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1.  A large spontaneous intrahepatic portosystemic shunt in a cirrhotic patient 
A spontaneous portosystemic shunt is a rare malformation of the vessels supplying the liver. This condition often leads to the development of hepatic encephalopathy due to excessive shunting of blood from the portal vein to the inferior vena cava. Some studies have suggested that the presence of spontaneous portosystemic shunts is inversely associated with the appearance of large esophageal varices. Spontaneous intrahepatic portosystemic shunts (SIPSS) are far less frequently observed than extrahepatic portosystemic shunts, which include spleno-gastric-renal shunts, mesenteric-caval shunts, and a large patent umbilical vein. Reported here is a case of decompensated liver cirrhosis with a large SIPSS without any incidence of overt hepatic encephalopathy.
PMCID: PMC4761588  PMID: 26989653
Spontaneous intrahepatic portosystemic shunt; liver cirrhosis; hepatic encephalopathy; embolization
2.  Incidence and risk factor analysis for sarcopenia in patients with cancer 
Oncology Letters  2015;11(2):1230-1234.
The objective of the present study was to investigate the incidence of and possible risk factors associated with sarcopenia among cancer patients. Patients with cancer were examined through the use of lumbar magnetic resonance imaging, and clinical data was collected between September and December, 2012, at Jilin Province Tumor Hospital (Changchun, China). The data was subsequently compared between patients with and without sarcopenia. Of the 113 treated cancer patients, 96 patients [39 males (L3 index, <52.4 cm2/m2) and 57 females (L3 index, <38.5 cm2/m2)] suffered from sarcopenia. Overall, the development of sarcopenia was not significantly associated with patient age or treatment, including surgery, chemotherapy or radiotherapy (P>0.05). The frequency of treatment-associated complications did not differ significantly between patients with or without sarcopenia. However, males were more inclined to develop sarcopenia than females (P=0.02). Patients with sarcopenia had significantly less lymphocytes than patients without sarcopenia (P=0.03). This was confirmed through multiple logistic regression analyses (P=0.046), which also identified that patients with cancer with an Eastern Cooperative Oncology Group score >2 had a significantly increased risk of developing sarcopenia. Finally, the serum albumin level in sarcopenia patients was 36.18±4.65 g/l, which was not significantly less than that of patients without sarcopenia (39.67±3.69 g/l; P=0.11). The incidence of sarcopenia among patients with cancer is high, particularly for males. Further research with larger sample sizes would be beneficial, with the aim of verifying the results obtained in the present study. During the treatment of patients with sarcopenia, precaution should continue to be taken to prevent associated complications, including infection, diarrhea and myelosuppression.
PMCID: PMC4734230  PMID: 26893724
lymphocytes; cluster of differentiation 4; cancer; sarcopenia; cluster of differentiation 8
3.  Serum Liver Fibrosis Markers for Predicting the Presence of Gastroesophageal Varices in Liver Cirrhosis: A Retrospective Cross-Sectional Study 
Background and Aims. A retrospective cross-sectional study was conducted to evaluate the role of hyaluronic acid (HA), laminin (LN), amino-terminal propeptide of type III procollagen (PIIINP), and collagen IV (CIV) in predicting the presence of gastroesophageal varices (GEVs) in patients with liver cirrhosis. Methods. We enrolled 118 patients with liver cirrhosis who underwent the tests for the four serum liver fibrosis markers and upper gastrointestinal endoscopy at the same admissions. The predictive values of the four serum liver fibrosis markers were evaluated by the areas under the receiving operator characteristics curves (AUROCs) with 95% confidence intervals (CIs). Results. The prevalence of GEVs was 88% (104/118). The AUROCs for HA, LN, PIIINP, and CIV levels in predicting the presence of GEVs were 0.553 (95% CI: 0.458 to 0.644, P = 0.5668), 0.490 (95% CI: 0.397 to 0.584, P = 0.9065), 0.622 (95% CI: 0.528 to 0.710, P = 0.1099), and 0.560 (95% CI: 0.466 to 0.652, P = 0.4909). The PIIINP level at a cut-off value of 31.25 had a sensitivity of 73.1% and a specificity of 57.1%. Conclusions. The present study did not recommend HA, LN, PIIINP, and CIV levels to evaluate the presence of GEVs in liver cirrhosis.
PMCID: PMC4684855  PMID: 26770190
4.  gga-miR-101-3p Plays a Key Role in Mycoplasma gallisepticum (HS Strain) Infection of Chicken 
Mycoplasma gallisepticum (MG), one of the most pathogenic Mycoplasma, has caused tremendous economic loss in the poultry industry. Recently, increasing evidence has suggested that micro ribonucleic acids (miRNAs) are involved in microbial pathogenesis. However, little is known about potential roles of miRNAs in MG infection of chicken. In the present study, using miRNA Solexa sequencing we have found that gga-miR-101-3p was up-regulated in the lungs of MG-infected chicken embryos. Moreover, gga-miR-101-3p regulated expression of the host enhancer of zeste homolog 2 (EZH2) through binding to the 3’ un-translated region (3’-UTR) of EZH2 gene. Over-expression of gga-miR-101-3p significantly inhibited EZH2 expression and hence inhibited proliferation of chicken embryonic fibroblast (DF-1 cells) by blocking the G1-to-S phase transition. Similar results were obtained in MG-infected chicken embryos and DF-1 cells, where gga-miR-101-3p was significantly up-regulated, while EZH2 was significantly down-regulated. This study reveals that gga-miR-101-3p plays an important role in MG infection through regulation of EZH2 expression and provides a new insight into the mechanisms of MG pathogenesis.
PMCID: PMC4691068  PMID: 26633386
chicken; Mycoplasma gallisepticum (HS strain); gga-miR-101-3p; EZH2
5.  Analysis of the correlation between serum resistin and the variability of erythropoietin responsiveness in patients with chronic kidney disease 
Chronic kidney disease (CKD) is commonly accompanied by inflammation and anemia; however, the pathogenesis of CKD is unclear. Expression of resistin, a cysteine-rich secretory plasma protein, is correlated with the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and lipoprotein-associated phospholipase A2, indicating that resistin may be involved in inflammatory events. In addition, inflammation inhibits the activity of erythropoietin (EPO) and, thus, erythropoiesis. The aim of the present study was to analyze the correlation between serum resistin and the variability of EPO responsiveness in CKD patients. The levels of serum creatinine (SCr), C-reactive protein (CRP), total cholesterol, triglycerides, IL-6 and serum resistin were measured in the samples obtained from 138 CKD patients and healthy control subjects. The levels of serum resistin in the CKD groups with and without hemodialysis were significantly higher than those observed in the normal control group (P<0.01) and the levels of serum resistin in the hemodialysis CKD group were higher than those observed in the CKD group without dialysis (P<0.01). The levels of serum resistin in patients in the randomly selected CKD group (with hemodialysis) were positively correlated with the duration of dialysis and the levels of SCr and CRP (P<0.05), however, were negatively correlated with the estimated glomerular filtration rate. The EPO resistance index (ERI) was identified to be associated with body mass index and the levels of CRP and resistin; furthermore, EPO reactivity was correlated with the level of resistin and ERI. The levels of serum resistin were correlated with the variability in EPO responsiveness that was observed in the CKD patients. Therefore, the measurement of serum resistin may aid with understanding the mechanisms, clinical diagnosis and treatment of CKD.
PMCID: PMC4665122  PMID: 26640574
chronic kidney disease; resistin; inflammation; anemia; erythropoietin; erythropoietin resistance index
6.  Association between D-dimer level and portal venous system thrombosis in liver cirrhosis: a retrospective observational study 
Objective: This study aimed to explore the association between D-dimer levels and presence of portal venous system thrombosis (PVST) in liver cirrhosis. Methods: All consecutive patients with a diagnosis of liver cirrhosis who underwent D-dimer test were retrospectively enrolled. Normal reference range of D-dimer level was 0-0.3 µg/mL. PVST was diagnosed on the basis of contrast-enhanced computed tomography and/or magnetic resonance imaging scans. Results: Of the 66 included patients, 24 were diagnosed with PVST. Mean D-dimer level was 0.51±0.72 µg/mL (range: 0.10-3.44). Mean D-dimer level was not significantly different between PVST and non-PVST groups (0.68±0.93 µg/mL versus 0.41±0.56 µg/mL, P=0.146). Area under the receiver operating curve for D-dimer level for predicting the presence of PVT was 0.606 (95% confidence interval: 0.478-0.724, P=0.1393). The optimal cut-off value for D-dimer was 0.22 with a sensitivity of 58.3% and a specificity of 69.0%. The subgroup analyses of patients without splenectomy or those with different Child-Pugh classes demonstrated no significant difference in the D-dimer level between PVST and non-PVST groups. Conclusion: D-dimer might not be useful to identify the presence of PVST in liver cirrhosis. However, given the retrospective nature of this study, further well-designed prospective study should be necessary to confirm this finding.
PMCID: PMC4658906  PMID: 26629017
Portal vein thrombosis; liver cirrhosis; D-dimer; fibrinolysis; pathogenesis
7.  Correlation of serum liver fibrosis markers with severity of liver dysfunction in liver cirrhosis: a retrospective cross-sectional study 
Hyaluronic acid (HA), laminin (LN), amino-terminal pro-peptide of type III pro-collagen (PIIINP), and collagen IV (CIV) are four major serum markers of liver fibrosis. This retrospective cross-sectional study aimed to evaluate the correlations of the four serum markers with the severity of liver dysfunction in cirrhotic patients. Between January 2013 and June 2014, a total of 228 patients with a clinical diagnosis with liver cirrhosis and without malignancy underwent the tests of HA, LN, PIIINP, and CIV levels. Laboratory data were collected. Child-Pugh and model for the end-stage of liver diseases (MELD) scores were calculated. Of them, 32%, 40%, and 18% had Child-Pugh class A, B, and C, respectively. MELD score was 7.58±0.50. HA (coefficient r: 0.1612, P=0.0203), LN (coefficient r: 0.2445, P=0.0004), and CIV (coefficient r: 0.2361, P=0.0006) levels significantly correlated with Child-Pugh score, but not PIIINP level. Additionally, LN (coefficient r: 0.2588, P=0.0002) and CIV (coefficient r: 0.1795, P=0.0108) levels significantly correlated with MELD score, but not HA or PIIINP level. In conclusions, HA, LN, and CIV levels might be positively associated with the severity of liver dysfunction in cirrhotic patients. However, given a relatively weak correlation between them, our findings should be cautiously interpreted and further validated.
PMCID: PMC4483917  PMID: 26131195
Liver; fibrosis; cirrhosis; Child-Pugh; MELD
8.  Shikonin induces apoptosis in the human gastric cancer cells HGC-27 through mitochondria-mediated pathway 
Pharmacognosy Magazine  2015;11(42):250-256.
Gastric cancer (GC) is one of the most frequently occurring digestive tract cancers and fewer chemotherapeutic drugs for GC have shown promising results. In this study, we investigated the anti-tumor activity of shikonin, a natural compound isolated from the Chinese plant Lithospermum erythrorhizon, against the human GC cell line HGC-27.
Materials and Methods:
HGC-27 cells treated with shikonin at a concentration of 30μM or above showed significant growth inhibition compared to control cells. Shikonin-treated cells also underwent apoptosis as detected by flow cytometric analysis and microscopic examination of cellular morphology. Further investigation into the underlying mechanism of apoptosis by western blot showed that the shikonin promoted the activation of poly-(ADP-ribose)-polymerase, caspase-3 and caspase-9 following 24 h or 48 h of treatment time, as well as the activation of caspase-8, but only after 48 h of treatment time. Furthermore, the levels of mitochondrial membrane potential, B-cell lymphoma 2 (Bcl-2) and Bcl-extra large were reduced following shikonin treatment while the level of Bax was increased. In addition, shikonin also caused a significant reduction of the protein Survivin, while having little effect on the expression on X-linked inhibitor of apoptosis protein.
Taken together, these results showed that the shikonin exhibited its anti-tumor activity against HGC-27 cells through inhibiting cell growth and promoting apoptosis by targeting mitochondrial-related signaling pathway. Our finding may represent a positive step in finding a natural and effective compound that could be important implication for future development of chemotherapeutic and/or chemopreventive agent against GC.
PMCID: PMC4378121  PMID: 25829762
Apoptosis; caspases; HGC-27 cells; mitochondrial pathway; shikonin
9.  The expression and functional evidence for voltage-dependent potassium channel Kv1.3 in lymphocytes during aging in spontaneously hypertensive rats 
Aims: Our previous studies showed that expression and functional profile of voltage-dependent potassium channels Kv1.3 were increased in lymphocytes of spontaneously hypertensive rats (SHR) compared to normotensive rats, suggesting a crucial role for lymphocyte Kv1.3 in the development of hypertension. Here, we further investigated whether the expression and functional profile of Kv1.3 was related to increased blood pressure in SHR with age of 4, 8, 16 and 24 wk. Methods: Systolic blood pressure was measured through pressure device around the tail. mRNA and protein expression were assessed by real-time PCR and western blot in lymphocytes of SHR. Current density of Kv channels in lymphocytes was measured by patch-clamp. Results: Systolic blood pressure was elevated in an age-dependent manner (ANOVA P < 0.05). mRNA and protein level of Kv1.3 were significantly increased in an age-dependent manner in lymphocyte of SHR (ANOVA P < 0.05). Moreover, the current density of Kv was dramatically enhanced in an age-dependent manner (ANOVA P < 0.05). Conclusion: The systolic blood pressure positively correlated with expression as well as current density of potassium channels in lymphocytes of SHR at age of 8, 16 and 24 wk. In conclusion, Kv1.3 channels were upregulated in an age-dependent manner in SHR and correlates with systolic blood pressure during aging. The present study implies that Kv1.3 blockers may be applied as a therapeutic treatment for the development of hypertension during aging.
PMCID: PMC4402843  PMID: 25932196
Spontaneously hypertensive rat; lymphocyte; Kv1.3; blood pressure; aging
10.  Transesophageal Echocardiography for Coronary Sinus Imaging in Partially Unroofed Coronary Sinus 
Partially unroofed coronary sinus (PUCS) is a rare congenital cardiac anomaly and prone to be misdiagnosed. The purpose of this study was to explore the value of transesophageal echocardiography (TEE) in CS imaging for the detection of PUCS and to develop a special two-dimensional TEE-based en face view of CS.
Twenty adult patients with suspected PUCS, showing a dilated coronary sinus and an enlarged right heart on transthoracic echocardiography (TTE), underwent TEE examination. In the mid-esophageal plane and close to an angle of 120°, the en face view of the CS successfully imaged the roof of the CS, which was beyond the realm of the atrial septum, and the interatrial septum was obtained simultaneously in the same view. Meanwhile, the 3D zoom mode could clearly display the comprehensive volume image and the adjacent structures of the PUCS. The results of TEE were compared with the findings of surgery or catheterization.
En face view of the CS was obtained successfully by 2DTEE in 20 patients. In addition, 3DTEE was used for imaging of PUCS in 11 of the 20 patients. PUCS was ultimately confirmed in 13 patients either by surgery or catheterization. The TEE for PUCS diagnosis was consistent with the surgical findings.
Transesophageal echocardiography can be successfully applied to obtain the comprehensive view of CS and its surrounding structures. The en face view of CS provided by 2DTEE may be helpful in better understanding PUCS and discriminating it from associated atrial septal defects.
PMCID: PMC4232913  PMID: 23909710
partially unroofed coronary sinus; transesophageal echocardiography; en face view
11.  New Sesquiterpene and Polymethoxy-Flavonoids from Artemisia annua L 
Pharmacognosy Magazine  2014;10(39):213-216.
Our previous study revealed that the polymethoxy-flavonoids, as main components of Artemisia annua, could improve the antimalarial activity of Artemisinin. Here, we described the isolation, elucidation, constituent analysis, flavonoids enrichment of the extracts of A. annua. A total of 20 compounds were isolated including a new sesquiterpene (compound 12) and five (1, 5, 6, 7, 15) afforded for the first time from A. annua. The elucidation of eight flavonoids may be a useful phytochemical data and chemical foundation for further mechanism studies on improving the anti-malarial action of artemisinin. Furthermore, the antitumor activities of the compounds were assayed using four different kinds of human cancer cell lines.
PMCID: PMC4159912  PMID: 25210306
Antitumor activities; artemisinin; polymethoxy-flavonoid; sesquiterpene
12.  Predicting response to bacillus Calmette-Guérin (BCG) in patients with carcinoma in situ of the bladder 
Urologic oncology  2013;32(1):45.e23-45.e30.
Currently, there is no reliable tool to predict response to intravesical bacillus Calmette-Guérin (BCG). Based on the fact that BCG is a Th1-polarizing immunotherapy, we attempt to correlate the pretreatment immunologic tumor microenvironment (Th1 or Th2) with response to therapy.
Materials and methods
Bladder cancer patients with initial diagnosis of carcinoma in situ (Tis) were stratified based on their response to BCG treatment. A total of 38 patients met inclusion criteria (20 patients who responded and 18 patients who did not respond). Immunohistochemical (IHC) methods known to assess the type of immunologic microenvironment (Th1 vs. Th2) were performed on tumor tissue obtained at initial biopsy/resection: the level of tumor eosinophil infiltration and degranulation (Th2 response); the number of tumor-infiltrating GATA-3+ (Th2-polarized) lymphocytes; and the number of tumor-infiltrating T-bet+ (Th1-polarized) lymphocytes. Results obtained from these metrics were correlated with response to treatment with BCG immunotherapy.
The IHC metrics of the tumor immune microenvironment prior to BCG treatment were each statistically significant predictors of responders (R) vs. nonresponders (NR). Eosinophil infiltration and degranulation was higher for R vs. NR: 1.02±0.17 vs. 0.5±0.12 (P = 0.01) and 1.1±0.15 vs. 0.56±0.15 (P = 0.04), respectively. Ratio of GATA-3+ (Th2-polarized) lymphocytes to T-bet+ (Th1-polarized) lymphocytes was higher for R vs. NR: 4.85±0.94 vs. 0.98±0.19 (P<0.001). The 3 markers were combined to create a Th2 signature biomarker, which was a statistically significant (P<0.0001) predictor of R vs. NR. All IHC markers demonstrated that a preexisting Th1 immunologic environment within the tumor was predictive of BCG failure.
The Th1 vs. Th2 polarization of bladder tumor immune microenvironment prior to treatment with BCG represents a prognostic metric of response to therapy. If a patient has a preexisting Th1 immunologic response within the tumor, there is no value in using a therapy intended to create a Th1 immunologic response. An algorithm integrating 3 IHC methods provided a sensitive and specific technique that may become a useful tool for pathologists and urologists to predict response to BCG in patients with carcinoma in situ of the bladder.
PMCID: PMC3953142  PMID: 24055426
Eosinophils; Tumor immune microenvironment; GATA-3; T-bet; Degranulation; Bladder cancer
13.  Neuroprotective Effect of Pseudoginsenoside-F11 on a Rat Model of Parkinson's Disease Induced by 6-Hydroxydopamine 
Pseudoginsenoside-F11 (PF11), a component of Panax quinquefolism (American ginseng), plays a lot of beneficial effects on disorders of central nervous system. In this paper, the neuroprotective effect of PF11 on Parkinson's disease (PD) and the possible mechanism were investigated in a rat PD model. PF11 was orally administered at 3, 6, and 12 mg/kg once daily for a period of 2 weeks before and 1 week after the unilateral lesion of left medial forebrain bundle (MFB) induced by 6-hydroxydopamine (6-OHDA). The results showed that PF11 markedly improved the locomotor, motor balance, coordination, and apomorphine-induced rotations in 6-OHDA-lesioned rats. The expression of tyrosine hydroxylase (TH) in substantia nigra (SN) and the content of extracellular dopamine (DA) in striatum were also significantly increased after PF11 treatment. Moreover, significant reduction in the levels of striatal extracellular hydroxyl radical (∙OH), detected as 2,3- and 2,5-dihydroxy benzoic acid (2,3- and 2,5-DHBA), and increase in the level of striatal extracellular ascorbic acid (AA) were observed in the PF11-treated groups compared with 6-OHDA-lesioned rats. Taken together, we propose that PF11 has potent anti-Parkinson property possibly through inhibiting free radical formation and stimulating endogenous antioxidant release.
PMCID: PMC3872412  PMID: 24386001
14.  Glutamine residues in Q-loops of multidrug resistance protein MRP1 contribute to ATP binding via interaction with metal cofactor 
Biochimica et biophysica acta  2011;1808(7):1790-1796.
Structural analyses of bacterial ATP-binding-cassette transporters revealed that the glutamine residue in Q-loop plays roles in interacting with: 1) a metal cofactor to participate in ATP binding; 2) a putative catalytic water molecule to participate in ATP hydrolysis; 3) other residues to transmit the conformational changes between nucleotide-binding-domains and transmembrane-domains, in ATP-dependent solute transport. We have mutated the glutamines at 713 and 1375 to asparagine, methionine or leucine to determine the functional roles of these residues in Q-loops of MRP1. All these single mutants significantly decreased Mg·ATP binding and increased the Km (Mg·ATP) and Vmax values in Mg·ATP-dependent leukotriene-C4 transport. However, the Vmax values of the double mutants Q713N/Q1375N, Q713M/Q1375M and Q713L/Q1375L were lower than that of wtMRP1, implying that the double mutants cannot efficiently bind Mg·ATP. Interestingly, MRP1 has higher affinity for Mn·ATP than for Mg·ATP and the Mn·ATP-dependent leukotriene-C4 transport activities of Q713N/Q1375N and Q713M/Q1375M are significantly higher than that of wtMRP1. All these results suggest that: 1) the glutamine residues in Q-loops contribute to ATP-binding via interaction with a metal cofactor; 2) it is most unlikely that these glutamine residues would play crucial roles in ATP hydrolysis and in transmitting the conformational changes between nucleotide-binding-domains and transmembrane-domains.
PMCID: PMC3091947  PMID: 21315686
15.  Inflatable hollow obturator prostheses for patients undergoing an extensive maxillectomy: a case report 
The presence of a large palatal or maxillary defect after partial or total maxillectomy for tumor, trauma or congenital deformation poses a challenge to prosthodontists, particularly when the use of an implant cannot be considered. This case report described the use of an air valve in a hollow silicone obturator to manufacture an inflatable obturator that could be extended further into undercut area to retain itself. The inflatable obturator exhibited adequate retention, stability and border sealing, thereby improving the masticatory, pronunciation and swallowing functions of patients. It may be a suitable alternative treatment option to an implant-retained obturator.
PMCID: PMC3421482  PMID: 22653550
inflatable; maxillectomy; maxillofacial defects; obturator; silicone
16.  Differences in adhesion and protrusion properties correlate with differences in migration speed under EGF stimulation 
BMC Biophysics  2012;5:8.
Cell migration plays an essential role in many biological processes, such as cancer metastasis, wound healing and immune response. Cell migration is mediated through protrusion and focal adhesion (FA) assembly, maturation and disassembly. Epidermal growth factor (EGF) is known to enhance migration rate in many cell types; however it is not known how FA maturation, FA dynamics and protrusion dynamics are regulated during EGF-induced migration. Here we use total internal reflection fluorescence (TIRF) microscopy and image analysis to quantify FA properties and protrusion dynamics under different doses of EGF stimulation.
EGF was found to broaden the distribution of cell migration rates, generating more fast and slow cells. Furthermore, groups based on EGF stimulation condition or cell migration speed were marked by characteristic signatures. When data was binned based on EGF stimulation conditions, FA intensity and FA number per cell showed the largest difference among stimulation groups. FA intensity decreased with increasing EGF concentration and FA number per cell was highest under intermediate stimulation conditions. No difference in protrusion behavior was observed. However, when data was binned based on cell migration speed, FA intensity and not FA number per cell showed the largest difference among groups. FA intensity was lower for fast migrating cells. Additionally, waves of protrusion tended to correlate with fast migrating cells.
Only a portion of the FA properties and protrusion dynamics that correlate with migration speed, correlate with EGF stimulation condition. Those that do not correlate with EGF stimulation condition constitute the most sensitive output for identifying why cells respond differently to EGF. The idea that EGF can both increase and decrease the migration speed of individual cells in a population has particular relevance to cancer metastasis where the microenvironment can select subpopulations based on some adhesion and protrusion characteristics, leading to a more invasive phenotype as would be seen if all cells responded like an “average” cell.
PMCID: PMC3414788  PMID: 22577847
17.  Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression 
Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used Taqman real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method, and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors.
PMCID: PMC3110064  PMID: 17882484
18.  Effects of Putative Catalytic Base Mutation E211Q on ABCG2-Mediated Methotrexate Transport 
Biochemistry  2009;48(38):9122-9131.
ABCG2 is a half ATP binding cassette (ABC) drug transporter that consists of a nucleotide binding domain (NBD) followed by a trans-membrane domain. This half ABC transporter is thought to form a homodimer in the plasma membrane where it transports anticancer drugs across the biological membranes in an ATP-dependent manner. Substitution of the putative catalytic residue E211 with a non-acidic amino acid glutamine (E211Q) completely abolished its ATPase activity and ATP-dependent methotrexate transport, suggesting that ATP hydrolysis is required for the ATP-dependent solute transport. However, whether one ATP hydrolysis or two ATP hydrolyses in the homodimer of ABCG2 with NBD·ATP·ATP·NBD sandwich structure is/are required for the ATP-dependent solute transport is not known yet. To address this question, we have made an YFP/ABCG2 fusion protein and expressed this 99 kDa fusion protein alone or along with the 70 kDa E211Q-mutated ABCG2 in BHK cells. Although membrane vesicles prepared from BHK cells expressing YFP/ABCG2 exert higher ATPase activity than that of wt ABCG2, the dATP-dependent methotrexate transport activities of these two proteins are the same. Interestingly, membrane vesicles prepared from BHK cells expressing both YFP/ABCG2 and E211Q-mutated ABCG2 (with a ratio of 1:1) form homodimers and heterodimer and exert 55% of wtABCG2 ATPase activity that can be further enhanced by anticancer drugs, suggesting that the wt NBD in the heterodimer of YFP/ABCG2 and E211Q may be able to hydrolyze ATP. Furthermore, the membrane vesicles containing both YFP/ABCG2 and E211Q exert ∼ 79% of wtABCG2-mediated methotrexate transport activity, implying that the heterodimer harboring YFP/ABCG2 and E211Q may be able to transport the anticancer drug methotrexate across the biological membranes.
PMCID: PMC2770347  PMID: 19691360
ABCG2; NBD; homodimers and heterodimer; ATPase; MTX; ATP-dependent MTX transport
19.  Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator 
Journal of cell science  2008;121(Pt 17):2814-2823.
The epithelial chloride channel CFTR is a glycoprotein that is modified by two N-linked oligosaccharides. The most common mutant CFTR protein in patients with cystic fibrosis, ΔF508, is misfolded and retained by ER quality control. As oligosaccharide moieties of glycoproteins are known to mediate interactions with ER lectin chaperones, we investigated the role of N-linked glycosylation in the processing of wild-type and ΔF508 CFTR. We found that N-glycosylation and ER lectin interactions are not major determinants of trafficking of wild-type and ΔF508 from the ER to the plasma membrane. Unglycosylated CFTR, generated by removal of glycosylation sites or treatment of cells with the N-glycosylation inhibitor tunicamycin, did not bind calnexin, but did traffic to the cell surface and exhibited chloride channel activity. Most importantly, unglycosylated Δ F508 CFTR still could not escape quality control in the early secretory pathway and remained associated with the ER. However, the absence of N-linked oligosaccharides did reduce the stability of wild-type CFTR, causing significantly more-rapid turnover in post-ER compartments. Surprisingly, the individual N-linked carbohydrates do not play equivalent roles and modulate the fate of the wild-type protein in different ways in its early biosynthetic pathway.
PMCID: PMC2677381  PMID: 18682497
CFTR; Glycosylation; Glycoprotein; Processing; Calnexin; EDEM
20.  Permeability of Wild-Type and Mutant Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels to Polyatomic Anions  
The Journal of General Physiology  1997;110(4):355-364.
Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (PX/PCl) sequence NO3− > Cl− > HCO3− > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (PX/PCl < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of ∼5.3 Å. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.
PMCID: PMC2229373  PMID: 9379168
pore size; channel selectivity; anion permeation; lyotropic sequence; cystic fibrosis

Results 1-20 (20)