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1.  Vinegar-baked Radix Bupleuri modulates the cell membrane constituents and inhibits the P-gp activity in rat hepatocytes 
Background
Vinegar-baked Radix Bupleuri (VBRB) enhances the effects of other drugs on the liver by increasing drug distribution to the liver, but the mechanism of action remains unclear. The present study was designed to determine the effects of VBRB on the membrane permeability, constituents, and P-glycoprotein (P-gp) activity of hepatocyte BRL cells, in order to interpret the liver targeting enhancing effects of VBRB.
Methods
The membrane permeability and P-gp expression were analyzed by flow cytometry. The membrane constituents were determined by an automatic biochemistry analyzer and thin-layer chromatography.
Results
The results showed that, compared with the control, VBRB enhanced the membrane permeability by 41-67% (P < 0.05), which occurred in the absence of any cytotoxicity. VBRB had marginal effects on the cholesterol content, but significantly affected the total protein contents and the lipid constituents of the cell membrane in a dose- and time-dependent manner. VBRB inhibited P-gp expression in the cell membrane by 59-86% (P < 0.01).
Conclusion
VBRB affects the constituents of BRL cells and increases its permeability, which may help explain its liver-targeting effects.
doi:10.1186/1472-6882-14-357
PMCID: PMC4192444  PMID: 25256115
Radix Bupleuri; P-gp; Phosphosphingolipid; Phosphatidylserine; Phosphatidylcholine; Phosphatidyl ethanolamine; Vinegar; Membrane constituents; Cholesterol
2.  Coxiella burnetii in Rodents on Heixiazi Island at the Sino-Russian Border 
This work is a molecular epidemiologic study to detect the incidence of Coxiella burnetii in rodents on Heixiazi Island at the Sino-Russian border of Heilongjiang Province. Liver tissues were extracted and processed to test the incidence of C. burnetii infection using polymerase chain reaction analysis. In total, 18% (66 of 370) of rodents tested positive for infection. The results of logistic regression analysis indicated that infection with C. burnetii was associated significantly with weight and month of capture, and infection was found in all rodent species that were observed; there was no significant difference of sex on the infection of C. burnetii. Though phylogenetic analysis disclosed heterogeneity in the nucleotide sequences isolated from the island rodents, the majority of observed strains were among the most common strains found worldwide. This is the first report on the incidence of C. burnetii in rodents on Heixiazi Island at the Sino-Russian border.
doi:10.4269/ajtmh.12-0580
PMCID: PMC3617867  PMID: 23382172
3.  Distinct Effects of Abelson Kinase Mutations on Myocytes and Neurons in Dissociated Drosophila Embryonic Cultures: Mimicking of High Temperature 
PLoS ONE  2014;9(1):e86438.
Abelson tyrosine kinase (Abl) is known to regulate axon guidance, muscle development, and cell-cell interaction in vivo. The Drosophila primary culture system offers advantages in exploring the cellular mechanisms mediated by Abl with utilizing various experimental manipulations. Here we demonstrate that single-embryo cultures exhibit stage-dependent characteristics of cellular differentiation and developmental progression in neurons and myocytes, as well as nerve-muscle contacts. In particular, muscle development critically depends on the stage of dissociated embryos. In wild-type (WT) cultures derived from embryos before stage 12, muscle cells remained within cell clusters and were rarely detected. Interestingly, abundant myocytes were spotted in Abl mutant cultures, exhibiting enhanced myocyte movement and fusion, as well as neuron-muscle contacts even in cultures dissociated from younger, stage 10 embryos. Notably, Abl myocytes frequently displayed well-expanded lamellipodia. Conversely, Abl neurons were characterized with fewer large veil-like lamellipodia, but instead had increased numbers of filopodia and darker nodes along neurites. These distinct phenotypes were equally evident in both homo- and hetero-zygous cultures (Abl/Abl vs. Abl/+) of different alleles (Abl1 and Abl4) indicating dominant mutational effects. Strikingly, in WT cultures derived from stage 10 embryos, high temperature (HT) incubation promoted muscle migration and fusion, partially mimicking the advanced muscle development typical of Abl cultures. However, HT enhanced neuronal growth with increased numbers of enlarged lamellipodia, distinct from the characteristic Abl neuronal morphology. Intriguingly, HT incubation also promoted Abl lamellipodia expansion, with a much greater effect on nerve cells than muscle. Our results suggest that Abl is an essential regulator for myocyte and neuron development and that high-temperature incubation partially mimics the faster muscle development typical of Abl cultures. Despite the extensive alterations by Abl mutations, we observed myocyte fusion events and nerve-muscle contact formation between WT and Abl cells in mixed WT and Abl cultures derived from labeled embryos.
doi:10.1371/journal.pone.0086438
PMCID: PMC3897706  PMID: 24466097
4.  Cdk5 Mediates Vimentin Ser56 Phosphorylation During GTP-Induced Secretion by Neutrophils 
Journal of cellular physiology  2012;227(2):10.1002/jcp.22782.
Secretion by neutrophils contributes to acute inflammation following injury or infection. Vimentin has been shown to be important for secretion by neutrophils but little is known about its dynamics during secretion, which is regulated by cyclin-dependent kinase 5 (Cdk5). In this study, we sought to examine the vimentin dynamics and its potential regulation by Cdk5 during neutrophil secretion. We show that vimentin is a Cdk5 substrate that is specifically phosphorylated at Ser56. In response to neutrophil stimulation with GTP, vimentin Ser56 was phosphorylated and colocalized with Cdk5 in the cytoplasmic compartment. Vimentin pSer56 and Cdk5 colocalization was consistent with coimmunoprecipitation from stimulated cells. Vimentin Ser56 phosphorylation occurred immediately after stimulation, and a remarkable increase in phosphorylation was noted later in the secretory process. Decreased GTP-induced vimentin Ser56 phosphorylation and secretion resulted from inhibition of Cdk5 activity by roscovitine or olomoucine or by depletion of Cdk5 by siRNA, suggesting that GTP-induced Cdk5-mediated vimentinSer56 phosphorylation may be related to GTP-inducedCdk5-mediated secretion by neutrophils. Indeed, inhibition of vimentin Ser56 phosphorylation led to a corresponding inhibition of GTP-induced secretion, indicating a link between these two events. While fMLP also induced vimentin Ser56 phosphorylation, such phosphorylation was unaffected by roscovitine, which nonetheless, inhibited secretion, suggesting that Cdk5 regulates fMLP-induced secretion via a mechanism independent of Cdk5-mediated vimentin Ser56 phosphorylation. These findings demonstrate the distinct involvement of Cdk5 in GTP- and fMLP-induced secretion by neutrophils, and support the notion that specific targeting of Cdk5 may serve to inhibit the neutrophil secretory process.
doi:10.1002/jcp.22782
PMCID: PMC3855454  PMID: 21465480 CAMSID: cams3771
5.  Huoxue Rongluo Tablet reduces matrix metalloproteinase-9 expression in infarcted brain tissue 
Neural Regeneration Research  2013;8(34):3216-3224.
Huoxue Rongluo Tablet was made of tall gastrodis tuber, dahurian angelica root, honeysuckle stem, grassleaf sweetflag rhizome, common flowering quince fruit, figwort root, red peony root and peach seed at a ratio of 3:2:6:2:3:3:3:3. Huoxue Rongluo Tablet is a well-established and common pre-scription for the treatment of cerebral infarction. In this study, a rat model of cerebral ischemia was established and the animals were intragastrically administered Huoxue Rongluo Tablet. This treat-ment reduced infarct volume, decreased matrix metalloproteinase-9 expression, and improved neurological function. Moreover, the effects of Huoxue Rongluo Tablet were better than those of buflomedil pyridoxal phosphate. These results indicate that Huoxue Rongluo Tablet is effective in treating cerebral infarction by regulating matrix metalloproteinase-9 protein expression.
doi:10.3969/j.issn.1673-5374.2013.34.005
PMCID: PMC4146180  PMID: 25206642
neural regeneration; traditional Chinese medicine; Huoxue Rongluo Tablet; cerebral infarction; neuroprotection; matrix metalloproteinase-9; buflomedil pyridoxal phosphate; grants-supported paper; neuroregeneration
6.  Effect of Land Use History and Pattern on Soil Carbon Storage in Arid Region of Central Asia 
PLoS ONE  2013;8(7):e68372.
The purpose of this study is to investigate variations in soil organic carbon (SOC) in arid areas due to differences in the cultivation history, land use, and soil salinization. The study area is the lower Sangong River basin on the piedmont of the northern TianShan mountains, which experiences heavy land-use activities. In 1982 and 2005,127(152) and 74 (161) samples in old (new) oasis were collected from each site at the surface soil (i.e., 0–20 cm). The data reveal that the mean value of the surface soil organic carbon content of the old oasis was higher than that of the new oasis by 4.01 g/kg in 1982 and 3.79 g/kg in 2005. Additionally, the soil organic carbon content decreased more rapidly in the newly reclaimed oasis than in the old oasis from 1982 to 2005. The spatial pattern of the SOC content was correlated with the exploitation time in the new oasis, the agricultural land use history, and the SOC content. The decreasing trend is clearer in the high SOC content area than in the low SOC content area. Farmland is the largest carbon pool in both the new and old oases. The carbon density of the old oasis was higher than that of the new oasis by 4.01 and 3.79 g/kg in 1982 and 2005 respectively. The loss of SOC in the agricultural watershed of the arid region in NW China is obvious. Improvements of land management practices, such as no tillage, straw returning to soil, and balanced fertilization techniques, should be adopted to increase the SOC content.
doi:10.1371/journal.pone.0068372
PMCID: PMC3707917  PMID: 23874601
7.  Crucial Role of Calcium-Sensing Receptor Activation in Cardiac Injury of Diabetic Rats 
PLoS ONE  2013;8(5):e65147.
Cardiac injury is a common pathological change frequently accompanied by diabetes mellitus. Recently, some evidence indicated that calcium-sensing receptor (CaSR) expressed in the cardiac tissue. However, the functional role of CaSR in diabetic cardiac injury remains unclear. The present study was designed to investigate the relationship between CaSR activation and diabetes-induced cardiac injury. Diabetic model was successfully established by administration of streptozotocin (STZ) in vivo, and cardiomyocyte injury was simulated by 25.5 mM glucose in vitro. Apoptotic rate, intracellular calcium concentration ([Ca2+]i) and the expression of Bcl-2, Bax, extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 were examined. We demonstrated a significant increase in left ventricular end-diastolic pressure (LVEDP) as well as decrease in maximum rate of left ventricular pressure rise and fall (±dp/dtmax), and left ventricular systolic pressure (LVSP), apoptosis of cardiomyocytes was also observed by TUNEL staining. In vitro, 25.5 mM glucose-induced apoptosis was detected by flow cytometry in neonatal rat cardiomyocytes. Further results showed that 25.5 mM glucose significantly increased [Ca2+]i, up-regulated the expression of Bax, P-ERK and P-JNK, and suppressed Bcl-2 expression. However, the above deleterious changes were further confirmed when co-treatment with CaSR agonist GdCl3 (300 µM). But the effects of GdCl3 were attenuated by 10 µM NPS-2390, a specific CaSR inhibitor. When CaSR was silence by siRNA transfection, the effects of high glucose were inhibited. These results suggest that CaSR activation could lead to the apoptosis of cardiomyocytes in diabetic cardiac injury through the induction of calcium overload, the activation of the mitochondrial, and mitogen-activated protein kinase pathway.
doi:10.1371/journal.pone.0065147
PMCID: PMC3661517  PMID: 23717692
8.  Seizure freedom in epilepsia partialis continua (EPC) through vagus nerve stimulation (VNS) therapy: A case report☆ 
Vagus nerve stimulation (VNS) is generally considered as a palliative treatment for patients with drug-resistant partial-onset epilepsy. We report a case in which a patient with drug-resistant epilepsia partialis continua (EPC), became seizure-free for 15 months with VNS combined with antiepileptic medication regimens. To our knowledge, similar cases have not been reported previously.
doi:10.1016/j.ebcr.2013.01.002
PMCID: PMC4150626
Epilepsia partialis continua; VNS
9.  Combination of peroxisome proliferator-activated receptor α/γ agonists may benefit type 2 diabetes patients with coronary artery disease through inhibition of inflammatory cytokine secretion 
Patients with type 2 diabetes mellitus (T2DM) have a higher risk of cardiovascular disease (CVD). Peroxisome proliferator-activated receptors (PPARs) play an important role in the regulation of energy homeostasis. Therefore, we aimed to observe the effects of combined PPARα/γ agonists on T2DM patients with coronary artery disease (CAD). Patients were randomly divided into a rosiglitazone (RSG) group (n=20), a bezafibrate (BEZ) group (n=20), a combination of RSG and BEZ group (n=20) and a control group (n=20). Plasma C-reactive protein (CRP) and monocyte chemoattractant protein-1 (MCP-1) were measured by enzyme-linked immunosorbent assay before and 12 weeks after treatment. Fasting blood glucose (FBG), fasting insulin, insulin resistance index (IRI), hemoglobin A1c (HbA1c), lipid levels and body mass index were also investigated. At the end of the treatment, FBG, insulin, IRI, HbA1c and triglyceride levels decreased and the level of high-density lipoprotein cholesterol increased in the RSG, BEZ and combination groups. A decrease in low-density lipoprotein cholesterol was only observed in the combination group. Although the total cholesterol levels in all groups decreased, no significant difference was noted. The levels of CRP and MCP-1 were reduced in patients in the RSG, BEZ and combination groups. In addition, RSG, BEZ and the combination of RSG and BEZ also inhibited MCP-1 secretion. The combination of RSG and BEZ was more efficient than RSG or BEZ alone in downregulating cytokines. In conclusion, our results suggest that a combination of RSG and BEZ may be more efficient than RSG or BEZ alone in the treatment of T2DM patients with CAD.
doi:10.3892/etm.2013.891
PMCID: PMC3570181  PMID: 23408783
diabetes mellitus; coronary heart disease; peroxisome proliferator-activated receptor α; peroxisome proliferator-activated receptor γ; inflammatory cytokine
10.  Dynamin 1 Regulates Amyloid Generation through Modulation of BACE-1 
PLoS ONE  2012;7(9):e45033.
Background
Several lines of investigation support the notion that endocytosis is crucial for Alzheimer’s disease (AD) pathogenesis. Substantial evidence have already been reported regarding the mechanisms underlying amyloid precursor protein (APP) traffic, but the regulation of beta-site APP-Cleaving Enzyme 1 (BACE-1) distribution among endosomes, TGN and plasma membrane remains unclear. Dynamin, an important adaptor protein that controls sorting of many molecules, has recently been associated with AD but its functions remain controversial. Here we studied possible roles for dynamin 1 (dyn1) in Aβ biogenesis.
Principal Findings
We found that genetic perturbation of dyn1 reduces both secreted and intracellular Aβ levels in cell culture. There is a dramatic reduction in BACE-1 cleavage products of APP (sAPPβ and βCTF). Moreover, dyn1 knockdown (KD) leads to BACE-1 redistribution from the Golgi-TGN/endosome to the cell surface. There is an increase in the amount of surface holoAPP upon dyn1 KD, with resultant elevation of α–secretase cleavage products sAPPα and αCTF. But no changes are seen in the amount of nicastrin (NCT) or PS1 N-terminal fragment (NTF) at cell surface with dyn1 KD. Furthermore, treatment with a selective dynamin inhibitor Dynasore leads to similar reduction in βCTF and Aβ levels, comparable to changes with BACE inhibitor treatment. But combined inhibition of BACE-1 and dyn1 does not lead to further reduction in Aβ, suggesting that the Aβ-lowering effects of dynamin inhibition are mainly mediated through regulation of BACE-1 internalization. Aβ levels in dyn1−/− primary neurons, as well as in 3-month old dyn1 haploinsufficient animals with AD transgenic background are consistently reduced when compared to their wildtype counterparts.
Conclusions
In summary, these data suggest a previously unknown mechanism by which dyn1 affects amyloid generation through regulation of BACE-1 subcellular localization and therefore its enzymatic activities.
doi:10.1371/journal.pone.0045033
PMCID: PMC3443198  PMID: 23024787
11.  Integrated Assessment of Heavy Metal Contamination in Sediments from a Coastal Industrial Basin, NE China 
PLoS ONE  2012;7(6):e39690.
The purpose of this study is to investigate the current status of metal pollution of the sediments from urban-stream, estuary and Jinzhou Bay of the coastal industrial city, NE China. Forty surface sediment samples from river, estuary and bay and one sediment core from Jinzhou bay were collected and analyzed for heavy metal concentrations of Cu, Zn, Pb, Cd, Ni and Mn. The data reveals that there was a remarkable change in the contents of heavy metals among the sampling sediments, and all the mean values of heavy metal concentration were higher than the national guideline values of marine sediment quality of China (GB 18668-2002). This is one of the most polluted of the world’s impacted coastal systems. Both the correlation analyses and geostatistical analyses showed that Cu, Zn, Pb and Cd have a very similar spatial pattern and come from the industrial activities, and the concentration of Mn mainly caused by natural factors. The estuary is the most polluted area with extremely high potential ecological risk; however the contamination decreased with distance seaward of the river estuary. This study clearly highlights the urgent need to make great efforts to control the industrial emission and the exceptionally severe heavy metal pollution in the coastal area, and the immediate measures should be carried out to minimize the rate of contamination, and extent of future pollution problems.
doi:10.1371/journal.pone.0039690
PMCID: PMC3387265  PMID: 22768107
12.  Inhaled NO Contributes to Lung Repair in Piglets with Acute Respiratory Distress Syndrome via Increasing Circulating Endothelial Progenitor Cells 
PLoS ONE  2012;7(3):e33859.
Background
Nitric oxide (NO) plays an important role in mobilization of endothelial progenitor cells (EPCs). We hypothesized that inhaled NO (iNO) would induce EPC mobilization and therefore promote lung repair in acute respiratory distress syndrome (ARDS).
Methodology/Principal Findings
Healthy piglets were randomized into four groups (n = 6): Control (Con; mechanical ventilation only); ARDS (established by oleic acid infusion and mechanical ventilation); ARDS plus granulocyte-colony stimulating factor (G-CSF; 10 µg/kg/d subcutaneously); ARDS plus NO inhalation (iNO; 10 ppm). EPCs and mobilizing cytokines were assayed at different time points (baseline, 0, 24, 72 and 168 h) and injury reparation was assessed at 168 h. Compared to the Con group, the levels of EPCs were increased in bone marrow but not in blood in the ARDS group at 24 h. Compared to the ARDS group, inhaled NO induced a rapid elevation in the number of CD34+KDR+, KDR+CD133+ and CD34+KDR+CD133+ EPCs in blood (2163±454 vs. 1094±416, 1302±413 vs. 429±244, 1140±494 vs. 453±273 cells/ml, respectively, P<0.05), and a reduction in the percentage of KDR+CD133+ cells in bone marrow. Lung CD34, CD133, VEGF, VEGF receptor 2, endothelial NO synthase mRNA, and VEGF and VEGF receptor 2 protein expression levels were augmented in the iNO group, but not in the G-CSF group, compared to ARDS. Furthermore, iNO treatment reduced vascular permeability, increased pulmonary vessel density, and alleviated pulmonary edema and inflammation compared to ARDS treatment. Plasma VEGF, stromal cell-derived factor-1 (SDF-1) and bone marrow NO2−/NO3− were significantly higher in the iNO group compared to the ARDS group at 72 h.
Conclusions
These results suggest that iNO induces mobilization of EPCs from bone marrow into circulation, contributes to vascular repair, and thereby alleviates lung damage.
doi:10.1371/journal.pone.0033859
PMCID: PMC3309020  PMID: 22448277
13.  KLF6SV1 siRNA inhibits proliferation of human lens epithelial cells 
Molecular Vision  2012;18:601-605.
Purpose
To investigate whether transfection with Krüppel-like factor 6 splice variant 1 (KLF6SV1) siRNA can inhibit proliferation of human lens epithelial cell (HLEC).
Methods
Plasmid containing KLF6SV1 siRNA was used to decrease the level of KLF6SV1 protein in HLEC. The expression of protein27 kinase inhibition protein 1 (p27kip1) and proliferation cell nuclear antigen (PCNA) was tested with western blot. Cell proliferation was assayed by 3-(4,5-dimethylthiazolyl-2-)-2,5-diphenyltetrazoliumbromide (MTT) assay and bromodeoxyuridine (BrdU) incorporation.
Results
KLF6SV1 siRNA can decrease KLF6SV1 expression which leads to increased levels of p27kip1 and decreased expression of PCNA in HLEC. Cells transfected with pKLF6SV1 siRNA showed less viability compared with the control group in vitro.
Conclusions
KLF6SV1 siRNA can effectively inhibit HLEC proliferation. It can be regarded as a novel target to treat posterior capsular opacity (PCO).
PMCID: PMC3298452  PMID: 22419853
14.  Astragalus Polysaccharides Lowers Plasma Cholesterol through Mechanisms Distinct from Statins 
PLoS ONE  2011;6(11):e27437.
To determine the efficacy and underlying mechanism of Astragalus polysaccharides (APS) on plasma lipids in hypercholesterolemia hamsters. The effect of APS (0.25g/kg/d) on plasma and liver lipids, fecal bile acids and neutral sterol, cholesterol absorption and synthesis, HMG-CoA reductase activity, and gene and protein expressions in the liver and small intestine was investigated in twenty-four hypercholesterolemia hamsters. Treatment periods lasted for three months. APS significantly lowered plasma total cholesterol by 45.8%, triglycerides by 30%, and low-density lipoprotein-cholesterol by 47.4%, comparable to simvastatin. Further examinations revealed that APS reduced total cholesterol and triglycerides in the liver, increased fecal bile acid and neutral sterol excretion, inhibited cholesterol absorption, and by contrast, increased hepatic cholesterol synthesis and HMG-CoA reductase activity. Plasma total cholesterol or low-density lipoprotein-cholesterol levels were significantly correlated with cholesterol absorption rates. APS up-regulated cholesterol-7α-hydroxylase and LDL-receptor gene expressions. These new findings identify APS as a potential natural cholesterol lowering agent, working through mechanisms distinct from statins.
doi:10.1371/journal.pone.0027437
PMCID: PMC3217967  PMID: 22110652
15.  Molecular cloning and preliminary function study of iron responsive element binding protein 1 gene from cypermethrin-resistant Culex pipiens pallens 
Parasites & Vectors  2011;4:215.
Background
Insecticide resistance jeopardizes the control of mosquito populations and mosquito-borne disease control, which creates a major public health concern. Two-dimensional electrophoresis identified one protein segment with high sequence homology to part of Aedes aegypti iron-responsive element binding protein (IRE-BP).
Method
RT-PCR and RACE (rapid amplification of cDNA end) were used to clone a cDNA encoding full length IRE-BP 1. Real-time quantitative RT-PCR was used to evaluate the transcriptional level changes in the Cr-IRE strain Aedes aegypti compared to the susceptible strain of Cx. pipiens pallens. The expression profile of the gene was established in the mosquito life cycle. Methyl tritiated thymidine (3H-TdR) was used to observe the cypermethrin resistance changes in C6/36 cells containing the stably transfected IRE-BP 1 gene of Cx. pipiens pallens.
Results
The complete sequence of iron responsive element binding protein 1 (IRE-BP 1) has been cloned from the cypermethrin-resistant strain of Culex pipiens pallens (Cr-IRE strain). Quantitative RT-PCR analysis indicated that the IRE-BP 1 transcription level was 6.7 times higher in the Cr-IRE strain than in the susceptible strain of 4th instar larvae. The IRE-BP 1 expression was also found to be consistently higher throughout the life cycle of the Cr-IRE strain. A protein of predicted size 109.4 kDa has been detected by Western blotting in IRE-BP 1-transfected mosquito C6/36 cells. These IRE-BP 1-transfected cells also showed enhanced cypermethrin resistance compared to null-transfected or plasmid vector-transfected cells as determined by 3H-TdR incorporation.
Conclusion
IRE-BP 1 is expressed at higher levels in the Cr-IRE strain, and may confer some insecticide resistance in Cx. pipiens pallens.
doi:10.1186/1756-3305-4-215
PMCID: PMC3223502  PMID: 22075242
16.  Identification and validation of rice reference proteins for western blotting 
Journal of Experimental Botany  2011;62(14):4763-4772.
Studies of rice protein expression have increased considerably with the development of rice functional genomics. In order to obtain reliable expression results in western blotting, information on appropriate reference proteins is necessary for data normalization. To date, no published study has identified and systematically validated reference proteins suitable for the investigation of rice protein expression. In this study, nine candidate proteins were selected and their specific antibodies were obtained through immunization of rabbits with either recombinant proteins expressed in Escherichia coli or synthesized peptides. Western blotting was carried out to detect the expression of target proteins in a set of 10 rice samples representing different rice tissues/organs at different developmental stages. The expression stability of the proteins was analysed using geNorm and Microcal Origin 6.0 software. The results indicated that heat shock protein (HSP) and elongation factor 1-α (eEF-1α) were the most constantly expressed among all rice proteins tested throughout all developmental stages, while the proteins encoded by conventional internal reference genes fluctuated in amount. Comparison among the profiling of translation and transcription [expressed sequence tags (EST) and massively parallel signature sequencing (MPSS)] revealed that a correlation existed. Based on the standard curves derived from the antigen–antibody reaction, the concentrations of HSP and eEF-1α proteins in rice leaves were ∼0.12%. Under the present experimental conditions, the lower limits of detection for HSP and eEF-1α proteins in rice were 0.24 ng and 0.06 ng, respectively. In conclusion, the reference proteins selected in this study, and the corresponding antibodies, can be used in qualitative and quantitative analysis of rice proteins.
doi:10.1093/jxb/err084
PMCID: PMC3192993  PMID: 21705388
Antibody-based proteomics; rice (Oryza sativa L.); reference gene; reference protein; western blotting
17.  Phosphatidylinositol-4-Phosphate 5-Kinases and Phosphatidylinositol 4,5-Bisphosphate Synthesis in the Brain* 
The Journal of Biological Chemistry  2010;285(37):28708-28714.
The predominant pathway for phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) synthesis is thought to be phosphorylation of phosphatidylinositol 4-phosphate at the 5 position of the inositol ring by type I phosphatidylinositol phosphate kinases (PIPK): PIPKIα, PIPKIβ, and PIPKIγ. PIPKIγ has been shown to play a role in PI(4,5)P2 synthesis in brain, and the absence of PIPKIγ is incompatible with postnatal life. Conversely, mice lacking PIPKIα or PIPKIβ (isoforms are referred to according to the nomenclature of human PIPKIs) live to adulthood, although functional effects in specific cell types are observed. To determine the contribution of PIPKIα and PIPKIβ to PI(4,5)P2 synthesis in brain, we investigated the impact of disrupting multiple PIPKI genes. Our results show that a single allele of PIPKIγ, in the absence of both PIPKIα and PIPKIβ, can support life to adulthood. In addition, PIPKIα alone, but not PIPKIβ alone, can support prenatal development, indicating an essential and partially overlapping function of PIPKIα and PIPKIγ during embryogenesis. This is consistent with early embryonic expression of PIPKIα and PIPKIγ but not of PIPKIβ. PIPKIβ expression in brain correlates with neuronal differentiation. The absence of PIPKIβ does not impact embryonic development in the PIPKIγ knock-out (KO) background but worsens the early postnatal phenotype of the PIPKIγ KO (death occurs within minutes rather than hours). Analysis of PIP2 in brain reveals that only the absence of PIPKIγ significantly impacts its levels. Collectively, our results provide new evidence for the dominant importance of PIPKIγ in mammals and imply that PIPKIα and PIPKIβ function in the generation of specific PI(4,5)P2 pools that, at least in brain, do not have a major impact on overall PI(4,5)P2 levels.
doi:10.1074/jbc.M110.132191
PMCID: PMC2937898  PMID: 20622009
Gene Knockout; Neural Metabolism; Neurobiology; Phosphatidylinositol; Phosphatidylinositol Signaling; Phosphatidylinositol 4,5-Bisphosphate; Phosphatidylinositol 4-Phosphate 5-Kinase
18.  Milk Fat Globule Protein-Epidermal Growth Factor-8: a Pivotal Relay Element within the Angiotensin II and Monocyte Chemoattractant Protein-1 Signaling Cascade Mediating Vascular Smooth Muscle Cells Invasion 
Circulation research  2009;104(12):1337-1346.
Advancing age induces aortic wall thickening that results from the concerted effects of numerous signaling proteins, many of which have yet to be identified. To search for novel proteins associated with aortic wall thickening, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 mo) and old (30 mo) rats and identified 50 proteins that significantly change in abundance with aging. One novel protein, the milk fat globule protein epidermal growth factor 8 (MFG-E8), increases 2.3-fold in abundance in old aorta. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II (Ang II) and monocyte chemoattractant protein-1 (MCP-1) within vascular smooth muscle cells (VSMCs) of the thickened aged aortic wall. Exposure of early passage VMSCs from young aorta to Ang II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMCs from old rats. Treatment of VSMCs with MFG-E8 increases MCP-1 expression and VSMCs invasion that are inhibited by the MCP-1 receptor blocker, vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMCs invasion capacity. The data indicate that arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the Ang II/MCP-1/VSMC invasion signaling cascade. Thus, targeting of MFG-E8 within this signaling axis pathway is a potential novel therapy for the prevention and treatment of the age-associated vascular diseases such as atherosclerosis.
doi:10.1161/CIRCRESAHA.108.187088
PMCID: PMC2764993  PMID: 19443842
MFG-E8; Angiotensin II; Monocyte chemoattractant protein-1; Vascular smooth muscle cells; Aging
19.  Resveratrol protects human lens epithelial cells against H2O2-induced oxidative stress by increasing catalase, SOD-1, and HO-1 expression 
Molecular Vision  2010;16:1467-1474.
Purpose
Oxidative damage induced by H2O2 treatment can irreversibly damage the lens epithelium, resulting in cell death and cataract. Whether the effects of oxidative stress could be attenuated in cultured human lens epithelial cells by incubation with resveratrol (RES) is still unknown. In the present study, we examined the function of resveratrol in protecting human lens epithelial B-3 (HLEB-3) cells against H2O2 induced cell death and cell apoptosis, its role in reducing H2O2 induced intracellular reactive oxygen species (ROS) accumulation, and investigated the mechanism by which resveratrol underlies the effect.
Methods
HLEB-3 cells, a human lens epithelial cell line, were exposed to 100 μM H2O2 with or without RES pre-treatment at different concentrations for different time duration. Cell viabilities were monitored by 4-[3-[4-iodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1) assay. The apoptosis rate and ROS generation were detected by flow cytometric analysis. Expression levels of superoxide dismutases-1 (SOD-1), catalase, and heme oxygenase-1 (HO-1) proteins were measured by western-blotting analysis. p38 and c-jun N terminal kinase (JNK) activation was also evaluated by western-blotting analysis.
Results
Resveratrol clearly reduced H2O2 induced cell apoptosis and ROS accumulation; protected HLEB-3 cells from H2O2 induced oxidative damage, and increased the expression levels of SOD-1, catalase, and HO-1. Further studies showed that RES also inhibited H2O2 induced p38 and JNK phosphorylation.
Conclusions
These findings suggested that RES protected HLEB-3 cells from H2O2 induced oxidative damage, presumably by inducing three antioxidative enzymes including catalase, SOD-1, and HO-1.
PMCID: PMC2925910  PMID: 20806083
20.  Loss of Mouse Ikbkap, a Subunit of Elongator, Leads to Transcriptional Deficits and Embryonic Lethality That Can Be Rescued by Human IKBKAP▿ † 
Molecular and Cellular Biology  2008;29(3):736-744.
Familial dysautonomia (FD), a devastating hereditary sensory and autonomic neuropathy, results from an intronic mutation in the IKBKAP gene that disrupts normal mRNA splicing and leads to tissue-specific reduction of IKBKAP protein (IKAP) in the nervous system. To better understand the roles of IKAP in vivo, an Ikbkap knockout mouse model was created. Results from our study show that ablating Ikbkap leads to embryonic lethality, with no homozygous Ikbkap knockout (Ikbkap−/−) embryos surviving beyond 12.5 days postcoitum. Morphological analyses of the Ikbkap−/− conceptus at different stages revealed abnormalities in both the visceral yolk sac and the embryo, including stunted extraembryonic blood vessel formation, delayed entry into midgastrulation, disoriented dorsal primitive neural alignment, and failure to establish the embryonic vascular system. Further, we demonstrate downregulation of several genes that are important for neurulation and vascular development in the Ikbkap−/− embryos and show that this correlates with a defect in transcriptional elongation-coupled histone acetylation. Finally, we show that the embryonic lethality resulting from Ikbkap ablation can be rescued by a human IKBKAP transgene. For the first time, we demonstrate that IKAP is crucial for both vascular and neural development during embryogenesis and that protein function is conserved between mouse and human.
doi:10.1128/MCB.01313-08
PMCID: PMC2630687  PMID: 19015235
21.  Sex differences in the mechanism of Met5-enkephalin-induced cardioprotection: role of PI3K/Akt 
Met5-enkephalin (ME)-induced cardioprotection occurs via epidermal growth factor receptor (EGFR) transactivation with the subsequent activation of phosphatidylinositol 3-kinase (PI3K). In the present study, we investigated whether there is a sex difference in ME-elicited PI3K signaling. Neonatal murine cardiomyocytes were isolated by collagenase digestion and subjected to 90 min hypoxia and 180 min reoxygenation at 37°C (n = 5 to 7 replicates). PI3K/Akt signaling was interrogated using pharmacological inhibitors and small interfering RNA (siRNA). Cell death was assessed by propidium iodide. More than 300 cells were examined for each treatment. The data are presented as means ± SE. There was not a sex difference in the basal content of total Akt. ME (100 μM) elicited comparable protection in both sexes. Wortmannin and the nonselective Akt inhibitor IV completely abolished ME-induced protection in male cardiomyocytes but only attenuated protection in female cardiomyocytes. Isoform-selective knockdown of Akt in males with siRNAs against Akt1/2 completely abolished ME-induced cardioprotection, whereas the siRNAs against Akt3 only attenuated protection of ∼40%. In contrast, in females the siRNAs against Akt1/2 attenuated and against Akt3 eliminated ME-induced cardioprotection. There is not a sex difference in the degree of ME-induced protection, and there is a sex difference in the cardioprotective signaling pathways after the administration of ME; ME-induced cardioprotection in males primarily utilizes a PI3K/Akt1/2 pathway and in females primarily utilizes a PI3K/Akt3 pathway. The incomplete loss of protection in females following the blockade of PI3K suggests that additional factors may facilitate the maintenance or function of activated Akt.
doi:10.1152/ajpheart.00845.2007
PMCID: PMC2596724  PMID: 17982014
peptides; opioid; pharmacological preconditioning; intracellular signaling
22.  A humanized IKBKAP transgenic mouse models a tissue specific human splicing defect 
Genomics  2007;90(3):389-396.
Familial dysautonomia (FD) is a severe hereditary sensory and autonomic neuropathy, and all patients with FD have a splice mutation in the IKBKAP gene. The FD splice mutation results in variable, tissue-specific skipping of exon 20 in IKBKAP mRNA, which leads to reduced IKAP protein levels. The development of therapies for FD will require suitable mouse models for preclinical studies. In this study, we report the generation and characterization of a mouse model carrying the complete human IKBKAP locus with the FD IVS20+6T>C splice mutation. We show that the mutant IKBKAP transgene is mis-spliced in this model in a tissue specific manner that replicates the pattern seen in FD patient tissues. Creation of this humanized mouse is the first step towards development of a complex phenotypic model of FD. These transgenic mice are an ideal model system for testing the effectiveness of therapeutic agents that target the mis-splicing defect. Lastly, these mice will permit direct studies of tissue-specific splicing and the identification of regulatory factors that play a role in complex gene expression.
doi:10.1016/j.ygeno.2007.05.012
PMCID: PMC1976430  PMID: 17644305

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