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1.  Arrestins function in cAR1 GPCR-mediated signaling and cAR1 internalization in the development of Dictyostelium discoideum 
Molecular Biology of the Cell  2014;25(20):3210-3221.
Evolutionarily conserved arrestin-like proteins are key components of the cAR1-mediated ERK2 activation that controls cAMP cell–cell signaling during Dictyostelium aggregation. They are also involved in ligand-induced cAR1 internalization, which is required for the switch of cAMP receptors during multicellular development.
Oscillation of chemical signals is a common biological phenomenon, but its regulation is poorly understood. At the aggregation stage of Dictyostelium discoideum development, the chemoattractant cAMP is synthesized and released at 6-min intervals, directing cell migration. Although the G protein–coupled cAMP receptor cAR1 and ERK2 are both implicated in regulating the oscillation, the signaling circuit remains unknown. Here we report that D. discoideum arrestins regulate the frequency of cAMP oscillation and may link cAR1 signaling to oscillatory ERK2 activity. Cells lacking arrestins (adcB−C−) display cAMP oscillations during the aggregation stage that are twice as frequent as for wild- type cells. The adcB−C− cells also have a shorter period of transient ERK2 activity and precociously reactivate ERK2 in response to cAMP stimulation. We show that arrestin domain–containing protein C (AdcC) associates with ERK2 and that activation of cAR1 promotes the transient membrane recruitment of AdcC and interaction with cAR1, indicating that arrestins function in cAR1-controlled periodic ERK2 activation and oscillatory cAMP signaling in the aggregation stage of D. discoideum development. In addition, ligand-induced cAR1 internalization is compromised in adcB−C− cells, suggesting that arrestins are involved in elimination of high-affinity cAR1 receptors from cell surface after the aggregation stage of multicellular development.
doi:10.1091/mbc.E14-03-0834
PMCID: PMC4196870  PMID: 25143405
2.  Occurrence of Different External Ear Deformities in Monozygotic Twins: Report of 2 Cases 
Summary:
Microtia is a spectrum of congenital deformities, which varies from barely discernable to anotia. Twinning is a well-known risk factor for congenital defects including external ear deformities. Monozygotic twins usually show identical appearances as well as congenital malformations. In special conditions as ear deformities, “mirror-image” may also occur. We report 2 cases of monozygotic twins with different ear deformities. The 8-year-old propositus with lobule type microtia and her identical female twin presented with facial symmetry. Patient A had sausage-type right microtia with absence of external auditory canal. The left external ear showed normal appearance. Patient B presented with left-sided preauricular skin tag and right-sided malformation of tragus with skin tag combined with hyperplasia of underlying cartilage. A granule-size skin tag was also noticed at crus of right helix. A 7-year-old male patient with right-sided conchal type microtia presented to an ear reconstruction center. The patient’s mother showed normal auricular appearance. Her monozygotic twin sister, whose son and daughter had normal ear appearance, was diagnosed with a leftsided lobule type microtia.
doi:10.1097/GOX.0000000000000142
PMCID: PMC4229265  PMID: 25426389
3.  Sequence analysis of mitochondrial ND1 gene can reveal the genetic structure and origin of Bactrocera dorsalis s.s. 
Background
The oriental fruit fly, Bactrocera dorsalis s.s., is one of the most important quarantine pests in many countries, including China. Although the oriental fruit fly has been investigated extensively, its origins and genetic structure remain disputed. In this study, the NADH dehydrogenase subunit 1 (ND1) gene was used as a genetic marker to examine the genetic diversity, population structure, and gene flow of B. dorsalis s.s. throughout its range in China and southeast Asia.
Results
Haplotype networks and phylogenetic analysis indicated two distinguishable lineages of the fly population but provided no strong support for geographical subdivision in B. philippinensis. Demographic analysis revealed rapid expansion of B. dorsalis s.s. populations in China and Southeast Asia in the recent years. The greatest amount of genetic diversity was observed in Manila, Pattaya, and Bangkok, and asymmetric migration patterns were observed in different parts of China. The data collected here further show that B. dorsalis s.s. in Yunnan, Guangdong, and Fujian Provinces, and in Taiwan might have different origins within southeast Asia.
Conclusions
Using the mitochondrial ND1 gene, the results of the present study showed B. dorsalis s.s. from different parts of China to have different genetic structures and origins. B. dorsalis s.s. in China and southeast Asia was found to have experienced rapid expansion in recent years. Data further support the existence of two distinguishable lineages of B. dorsalis s.s. in China and indicate genetic diversity and gene flow from multiple origins.
The sequences in this paper have been deposited in GenBank/NCBI under accession numbers KC413034–KC413367.
doi:10.1186/1471-2148-14-55
PMCID: PMC3998037  PMID: 24655832
Genetic structure; Origin; Bactrocera dorsalis s.s; Mitochondrial ND1
4.  Meta-Analysis of the rs4779584 Polymorphism and Colorectal Cancer Risk 
PLoS ONE  2014;9(2):e89736.
Purpose
Several researchers have suggested that the rs4779584 (15q13.3) polymorphism is associated with an increased risk of developing colorectal cancer (CRC). However, past results remain inconclusive. We addressed this controversy by performing a meta-analysis of the relationship between rs4779584 of GREM1-SCG5 and colorectal cancer.
Methods
We selected 12 case-control studies involving 11,769 cases of CRC and 14,328 healthy controls. The association between the rs4779584 polymorphism and CRC was examined by the overall odds ratio (OR) with a 95% confidence interval (CI). We used different genetic model analyses, sensitivity analyses, and assessments of bias in our meta-analysis.
Results
GREM1-SCG5 rs4779584 polymorphisms were associated with CRC in all of the genetic models that were examined in this meta-analysis of 12 case-control studies.
Conclusion
GREM1-SCG5 rs4779584 polymorphisms may increase the risk of developing colorectal cancer.
doi:10.1371/journal.pone.0089736
PMCID: PMC3933649  PMID: 24586997
5.  In vitro study on the feasibility of magnetic stent hyperthermia for the treatment of cardiovascular restenosis 
Thermal treatment or hyperthermia has received considerable attention in recent years due to its high efficiency, safety and relatively few side-effects. In this study, we investigated whether it was possible to utilize targeted thermal or instent thermal treatments for the treatment of restenosis following percutaneous transluminal coronary angioplasty (PTCA) through magnetic stent hyperthermia (MSH). A 316L stainless steel stent and rabbit vascular smooth muscle cells (VSMCs) were used in the present study, in which the inductive heating characteristics of the stent under alternative magnetic field (AMF) exposure, as well as the effect of MSH on the proliferation, apoptosis, cell cycle and proliferating cell nuclear antigen (PCNA) expression of the rabbit VSMCs, were evaluated. The results demonstrated that 316L stainless steel coronary stents possess ideal inductive heating characteristics under 300 kHz AMF exposure. The heating properties were shown to be affected by the field intensity of the AMF, as well as the orientation the stent axis. MSH had a significant effect on the proliferation and apoptosis of VSMCs, and the effect was temperature-dependent. While a mild temperature of 43°C demonstrated negligible effects on the growth of VSMCs, MSH treatment above 47°C effectively inhibited the VSMC proliferation and induced apoptosis. Furthermore, a 47°C treatment exhibited a significant and long-term inhibitory effect on VSMC migration. The results strongly suggested that MSH may be potentially applied in the clinic as an alternative approach for the prevention and treatment of restenosis.
doi:10.3892/etm.2013.1177
PMCID: PMC3786833  PMID: 24137187
magnetic stent hyperthermia; percutaneous transluminal coronary angioplasty; restenosis; vascular smooth muscle cells; alternative magnetic field
6.  Genistein interferes with SDF-1- and HIV-mediated actin dynamics and inhibits HIV infection of resting CD4 T cells 
Retrovirology  2013;10:62.
Background
Binding of HIV to the chemokine coreceptor CXCR4 mediates viral fusion and signal transduction that promotes actin dynamics critical for HIV infection of blood resting CD4 T cells. It has been suggested that this gp120-mediated actin activity resembles the chemotactic actin dynamics mediated by chemokines such as SDF-1. To determine whether inhibiting SDF-1-mediated chemotactic activity can also inhibit HIV infection, we screened several inhibitors known to reduce SDF-1-mediated chemotaxis of T cells.
Results
We found that a tyrosine kinase inhibitor, genistein, inhibited both SDF-1-mediated chemotaxis and HIV infection of resting CD4 T cells. Genistein was also found to interfere with SDF-1- and HIV-mediated actin dynamics in CD4 T cells. This reduction in actin activity correlates with genistein-mediated inhibition of viral DNA accumulation in resting CD4 T cells. In addition, we also tested two other tyrosine kinase inhibitors, sunitinib and AG1478. Sunitinib, but not AG1478, inhibited HIV infection of resting CD4 T cells. We further tested the safety of genistein in 3 Chinese rhesus macaques (Macaca mulatta), and each animal was given a monotherapy of genistein at 10 mg/kg orally for 12 weeks. No adverse drug effects were observed in these animals.
Conclusions
Our results suggest that novel therapeutic strategies can be developed based on targeting cellular proteins involved in HIV-dependent signaling. This approach can interfere with HIV-mediated actin dynamics and inhibit HIV infection.
doi:10.1186/1742-4690-10-62
PMCID: PMC3693989  PMID: 23782904
HIV-1; Actin; SDF-1; Genistein; Sunitinib; Cofilin; LIMK1
7.  Contemporary environmental variation determines microbial diversity patterns in acid mine drainage 
The ISME Journal  2012;7(5):1038-1050.
A wide array of microorganisms survive and thrive in extreme environments. However, we know little about the patterns of, and controls over, their large-scale ecological distribution. To this end, we have applied a bar-coded 16S rRNA pyrosequencing technology to explore the phylogenetic differentiation among 59 microbial communities from physically and geochemically diverse acid mine drainage (AMD) sites across Southeast China, revealing for the first time environmental variation as the major factor explaining community differences in these harsh environments. Our data showed that overall microbial diversity estimates, including phylogenetic diversity, phylotype richness and pairwise UniFrac distance, were largely correlated with pH conditions. Furthermore, multivariate regression tree analysis also identified solution pH as a strong predictor of relative lineage abundance. Betaproteobacteria, mostly affiliated with the ‘Ferrovum' genus, were explicitly predominant in assemblages under moderate pH conditions, whereas Alphaproteobacteria, Euryarchaeota, Gammaproteobacteria and Nitrospira exhibited a strong adaptation to more acidic environments. Strikingly, such pH-dependent patterns could also be observed in a subsequent comprehensive analysis of the environmental distribution of acidophilic microorganisms based on 16S rRNA gene sequences previously retrieved from globally distributed AMD and associated environments, regardless of the long-distance isolation and the distinct substrate types. Collectively, our results suggest that microbial diversity patterns are better predicted by contemporary environmental variation rather than geographical distance in extreme AMD systems.
doi:10.1038/ismej.2012.139
PMCID: PMC3635239  PMID: 23178673
acid mine drainage; biogeography; contemporary environmental variation; geographical distance; microbial diversity; pyrosequencing
9.  Antitumor effect and immune response induced by local hyperthermia in B16 murine melanoma: Effect of thermal dose 
Oncology Letters  2012;4(4):711-718.
This study aimed at investigating the antitumor effect and immune response induced by local high-temperature hyperthermia at different thermal doses in B16 murine melanoma. The screened optimal thermal dose (50°C, 15 min) which was demonstrated to be the most effective in immune response activation was applied to the treatment of lung metastasis. The optimal thermal dose was determined by evaluating the tumor volume change, survival period of tumor-bearing mice, and immune indices including interleukin (IL)-2, interferon (IFN)-γ and TNF-α mRNA expression in the spleen of mice subjected to local hyperthermia at various thermal doses. The activation of the immune response was further investigated by rechallenging the cured mice 60 days after hyperthermia treatment. The screened optimal thermal dose combined with immunoadjuvant compound 48/80 was applied for melanoma lung metastasis. While local hyperthermia effectively inhibited B16 melanoma tumor growth and prolonged the survival period of tumor-bearing mice, the antitumor immunity was significantly enhanced and the effect was thermal dose-dependent. Higher temperatures (≥50°C) induced a significant effect even with a short treatment time (≤15 min). No tumor regrowth was observed for rechallenged B16 melanoma in mice following treatment with local hyperthermia at a higher temperature. Local hyperthermia by optimal thermal dose in combination with immunoadjuvant compound 48/80 is an effective approach for the treatment of B16 melanoma lung metastasis. This study indicated that the use of a local high-temperature hyperthermia protocol inhibits tumor growth and stimulates a favorable antitumor immune response against malignant melanoma. The results of these experiments may have clinical significance for the treatment of melanoma.
doi:10.3892/ol.2012.804
PMCID: PMC3506660  PMID: 23205088
local hyperthermia; B16 melanoma; lung metastasis; thermal dose; immunity-response
10.  A shortcut from GPCR signaling to Rac-mediated actin cytoskeleton through an ELMO/DOCK complex 
Small GTPases  2012;3(3):183-185.
Chemotaxis, chemoattractant-guided directional cell migration, plays major roles in human innate immunity and in development of a model organism Dictyostelium discoideum. Human leukocytes and D. disscoideum share remarkable similarities in the molecular mechanisms that control chemotaxis. These cells use G-Protein-Coupled Receptors (GPCRs), such as chemokine receptors, to control a signaling network that carries out chemotactic gradient sensing and directs cell migration. Diverse chemokines bind to their receptors to activate small G protein Rac through an evolutionarily conserved mechanism. Elmo and Dock180 proteins form ELMO/Dock180 complexes functioning as guanine nucleotide exchange factors (GEFs) for Rac activation. However, the linkage between GPCR to Elmo/Dock180 for Rac activation that controls F-actin dynamics remained unclear. Recently, we discovered a novel function of an ELMO protein in Dictyostelium discoideum linking GPCR signaling from Gβ to actin dynamics through regulating Rac activation during chemotaxis.
doi:10.4161/sgtp.20271
PMCID: PMC3442806  PMID: 22647486
ELMO protein; G-protein-coupled receptor; Gβ subunit; Rac; chemotaxis; guanine nucleotide exchange factor (GEF); heterotrimeric G protein; small G protein; the actin cytoskeleton
11.  Curcumin attenuates Nrf2 signaling defect, oxidative stress in muscle and glucose intolerance in high fat diet-fed mice 
World Journal of Diabetes  2012;3(5):94-104.
AIM: To investigate the signaling mechanism of anti-oxidative action by curcumin and its impact on glucose disposal.
METHODS: Male C57BL/6J mice were fed with either a normal diet (n = 10) or a high fat diet (HFD) (n = 20) to induce obesity and insulin resistance. After 16 wk, 10 HFD-fed mice were further treated with daily curcumin oral gavage at the dose of 50 mg/kg body weight (BW) (HFD + curcumin group). After 15 d of the curcumin supplementation, an intraperitoneal glucose tolerance test was performed. Fasting blood samples were also collected for insulin and glucose measurements. Insulin-sensitive tissues, including muscle, adipose tissue and the liver, were isolated for the assessments of malondialdehyde (MDA), reactive oxygen species (ROS) and nuclear factor erythroid-2-related factor-2 (Nrf2) signaling.
RESULTS: We show here that in a HFD mouse model, short-term curcumin gavage attenuated glucose intolerance without affecting HFD-induced BW gain. Curcumin also attenuated HFD-induced elevations of MDA and ROS in the skeletal muscle, particularly in its mitochondrial fraction, but it had no such an effect in either adipose tissue or the liver of HFD-fed mice. Correspondingly, in skeletal muscle, the levels of total or nuclear content of Nrf2, as well as its downstream target, heme oxygenase-1, were reduced by HFD-feeding. Curcumin intervention dramatically reversed these defects in Nrf2 signaling. Further analysis of the relationship of oxidative stress with glucose level by a regression analysis showed a positive and significant correlation between the area under the curve of a glucose tolerance test with MDA levels either in muscle or muscular mitochondria.
CONCLUSION: These findings suggest that the short-term treatment of curcumin in HFD-fed mice effectively ameliorates muscular oxidative stress by activating Nrf2 function that is a novel mechanism for its effect in improving glucose intolerance.
doi:10.4239/wjd.v3.i5.94
PMCID: PMC3360224  PMID: 22645638
Oxidative stress; Insulin resistance; Glucose tolerance; Nuclear factor erythroid-2-related factor-2; Curcumin; Mitochondria
12.  Role of nuclear factor (erythroid-derived 2)-like 2 in metabolic homeostasis and insulin action: A novel opportunity for diabetes treatment? 
World Journal of Diabetes  2012;3(1):19-28.
Redox balance is fundamentally important for physiological homeostasis. Pathological factors that disturb this dedicated balance may result in oxidative stress, leading to the development or aggravation of a variety of diseases, including diabetes mellitus, cardiovascular diseases, metabolic syndrome as well as inflammation, aging and cancer. Thus, the capacity of endogenous free radical clearance can be of patho-physiological importance; in this regard, the major reactive oxygen species defense machinery, the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) system needs to be precisely modulated in response to pathological alterations. While oxidative stress is among the early events that lead to the development of insulin resistance, the activation of Nrf2 scavenging capacity leads to insulin sensitization. Furthermore, Nrf2 is evidently involved in regulating lipid metabolism. Here we summarize recent findings that link the Nrf2 system to metabolic homeostasis and insulin action and present our view that Nrf2 may serve as a novel drug target for diabetes and its complications.
doi:10.4239/wjd.v3.i1.19
PMCID: PMC3258535  PMID: 22253942
Nuclear factor (erythroid-derived 2)-like 2; Oxidative stress; Insulin resistance; Metabolism; Diabetic drug
13.  Mechanistic Analysis of a DNA Damage-Induced, PTEN-Dependent Size Checkpoint in Human Cells ▿  
Molecular and Cellular Biology  2011;31(13):2756-2771.
Following DNA damage, human cells undergo arrests in the G1 and G2 phases of the cell cycle and a simultaneous arrest in cell size. We previously demonstrated that the cell size arrest can be uncoupled from the cell cycle arrest by mutational inactivation of the PTEN tumor suppressor gene. Here we show that the cell size checkpoint is inducible by DNA-damaging chemotherapeutic agents as well as by ionizing radiation and is effectively regulated by PTEN but not by its oncogenic counterpart, PIK3CA. Mutational analysis of PTEN and pharmacological inhibition of Akt revealed that modulation of Akt phosphorylation is unnecessary for cell size checkpoint control. To discover putative PTEN regulators and/or effectors involved in size checkpoint control, we employed a novel endogenous epitope tagging (EET) approach, which revealed that endogenous PTEN interacts at the membrane with an actin-remodeling complex that includes actin, gelsolin, and EPLIN. Pharmacological inhibition of actin remodeling in PTEN+/+ cells recapitulated the lack of size checkpoint control seen in PTEN−/− cells. Taken together, these results provide further support for the existence of a DNA damage-inducible size checkpoint that is regulated by a major tumor suppressor, and they provide a novel Akt-independent mechanism by which PTEN controls cell size.
doi:10.1128/MCB.01323-10
PMCID: PMC3133370  PMID: 21536651
14.  Signaling network from GPCR to the actin cytoskeleton during chemotaxis 
Bioarchitecture  2012;2(1):15-18.
Chemotaxis is crucial for many physiological processes including the recruitment of leukocytes to sites of infection, trafficking of lymphocytes in the human body, and metastasis of cancer cells. A family of small proteins, chemokines, serves as the signals, and a family of G-protein coupled receptors (GPCRs) detects chemokines and direct cell migration. One of the basic questions in chemotaxis of eukaryotes is how a GPCR transduces signals to control the assembly of the actin network that generates directional force for cell migration. Over the past decade, a variety of signaling components have been implicated to transduce the GPCR signaling to the actin cytoskeleton. Studies in a lower eukaryotic organism, Dictyostelium discoideum, have allowed us to discover evolutionary conversed components involved in the GPCR-controlled actin network during chemotaxis. However, complete pathways linking GPCR to the actin network are still far from clear. Here we first summarize the previous studies on these components, and then update with our finding showing a new pathway, consisting of a GPCR, Gβγ, Elmo/Dock, Rac and Arp2/3 and actin. We suggest that this pathway serves as a direct linkage between the GPCR/G-protein, the chemoattractant sensing machinery, and the actin cytoskeleton, the machinery of cell movement during chemotaxis of eukaryotic cells.
PMCID: PMC3383712  PMID: 22754623
Dictyostelium; Dock; Elmo; GPCR; actin; chemotaxis; cytoskeleton; signaling
15.  Pharmacokinetic and pharmacodynamic profiles of recombinant human erythropoietin-loaded poly(lactic-co-glycolic acid) microspheres in rats 
Acta Pharmacologica Sinica  2011;33(1):137-144.
Aim:
To characterize the pharmacokinetic and pharmacodynamic profiles of the recombinant human erythropoietin (rhEPO)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres in rats.
Methods:
The rhEPO-loaded microspheres were prepared using a solid-in-oil-in-water emulsion method. Pharmacokinetics and pharmacodynamics of the rhEPO-loaded microspheres were evaluated in male Sprague-Dawley rats. The serum rhEPO level was determined with ELISA. The level of anti-rhEPO antibody in the serum was measured to assess the immunogenicity of rhEPO released from the microspheres.
Results:
rhEPO was almost completely released from the PLGA microspheres in vitro, following zero-order release kinetics over approximately 30 d. After intramuscular injection (10 000 or 30 000 IU rhEPO/kg) in the rats, the serum rhEPO concentration reached maximum levels on d 1, then decreased gradually and was maintained at nearly steady levels for approximately 4 weeks. Furthermore, the release of rhEPO from the PLGA microspheres was found to be controlled mainly by a dissolution/diffusion mechanism. A good linear correlation (R2=0.98) was obtained between the in vitro and in vivo release data. A single intramuscular injection of the rhEPO-loaded PLGA microspheres (10 000 or 30 000 IU rhEPO/kg) in the rats resulted in elevated hemoglobin and red blood cell concentrations for more than 28 d. Moreover, the immunogenicity of rhEPO released from the PLGA microspheres was comparable with that of the unencapsulated rhEPO.
Conclusion:
The results prove the feasibility of using the PLGA-based microspheres to deliver rhEPO for approximately 1 month.
doi:10.1038/aps.2011.157
PMCID: PMC4010276  PMID: 22139004
recombinant human erythropoietin; poly(lactic-co-glycolic acid); microspheres; pharmacokinetics; pharmacodynamics
16.  Magnetic fluid hyperthermia induced by radiofrequency capacitive field for the treatment of transplanted subcutaneous tumors in rats 
Magnetic fluid hyperthermia (MFH) induced by a magnetic field has become a new heating technology for the treatment of malignant tumors due to its ability to heat the tumor tissue precisely and properly, and due to its significant therapeutic effects. In this study, MFH induced by radiofrequency capacitive field (RCF) for the treatment of transplanted subcutaneous tumors in rats, was investigated. A total of 50 rats bearing subcutaneous tumors were randomly divided into five groups, including i) a pseudo-treatment (PT) control group, ii) magnetic fluid (MF) group, iii) pure hyperthermia (PH) group, iv) magnetic fluid hyperthermia 1 (MFH1) group, and v) magnetic fluid hyperthermia 2 (MFH2) group. Tumors were irradiated for 30 min in the MFH1 group 24 h following injection of MF. Tumors were irradiated for 30 min in the MFH2 group 24 h following injection of MF, and irradiation was repeated for 30 min 72 h following injection of MF. Tumor volumes, tumor volume inhibition ratios and survival times in the rat model were examined. Temperatures of tumor cores and rims both rapidly reached the desired temperature (∼50°C) for tumor treatment within 5 to 10 min in the MFH1 and MFH2 groups, and we maintained this temperature level by manually adjusting the output power (70–130 W). Tumor volumes of the MFH1 and MFH2 groups were reduced compared to those of the PT, MF and PH groups. The inhibitory effect on tumor growth in the MFH2 group (91.57%) was higher compared to that in the MFH1 group (85.21%) and the other groups. The survival time of the MFH2 group (51.62±2.28 days) and MFH1 group (43.10±1.57 days) was increased compared to that of the PH, MF and PT groups. The results obtained show that MFH induced by RCF may serve as a potential and promising method for the treatment of tumors.
doi:10.3892/etm.2011.397
PMCID: PMC3438723  PMID: 22969882
magnetic fluid hyperthermia; radiofrequency capacitive field; transplanted subcutaneous tumor; rat
17.  Nkx6.2 synergizes with Cdx-2 in stimulating proglucagon gene expression 
World Journal of Diabetes  2011;2(5):66-74.
AIM: To investigate whether the transactivator of the proglucagon gene (Gcg), Cdx-2, synergizes with other transcription factors in stimulating Gcg expression and the trans-differentiation of Gcg-expressing cells.
METHODS: We conducted affinity chromatography to identify proteins that interact with Cdx-2, using GST-tagged Cdx-2 against cell lysates from pancreatic InR1-G9 and intestinal GLUTag cell lines. This was followed by a mass-spectrometry analysis. From a potential Cdx-2 interaction protein identified, we examined its expression in pancreatic and gut endocrine cells, confirmed its interaction with Cdx-2 by GST-pull down and determined its effect in provoking Gcg expression in cell lines that do not express endogenous Gcg.
RESULTS: We identified 18 potential Cdx-2 interacting proteins. One of them is Nkx6.2. This homeodomain (HD) protein is expressed in pancreatic α and intestinal endocrine L cells but not in insulin producing cell lines, including In111. Nkx6.2, but not Nkx6.1, was shown to interact with Cdx-2, detected by GST-pull down. Furthermore, Nkx6.2 was found to synergize with Cdx-2 in provoking Gcg expression when they were ectopically expressed in the In111 cell line. Finally, when Cdx-2 and Nkx6.2 were co-transfected into the undifferentiated rat intestinal IEC-6 cell line, it produced detectable amount of Gcg mRNA.
CONCLUSION: Cdx-2 recruits Nkx6.2 in exerting its effect in stimulating Gcg expression. Our observations further support the notion that multiple HD proteins, including Cdx-2 and Nkx6.2, are involved in the regulation of Gcg expression and the genesis of Gcg-producing cells.
doi:10.4239/wjd.v2.i5.66
PMCID: PMC3116010  PMID: 21691557
Cdx-2; Nkx6.2; Homeodomain; Proglucagon; Affinity chromatograph
18.  Cutting Edge: Evidence for Ligand-Independent Multimerization of the IL-17 Receptor1 
IL-17 and its receptor are founding members of a novel inflammatory cytokine family. To date, only one IL-17 receptor subunit has been identified, termed IL-17RA. All known cytokine receptors consist of a complex of multiple subunits. Although IL-17-family cytokines exist as homodimers, the configuration and stoichiometry of the IL-17R complex remain unknown. We used fluorescence resonance energy transfer (FRET) to determine whether IL-17RA subunits multimerize, and, if so, whether they are preassembled in the plasma membrane. HEK293 cells coexpressing IL-17RA fused to cyan or yellow fluorescent proteins (CFP or YFP) were used to evaluate FRET before and after IL-17A or IL-17F treatment. In the absence of ligand, IL-17RA molecules exhibited significant specific FRET efficiency, demonstrating that they exist in a multimeric, preformed receptor complex. Strikingly, treatment with IL-17A or IL-17F markedly reduced FRET efficiency, suggesting that IL-17RA subunits within the IL-17R complex undergo a conformational change upon ligand binding.
PMCID: PMC2973994  PMID: 16393951
19.  Cutting Edge: Identification of a Pre-Ligand Assembly Domain (PLAD) and Ligand Binding Site in the IL-17 Receptor1 
IL-17 is the hallmark cytokine of the newly described “Th17” lymphocyte population. The composition, subunit dynamics, and ligand contacts of the IL-17 receptor are poorly defined. We previously demonstrated that the IL-17RA subunit oligomerizes in the membrane without a ligand. In this study, computational modeling identified two fibronectin-III-like (FN) domains in IL-17RA connected by a nonstructured linker, which we predicted to mediate homotypic interactions. In yeast two-hybrid, the membrane-proximal FN domain (FN2), but not the membrane-distal domain (FN1), formed homomeric interactions. The ability of FN2 to drive ligand-independent multimerization was verified by coimmunoprecipitation and fluorescence resonance energy transfer microscopy. Thus, FN2 constitutes a “pre-ligand assembly domain” (PLAD). Further studies indicated that the FN2 linker domain contains the IL-17 binding site, which was never mapped. However, the FN1 domain is also required for high affinity interactions with IL-17. Therefore, although the PLAD is located entirely within FN2, effective ligand binding also involves contributions from the linker and FN1.
PMCID: PMC2973996  PMID: 17982023
20.  Genome-wide Association Analyses Suggested a Novel Mechanism for Smoking Behavior Regulated by IL15 
Molecular psychiatry  2009;14(7):668-680.
Cigarette smoking is the leading preventable cause of death in the US. Although smoking behavior has a significant genetic determination, the specific genes and associated mechanisms underlying smoking behavior are largely unknown. Here, we performed a genome-wide association study on smoking behavior in 840 Caucasians, including 417 males and 423 females, in which we examined ∼380,000 SNPs. We found that a cluster of nine SNPs upstream from the IL15 gene were associated with smoking status in males, with the most significant SNP, rs4956302, achieving a p value (8.80×10−8) of genome-wide significance. Another SNP, rs17354547, that is highly conserved across multiple species, achieved a p value of 5.65×10−5. These two SNPs, together with two additional SNPs (rs1402812 and rs4956396) were selected from the above nine SNPs for replication in an African-American sample containing 1,251 subjects, including 412 males and 839 females. The SNP rs17354547 was successfully replicated in the male subgroup of the replication sample; it was associated with smoking quantity (SQ), the Heaviness of Smoking Index (HSI) and the Fagerstrom Test for Nicotine Dependence (FTND), with p values of 0.031, 0.0046 and 0.019, respectively. In addition, a haplotype formed by rs17354547, rs1402812 and rs4956396 was also associated with SQ, HSI and FTND, achieving p values of 0.039, 0.0093 and 0.0093, respectively. To further confirm our findings, we performed an in silico replication study of the nine SNPs in a Framingham Heart Study sample containing 7,623 Caucasians from 1,731 families, among which, 3,491 subjects are males and 4,132 are females. Again, male-specific association with smoking status was observed, for which seven of the nine SNPs achieved significant p values (p<0.05) and two achieved marginally significant p values (p<0.10) in males. Several of the nine SNPs, including the highly conserved one across species, rs17354547, are located at potential transcription factor binding sites, suggesting transcription regulation as a possible function for these SNPs. Through this function, the SNPs may modulate gene expression of IL15, a key cytokine regulating immune function. As the immune system has long been recognized to influence drug addiction behavior, our association findings suggest a novel mechanism for smoking addiction involving immune modulation via the IL15 pathway.
doi:10.1038/mp.2009.3
PMCID: PMC2700850  PMID: 19188921
smoking; nicotine addiction; IL15; genomewide association; genetics
21.  Chemotaxis, chemokine receptors and human disease 
Cytokine  2008;44(1):1-8.
Cell migration is involved in diverse physiological processes including embryogenesis, immunity, and diseases such as cancer and chronic inflammatory disease. The movement of many cell types is directed by extracellular gradients of diffusible chemicals. This phenomenon, referred to as "chemotaxis", was first described in 1888 by Leber who observed the movement of leukocytes toward sites of inflammation. We now know that a large family of small proteins, chemokines, serves as the extracellular signals and a family of G-protein-coupled receptors (GPCRs), chemokine receptors, detects gradients of chemokines and guides cell movement in vivo. Currently, we still know little about the molecular machineries that control chemokine gradient sensing and migration of immune cells. Fortunately, the molecular mechanisms that control these fundamental aspects of chemotaxis appear to be evolutionarily conserved, and studies in lower eukaryotic model systems allowed us to form concepts, uncover molecular components, develop new techniques, and test models of chemotaxis. These studies have helped our current understanding of this complicated cell behavior. In this review, we wish to mention landmark discoveries in the chemotaxis research field that shaped our current understanding of this fundamental cell behavior and lay out key questions that remain to be addressed in the future.
doi:10.1016/j.cyto.2008.06.017
PMCID: PMC2613022  PMID: 18722135
chemotaxis; chemokine; GPCR; actin; inflammation
22.  The Elmo family forms an ancient group of actin-regulating proteins 
The Elmo protein family members are important mediators of small G protein activity, regulating actin-mediated processes such as chemotaxis and engulfment. Until recently,1 Elmo function has not been explored in professional phagocytes such as Dictyostelium discoideum. We discuss the significance of this family with respect to pathways that regulate Rac signaling, we present a comparison of Elmo proteins between representative taxa, and discuss our findings on ElmoA, one of six Elmo proteins found in D. discoideum.
PMCID: PMC2734041  PMID: 19721884
Elmo; TIRFM; Dictyostelium; actin; myosin; small G-protein; chemotaxis
24.  Fluorescence Resonance Energy Transfer Imaging Reveals that Chemokine-Binding Modulates Heterodimers of CXCR4 and CCR5 Receptors 
PLoS ONE  2008;3(10):e3424.
Background
Dimerization has emerged as an important feature of chemokine G-protein-coupled receptors. CXCR4 and CCR5 regulate leukocyte chemotaxis and also serve as a co-receptor for HIV entry. Both receptors are recruited to the immunological synapse during T-cell activation. However, it is not clear whether they form heterodimers and whether ligand binding modulates the dimer formation.
Methodology/Principal Findings
Using a sensitive Fluorescence Resonance Energy Transfer (FRET) imaging method, we investigated the formation of CCR5 and CXCR4 heterodimers on the plasma membrane of live cells. We found that CCR5 and CXCR4 exist as constitutive heterodimers and ligands of CCR5 and CXCR4 promote different conformational changes within these preexisting heterodimers. Ligands of CCR5, in contrast to a ligand of CXCR4, induced a clear increase in FRET efficiency, indicating that selective ligands promote and stabilize a distinct conformation of the heterodimers. We also found that mutations at C-terminus of CCR5 reduced its ability to form heterodimers with CXCR4. In addition, ligands induce different conformational transitions of heterodimers of CXCR4 and CCR5 or CCR5STA and CCR5Δ4.
Conclusions/Significance
Taken together, our data suggest a model in which CXCR4 and CCR5 spontaneously form heterodimers and ligand-binding to CXCR4 or CCR5 causes different conformational changes affecting heterodimerization, indicating the complexity of regulation of dimerization/function of these chemokine receptors by ligand binding.
doi:10.1371/journal.pone.0003424
PMCID: PMC2566588  PMID: 18923649
25.  Identification of PLCL1 Gene for Hip Bone Size Variation in Females in a Genome-Wide Association Study 
PLoS ONE  2008;3(9):e3160.
Osteoporosis, the most prevalent metabolic bone disease among older people, increases risk for low trauma hip fractures (HF) that are associated with high morbidity and mortality. Hip bone size (BS) has been identified as one of the key measurable risk factors for HF. Although hip BS is highly genetically determined, genetic factors underlying the trait are still poorly defined. Here, we performed the first genome-wide association study (GWAS) of hip BS interrogating ∼380,000 SNPs on the Affymetrix platform in 1,000 homogeneous unrelated Caucasian subjects, including 501 females and 499 males. We identified a gene, PLCL1 (phospholipase c-like 1), that had four SNPs associated with hip BS at, or approaching, a genome-wide significance level in our female subjects; the most significant SNP, rs7595412, achieved a p value of 3.72×10−7. The gene's importance to hip BS was replicated using the Illumina genotyping platform in an independent UK cohort containing 1,216 Caucasian females. Two SNPs of the PLCL1 gene, rs892515 and rs9789480, surrounded by the four SNPs identified in our GWAS, achieved p values of 8.62×10−3 and 2.44×10−3, respectively, for association with hip BS. Imputation analyses on our GWAS and the UK samples further confirmed the replication signals; eight SNPs of the gene achieved combined imputed p values<10−5 in the two samples. The PLCL1 gene's relevance to HF was also observed in a Chinese sample containing 403 females, including 266 with HF and 177 control subjects. A SNP of the PLCL1 gene, rs3771362 that is only ∼0.6 kb apart from the most significant SNP detected in our GWAS (rs7595412), achieved a p value of 7.66×10−3 (odds ratio = 0.26) for association with HF. Additional biological support for the role of PLCL1 in BS comes from previous demonstrations that the PLCL1 protein inhibits IP3 (inositol 1,4,5-trisphosphate)-mediated calcium signaling, an important pathway regulating mechanical sensing of bone cells. Our findings suggest that PLCL1 is a novel gene associated with variation in hip BS, and provide new insights into the pathogenesis of HF.
doi:10.1371/journal.pone.0003160
PMCID: PMC2522269  PMID: 18776929

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