Pi uptake of the yeast mutant with Km values ranging from 25.7 to 116.3 µM, demonstrating that most of the GmPTs are high-affinity Pi transporters. Further results from qRT-PCR showed that the expressions of the 14 GmPTs differed not only in response to P availability in different tissues, but also to other nutrient stresses, including N, K and Fe deficiency, suggesting that besides functioning in Pi uptake and translocation, GmPTs might be involved in synergistic regulation of mineral nutrient homeostasis in soybean.
The comprehensive analysis of Pi transporter function in yeast and expression responses to nutrition starvation of Pht1 family genes in soybean revealed their involvement in other nutrient homeostasis besides P, which could help to better understand the regulation network among ion homeostasis in plants.
PMCID: PMC3485015  PMID: 23133521
20.  Comparative Analysis of PvPAP Gene Family and Their Functions in Response to Phosphorus Deficiency in Common Bean 
PLoS ONE  2012;7(5):e38106.
Purple acid phosphatases (PAPs) play a vital role in adaptive strategies of plants to phosphorus (P) deficiency. However, their functions in relation to P efficiency are fragmentary in common bean.
Principal Findings
Five PvPAPs were isolated and sequenced in common bean. Phylogenetic analysis showed that PvPAPs could be classified into two groups, including a small group with low molecular mass, and a large group with high molecular mass. Among them, PvPAP3, PvPAP4 and PvPAP5 belong to the small group, while the other two belong to the large group. Transient expression of 35S:PvPAPs-GFP on onion epidermal cells verified the variations of subcellular localization among PvPAPs, suggesting functional diversities of PvPAPs in common bean. Quantitative PCR results showed that most PvPAPs were up-regulated by phosphate (Pi) starvation. Among them, the expression of the small group PvPAPs responded more to Pi starvation, especially in the roots of G19833, the P-efficient genotype. However, only overexpressing PvPAP1 and PvPAP3 could result in significantly increased utilization of extracellular dNTPs in the transgenic bean hairy roots. Furthermore, overexpressing PvPAP3 in Arabidopsis enhanced both plant growth and total P content when dNTPs were supplied as the sole external P source.
The results suggest that PvPAPs in bean varied in protein structure, response to P deficiency and subcellular localization. Among them, both PvPAP1 and PvPAP3 might function as utilization of extracellular dNTPs.
PMCID: PMC3360649  PMID: 22662274
21.  Co-Inoculation with Rhizobia and AMF Inhibited Soybean Red Crown Rot: From Field Study to Plant Defense-Related Gene Expression Analysis 
PLoS ONE  2012;7(3):e33977.
Soybean red crown rot is a major soil-borne disease all over the world, which severely affects soybean production. Efficient and sustainable methods are strongly desired to control the soil-borne diseases.
Principal Findings
We firstly investigated the disease incidence and index of soybean red crown rot under different phosphorus (P) additions in field and found that the natural inoculation of rhizobia and arbuscular mycorrhizal fungi (AMF) could affect soybean red crown rot, particularly without P addition. Further studies in sand culture experiments showed that inoculation with rhizobia or AMF significantly decreased severity and incidence of soybean red crown rot, especially for co-inoculation with rhizobia and AMF at low P. The root colony forming unit (CFU) decreased over 50% when inoculated by rhizobia and/or AMF at low P. However, P addition only enhanced CFU when inoculated with AMF. Furthermore, root exudates of soybean inoculated with rhizobia and/or AMF significantly inhibited pathogen growth and reproduction. Quantitative RT-PCR results indicated that the transcripts of the most tested pathogen defense-related (PR) genes in roots were significantly increased by rhizobium and/or AMF inoculation. Among them, PR2, PR3, PR4 and PR10 reached the highest level with co-inoculation of rhizobium and AMF.
Our results indicated that inoculation with rhizobia and AMF could directly inhibit pathogen growth and reproduction, and activate the plant overall defense system through increasing PR gene expressions. Combined with optimal P fertilization, inoculation with rhizobia and AMF could be considered as an efficient method to control soybean red crown rot in acid soils.
PMCID: PMC3307780  PMID: 22442737
22.  Comparative Proteomic Approach Identifies Pkm2 and Cofilin-1 as Potential Diagnostic, Prognostic and Therapeutic Targets for Pulmonary Adenocarcinoma 
PLoS ONE  2011;6(11):e27309.
Lung cancer is the leading cause of cancer-related death in the world. Non-small cell lung carcinomas (Non-SCLC) account for almost 80% of lung cancers, of which 40% were adenocarcinomas. For a better understanding of the molecular mechanisms behind the development and progression of lung cancer, particularly lung adenocarcinoma, we have used proteomics technology to search for candidate prognostic and therapeutic targets in pulmonary adenocarcinoma. The protein profile changes between human pulmonary adenocarcinoma tissue and paired surrounding normal tissue were analyzed using two-dimensional polyacrylamide gel electrophoresis (2-DE) based approach. Differentially expressed protein-spots were identified with ESI-Q-TOF MS/MS instruments. As a result, thirty two differentially expressed proteins (over 2-fold, p<0.05) were identified in pulmonary adenocarcinoma compared to normal tissues. Among them, two proteins (PKM2 and cofilin-1), significantly up-regulated in adenocarcinoma, were selected for detailed analysis. Immunohistochemical examination indicated that enhanced expression of PKM2 and cofilin-1 were correlated with the severity of epithelial dysplasia, as well as a relatively poor prognosis. Knockdown of PKM2 expression by RNA interference led to a significant suppression of cell growth and induction of apoptosis in pulmonary adenocarcinoma SPC-A1 cells in vitro, and tumor growth inhibition in vivo xenograft model (P<0.05). In addition, the shRNA expressing plasmid targeting cofilin-1 significantly inhibited tumor metastases and prolonged survival in LL/2 metastatic model. While additional works are needed to elucidate the biological significance and molecular mechanisms of these altered proteins identified in this study, PKM2 and cofilin-1 may serve as potential diagnostic and prognostic biomarkers, as well as therapeutic targets for pulmonary adenocarcinoma.
PMCID: PMC3210781  PMID: 22087286
23.  Genetic improvement for phosphorus efficiency in soybean: a radical approach 
Annals of Botany  2010;106(1):215-222.
Low phosphorus (P) availability is a major constraint to soybean growth and production. Developing P-efficient soybean varieties that can efficiently utilize native P and added P in the soils would be a sustainable and economical approach to soybean production.
This review summarizes the possible mechanisms for P efficiency and genetic strategies to improve P efficiency in soybean with examples from several case studies. It also highlights potential obstacles and depicts future perspectives in ‘root breeding’.
This review provides new insights into the mechanisms of P efficiency and breeding strategies for this trait in soybean. Root biology is a new frontier of plant biology. Substantial efforts are now focusing on increasing soybean P efficiency through ‘root breeding’. To advance this area, additional collaborations between plant breeders and physiologists, as well as applied and theoretical research are needed to develop more soybean varieties with enhanced P efficiency through root modification, which might contribute to reduced use of P fertilizers, expanding agriculture on low-P soils, and achieving more sustainable agriculture.
PMCID: PMC2889788  PMID: 20228090
Soybean; genetic improvement; phosphorus efficiency; root breeding
24.  QTL analysis of root traits as related to phosphorus efficiency in soybean 
Annals of Botany  2010;106(1):223-234.
Background and Aims
Low phosphorus (P) availability is a major constraint to soybean growth and production, especially in tropical and subtropical areas. Root traits have been shown to play critical roles in P efficiency in crops. Identification of the quantitative trait loci (QTLs) conferring superior root systems could significantly enhance genetic improvement in soybean P efficiency.
A population of 106 F9 recombinant inbred lines (RILs) derived from a cross between BD2 and BX10, which contrast in both P efficiency and root architecture, was used for mapping and QTL analysis. Twelve traits were examined in acid soils. A linkage map was constructed using 296 simple sequence repeat (SSR) markers with the Kosambi function, and the QTLs associated with these traits were detected by composite interval mapping and multiple-QTL mapping.
Key Results
The first soybean genetic map based on field data from parental genotypes contrasting both in P efficiency and root architecture was constructed. Thirty-one putative QTLs were detected on five linkage groups, with corresponding contribution ratios of 9·1–31·1 %. Thirteen putative QTLs were found for root traits, five for P content, five for biomass and five for yield traits. Three clusters of QTLs associated with the traits for root and P efficiency at low P were located on the B1 linkage group close to SSR markers Satt519 and Satt519-Sat_128, and on the D2 group close to Satt458; and one cluster was on the B1 linkage group close to Satt519 at high P.
Most root traits in soybean were conditioned by more than two minor QTLs. The region closer to Satt519 on the B1 linkage group might have great potential for future genetic improvement for soybean P efficiency through root selection.
PMCID: PMC2889805  PMID: 20472699
Quantitative trait loci (QTLs); soybean; Glycine max; root traits; phosphorus efficiency
25.  Cetuximab/C225 -Induced Intracellular Trafficking of EGF Receptor 
Cancer research  2009;69(15):6179-6183.
The monoclonal antibody C225 interacts with the ectodomain of the EGF receptor to block ligand binding and initiates receptor endocytosis and intracellular trafficking. The data herein show that C225-dependent EGF receptor trafficking relocalizes the receptor to the endoplasmic reticulum (ER) and nucleus. This mechanism, which also involves interaction of the C225-internalized receptor with the Sec61 translocon within the endoplasmic reticulum (ER), is, in most respect, analogous to the pathway previously described for EGF-induced trafficking to the ER and nucleus (Liao HJ and Carpenter G. Mol Biol Cell 2007; 18: 1064-1072), However, while inhibition of receptor tyrosine kinase activity blocks EGF-induced nuclear localization of the receptor, the same kinase inhibitors stimulate C225-dependent nuclear localization of EGF receptor in the nucleus. In contrast, the kinase inhibitor Lapatinib fails to stimulate nuclear accumulation of the receptor in C225-treated cells and does not provoke receptor dimerization as do inhibitors that recognizing the open conformation of the receptor kinase. This suggests that inhibitor-dependent receptor dimerization may facilitate C225-induced receptor trafficking.
PMCID: PMC2753411  PMID: 19602593

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