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Articles by J Liu
Total Records ( 39 ) for J Liu
  C Peng , W Chen , Y Han , T Sanders , G Chew , J Liu , E Hawrot , C Chi and C. Wang
 

As part of continuing studies of the identification of gene organization and cloning of novel -conotoxins, the first 4/4-conotoxin identified in a vermivorous Conus species, designated Qc1.2, was originally obtained by cDNA and genomic DNA cloning from Conus quercinus collected in the South China Sea. The predicted mature toxin of Qc1.2 contains 14 amino acid residues with two disulfide bonds (I-III, II-IV connectivity) in a native globular configuration. The mature peptide of Qc1.2 is supposed to contain an N-terminal post-translationally processed pyroglutamate residue and a free carboxyl C-terminus. This peptide was chemically synthesized and refolded for further characterization of its functional properties. The synthetic Qc1.2 has two interconvertible conformations in aqueous solution, which may be due to the cis-trans isomerization of the two successive Pro residues in its first Cys loop. Using the Xenopus oocyte heterologous expression system, Qc1.2 was shown to selectively inhibit both rat neuronal 3β2 and 3β4 subtypes of nicotinic acetylcholine receptors with low potency. A block of ~63% and 37% of the ACh-evoked currents was observed, respectively, and the toxin dissociated rapidly from the receptors. Compared with other characterized -conotoxin members, the unusual structural features in Qc1.2 that confer to its receptor recognition profile are addressed.

  P Jiang , J Liu , W Li , X Zeng and J. Tang
 

The objective of this study was to identify whether polymorphic variants of p53 at codon 72 and p21 at codon 31 were associated with increased risk for cervical cancer, either independently or jointly, among Chinese women from southern Han. We genotyped p53 codon 72 and p21 codon 31 polymorphisms of peripheral blood DNA from 104 cervical cancer patients and 160 controls. Genotyping was confirmed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) and direct DNA sequencing. We observed an increased risk of cervical cancer associated with the p53 Arg/Arg (OR, 2.25; 95% CI, 1.11–4.54) or p21 Ser/Ser (OR, 2.09; 95% CI, 1.04–4.19) genotype, compared with the p53 Pro/Pro or p21 Arg/Arg genotype, respectively. In additional, interaction between these p53 and p21 polymorphisms increased the risk of cervical cancer in a multiplicative manner, with the OR being 3.96 (95% CI, 1.51–10.41) for subjects carrying both p53 Arg/Arg and p21 Ser/Ser genotypes. These findings suggest that there is a significant association between the genetic polymorphism of p53, p21, and the risk of cervical cancer among Chinese southern women, and there is a possible gene–gene interaction in the incidence of cervical cancer.

  C Peng , W Chen , T Sanders , G Chew , J Liu , E Hawrot and C. Chi
 

-Conotoxins are small disulfide-constrained peptides that act as potent and selective antagonists on specific subtypes of nicotinic acetylcholine receptors (nAChRs). We previously cloned two -conotoxins, Mr1.1 from the molluscivorous Conus marmoreus and Lp1.4 from the vermivorous Conus leopardus. Both of them have the typical 4/7-type framework of the subfamily of -conotoxins that act on neuronal nAChRs. In this work, we chemically synthesized these two toxins and characterized their functional properties. The synthetic Mr1.1 could primarily inhibit acetylcholine (ACh)-evoked currents reversibly in the oocyte-expressed rat 7 nAChR, whereas Lp1.4 was an unexpected specific blocker of the mouse fetal muscle 1β1 receptor. Although their inhibition affinities were relatively low, their unique receptor recognition profiles make them valuable tools for toxin-receptor interaction studies. Mr1.1 could also suppress the inflammatory response to pain in vivo, suggesting that it should be further investigated with respect to its molecular role in analgesia and its mechanism or therapeutic target for the treatment of pain.

  N Ji , J Yuan , J Liu and S. Tian
 

Expression of breast cancer resistance protein/ATP-binding cassette sub-family G member 2 (BCRP/ABCG2) is the major cause of chemotherapy failure. It is important to establish and characterize the multidrug resistance cells and to investigate the mechanism of multidrug resistance. Multidrug-resistant cells expressing BCRP/ABCG2 based on human breast cancer MCF-7/wt cells were developed by gradually increasing application of low concentration of mitoxantrone. Real-time quantitative PCR, western blot, and immunofluorescence assay were employed to analyze BCRP mRNA and protein expression. Drug accumulation in the cells was measured by flow cytometry and DNA methyltransferases were analyzed by western blot. The results indicated that the inhibitory ratio of cell proliferative growth exhibited an exponential relation with the concentration of mitoxantrone. The IC50 of MCF-7/wt cells to mitoxantrone was found to be 0.42 µM. 3-(4,5-Dimethylthlthiazol-2-YI)-2,5-Diphenyltetrazolium Bromide assay indicated that the mitoxantrone-resistant cells at different stages exhibited cross-resistance to adriamycin and taxol. BCRP/ABCG2 mRNA and protein levels in the mitoxantrone-resistant cells at different stages increased with increasing concentration of mitoxantrone. Intracellular accumulation of mitoxantrone in the cells decreased with the increase of the BCRP/ABCG2 expression levels. DNA methyltransferase 1 (DNMT1) and DNA methyltransferase 3a (DNMT3a) expressions in the cells at different stages decreased slightly, whereas DNA methyltransferase 3b (DNMT3b) expression decreases significantly. BCRP/ABCG2 overexpression and its drug-efflux function in the drug-resistant cells are the main factors to produce multidrug resistance. Our results suggest that multidrug resistance is related to overexpression of BCRP/ABCG2 and the decrease of DNA methyltransferases, especially DNMT3b.

  Y Zhao , J Liu , Q Hong , C Yang , L Chen , Y Chen , Q Wang , K Zhao and W. Jin
 

Overexpression of multidrug resistance 1 (MDR1) in cancer remains one of the major causes for the failure of chemotherapy. In the present study, we found that MyoD and PEA3 could activate P-glycoprotein (P-gp) expression in SGC7901 cells. Knockdown of MyoD and PEA3 attenuated MDR1 expression and increased the sensitivity of multidrug resistant cancer cells to cytotoxic drugs that were transported by P-gp in SGC7901/VCR cells. MyoD or PEA3 could bind to the E-box and PEA3 sites on the MDR1 promoter and activate its transcription. The regulation of MDR1 expression by MyoD and PEA3 may provide potential ways to overcome MDR in cancer treatment.

  J Liu , X Wu , J. L Franklin , J. L Messina , H. S Hill , D. R Moellering , R. G Walton , M Martin and W. T. Garvey
 

Tribbles homolog 3 (TRIB3) was found to inhibit insulin-stimulated Akt phosphorylation and modulate gluconeogenesis in rodent liver. Currently, we examined a role for TRIB3 in skeletal muscle insulin resistance. Ten insulin-sensitive, ten insulin-resistant, and ten untreated type 2 diabetic (T2DM) patients were metabolically characterized by hyperinsulinemic euglycemic glucose clamps, and biopsies of vastus lateralis were obtained. Skeletal muscle samples were also collected from rodent models including streptozotocin (STZ)-induced diabetic rats, db/db mice, and Zucker fatty rats. Finally, L6 muscle cells were used to examine regulation of TRIB3 by glucose, and stable cell lines hyperexpressing TRIB3 were generated to identify mechanisms underlying TRIB3-induced insulin resistance. We found that 1) skeletal muscle TRIB3 protein levels are significantly elevated in T2DM patients; 2) muscle TRIB3 protein content is inversely correlated with glucose disposal rates and positively correlated with fasting glucose; 3) skeletal muscle TRIB3 protein levels are increased in STZ-diabetic rats, db/db mice, and Zucker fatty rats; 4) stable TRIB3 hyperexpression in muscle cells blocks insulin-stimulated glucose transport and glucose transporter 4 (GLUT4) translocation and impairs phosphorylation of Akt, ERK, and insulin receptor substrate-1 in insulin signal transduction; and 5) TRIB3 mRNA and protein levels are increased by high glucose concentrations, as well as by glucose deprivation in muscle cells. These data identify TRIB3 induction as a novel molecular mechanism in human insulin resistance and diabetes. TRIB3 acts as a nutrient sensor and could mediate the component of insulin resistance attributable to hyperglycemia (i.e., glucose toxicity) in diabetes.

  W Niu , P. J Bilan , S Ishikura , J. D Schertzer , A Contreras Ferrat , Z Fu , J Liu , S Boguslavsky , K. P Foley , Z Liu , J Li , G Chu , T Panakkezhum , G. D Lopaschuk , S Lavandero , Z Yao and A. Klip
 

Muscle contraction stimulates glucose uptake acutely to increase energy supply, but suitable cellular models that faithfully reproduce this complex phenomenon are lacking. To this end, we have developed a cellular model of contracting C2C12 myotubes overexpressing GLUT4 with an exofacial myc-epitope tag (GLUT4myc) and explored stimulation of GLUT4 traffic by physiologically relevant agents. Carbachol (an acetylcholine receptor agonist) induced a gain in cell surface GLUT4myc that was mediated by nicotinic acetylcholine receptors. Carbachol also activated AMPK, and this response was sensitive to the contractile myosin ATPase inhibitor N-benzyl-p-toluenesulfonamide. The gain in surface GLUT4myc elicited by carbachol or by the AMPK activator 5-amino-4-carboxamide-1 β-ribose was sensitive to chemical inhibition of AMPK activity by compound C and partially reduced by siRNA-mediated knockdown of AMPK catalytic subunits or LKB1. In addition, the carbachol-induced gain in cell surface GLUT4myc was partially sensitive to chelation of intracellular calcium with BAPTA-AM. However, the carbachol-induced gain in cell surface GLUT4myc was not sensitive to the CaMKK inhibitor STO-609 despite expression of both isoforms of this enzyme and a rise in cytosolic calcium by carbachol. Therefore, separate AMPK- and calcium-dependent signals contribute to mobilizing GLUT4 in response to carbachol, providing an in vitro cell model that recapitulates the two major signals whereby acute contraction regulates glucose uptake in skeletal muscle. This system will be ideal to further analyze the underlying molecular events of contraction-regulated GLUT4 traffic.

  L He , H Zeng , F Li , J Feng , S Liu , J Liu , J Yu , J Mao , T Hong , A. F Chen , X Wang and G. Wang
 

Hyperhomocysteinemia (HHcy) has been associated with impaired vascular endothelial function. Our previous study demonstrated significantly higher secretion of the chemokine monocyte chemoattractant protein-1 from monocytes in response to lipopolysaccharide in patients with HHcy. In the present study, we investigated whether coronary endothelial function was damaged in patients with chronic HHcy (plasma level of homocysteine >15 µmol/l) and, if so, whether this impaired endothelial function is induced by the uncoupling of endothelial nitric oxide synthase (eNOS). When tetrahydrobiopterin levels are inadequate, eNOS is no longer coupled to l-arginine oxidation, which results in reactive oxygen species rather than nitric oxide production, thereby inducing vascular endothelial dysfunction. The 71 participants were divided into two groups, control (n = 50) and HHcy (n = 21). Quantification of coronary flow velocity reserve (CFVR) was after rest and after adenosine administration done by noninvasive Doppler echocardiography. Plasma levels of nitric oxide and tetrahydrobiopterin were significantly lower in patients with HHcy than in controls (99.54 ± 32.23 vs. 119.50 ± 37.68 µmol/l and 1.43 ± 0.46 vs. 1.73 ± 0.56 pmol/ml, all P < 0.05). Furthermore, CFVR was significantly lower in the HHcy than the control group (2.76 ± 0.49 vs. 3.09 ± 0.52, P < 0.05). In addition, plasma level of homocysteine was negatively correlated with CFVR. Chronic HHcy may contribute to coronary artery disease by inducing dysfunction of the coronary artery endothelium. The uncoupling of eNOS induced by HHcy in patients with chronic HHcy may explain this adverse effect in part.

  L. J Sobel , R Bansal , T. V Maia , J Sanchez , L Mazzone , K Durkin , J Liu , X Hao , I Ivanov , A Miller , L. L Greenhill and B. S. Peterson
  Objective

Disturbances in the basal ganglia portions of cortico-striato-thalamo-cortical circuits likely contribute to the symptoms of attention deficit hyperactivity disorder (ADHD). The authors examined the morphologic features of the basal ganglia nuclei (caudate, putamen, and globus pallidus) in children with ADHD.

Method

A total of 104 individuals (combined-type ADHD patients: N=47; healthy comparison subjects: N=57), aged 7 to 18 years, were examined in a cross-sectional case-control study using anatomical magnetic resonance imaging. Conventional volumes and the surface morphology for the basal ganglia were measured.

Results

Overall volumes were significantly smaller only in the putamen. Analysis of the morphological surfaces revealed significant inward deformations in each of the three nuclei, localized primarily in portions of these nuclei that are components of limbic, associative, and sensorimotor pathways in the cortico-striato-thalamo-cortical circuits in which these nuclei reside. The more prominent these inward deformations were in the patient group, the more severe the ADHD symptoms. Surface analyses also demonstrated significant outward deformations of all basal ganglia nuclei in the ADHD children treated with stimulants compared with those ADHD youth who were untreated. These stimulant-associated enlargements were in locations similar to the reduced volumes detected in the ADHD group relative to the comparison group. The outward deformations associated with stimulant medications attenuated the statistical effects of the primary group comparisons.

Conclusions

These findings potentially represent evidence of anatomical dysregulation in the circuitry of the basal ganglia in children with ADHD and suggest that stimulants may normalize morphological features of the basal ganglia in children with the disorder.

  J Kim , J Liu , N Colabianchi and R. R. Pate
 

Objective  To test whether physical activity and sedentary behaviors are associated with perceived and structural neighborhood conditions.

Design  Secondary analysis of data from wave I (1994-1995) of the National Longitudinal Study of Adolescent Health in the United States.

Setting  In-home interviews of adolescents and their parents.

Participants  Thirteen thousand six hundred sixty-eight adolescents (6794 adolescent boys and 6874 adolescent girls; grades 7-12).

Main Exposures  Perceived neighborhood conditions (public nuisance, acquaintanceship, and informal control measured at both individual and census tract levels) and structural neighborhood characteristics (concentrated poverty, immigrant concentration, and residential stability at the census tract level).

Outcome Measures  Failure to meet moderate to vigorous physical activity (MVPA) guidelines (<5 bouts of MVPA per week) and excessive screen time (>2 hours per day).

Results  Individual-level perception of intermediate (adjusted odds ratio [OR], 0.76; 95% confidence interval [CI], 0.68-0.84) or high (OR, 0.56; 95% CI, 0.50-0.61) acquaintanceship and high informal control (OR, 0.90; 95% CI, 0.82-0.99) in the neighborhood were negatively associated with failure to meet the MVPA guidelines compared with lower levels. At the census tract level, high informal control (OR, 1.16; 95% CI, 1.04-1.29) and intermediate (OR, 1.13; 95% CI, 1.01-1.26) and high (OR, 1.22; 95% CI, 1.06-1.40) concentrated poverty compared with lower levels were also associated with failing to meet the MVPA guidelines (when adjusted for sociodemographic characteristics). Compared with the lowest levels, high public nuisance (OR, 1.19; 95% CI, 1.04-1.36) and immigrant concentration (OR, 1.18; 95% CI, 1.01-1.38) at the census tract level were associated with excessive screen time.

Conclusion  These findings suggest that both perceptual and structural neighborhood factors should be considered to understand adolescents' physical activity and sedentary behaviors.

  J Liu , H Zhang , Z Li , T. K Hailemariam , M Chakraborty , K Jiang , D Qiu , H. H Bui , D. A Peake , M. S Kuo , R Wadgaonkar , G Cao and X. C. Jiang
 

Background— It has been proposed that plasma sphingomyelin (SM) plays a very important role in plasma lipoprotein metabolism and atherosclerosis. Sphingomyelin synthase (SMS) is the last enzyme for SM de novo biosynthesis. Two SMS genes, SMS1 and SMS2, have been cloned and characterized.

Methods and Results— To evaluate the in vivo role of SMS2 in SM metabolism, we prepared SMS2 knockout (KO) and SMS2 liver-specific transgenic (LTg) mice and studied their plasma SM and lipoprotein metabolism. On a chow diet, SMS2 KO mice showed a significant decrease in plasma SM levels (25%, P<0.05), but no significant changes in total cholesterol, total phospholipids, or triglyceride, compared with wild-type (WT) littermates. On a high-fat diet, SMS2 KO mice showed a decrease in plasma SM levels (28%, P<0.01), whereas SMS2LTg mice showed a significant increase in those levels (29%, P<0.05), but no significant changes in other lipids, compared with WT littermates. Atherogenic lipoproteins from SMS2LTg mice displayed a significantly stronger tendency toward aggregation after mammalian sphingomyelinase treatment, compared with controls. Moreover, SMS2 deficiency significantly increased plasma apoE levels (2.0-fold, P<0.001), whereas liver-specific SMS2 overexpression significantly decreased those levels (1.8-fold, P<0.01). Finally, SMS2 KO mouse plasma promoted cholesterol efflux from macrophages, whereas SMS2LTg mouse plasma prevented it.

Conclusions— We therefore believe that regulation of liver SMS2 activity could become a promising treatment for atherosclerosis.

  J Liu , N Bandyopadhyay , S Ranka , M Baudis and T. Kahveci
 

Motivation: One of the mutational processes that has been monitored genome-wide is the occurrence of regional DNA copy number alterations (CNAs), which may lead to deletion or over-expression of tumor suppressors or oncogenes, respectively. Understanding the relationship between CNAs and different cancer types is a fundamental problem in cancer studies.

Results: This article develops an efficient method that can accurately model the progression of the cancer markers and reconstruct evolutionary relationship between multiple types of cancers using comparative genomic hybridization (CGH) data. Such modeling can lead to better understanding of the commonalities and differences between multiple cancer types and potential therapies. We have developed an automatic method to infer a graph model for the markers of multiple cancers from a large population of CGH data. Our method identifies highly related markers across different cancer types. It then builds a directed acyclic graph that shows the evolutionary history of these markers based on how common each marker is in different cancer types. We demonstrated the use of this model in determining the importance of markers in cancer evolution. We have also developed a new method to measure the evolutionary distance between different cancers based on their markers. This method employs the graph model we developed for the individual markers to measure the distance between pairs of cancers. We used this measure to create an evolutionary tree for multiple cancers. Our experiments on Progenetix database show that our markers are largely consistent to the reported hot-spot imbalances and most frequent imbalances. The results show that our distance measure can accurately reconstruct the evolutionary relationship between multiple cancer types.

  L Zhang , S. F Messner , J Liu and Y. A. Zhuo
 

Western research has investigated individual correlates of fear of crime with a primary focus on people's vulnerability. This vulnerability model examines the possible effects on fear of indicators of people's physical vulnerability (e.g. age and gender) and social vulnerability (e.g. income and education). As is well documented in the research on China, guanxi is a unique aspect of social capital in Chinese society. The present study argues that guanxi in the immediate neighbourhood is an important indicator of the social vulnerability of individuals in urban China. We accordingly hypothesize that residents who have strong neighbourhood guanxi are less likely to be fearful of crime. This hypothesis is assessed with data collected from a recent survey in the city of Tianjin, China. The results of multilevel analysis show that guanxi in the neighbourhood is a significant predictor of fear of crime in contemporary urban China when other important factors are controlled.

  V. V Levina , B Nolen , Y Su , A. K Godwin , D Fishman , J Liu , G Mor , L. G Maxwell , R. B Herberman , M. J Szczepanski , M. E Szajnik , E Gorelik and A. E. Lokshin
 

There is increasing evidence that prolactin (PRL), a hormone/cytokine, plays a role in breast, prostate, and colorectal cancers via local production or accumulation. Elevated levels of serum PRL in ovarian and endometrial cancers have been reported, indicating a potential role for PRL in endometrial and ovarian carcinogenesis. In this study, we show that serum PRL levels are significantly elevated in women with a strong family history of ovarian cancer. We show dramatically increased expression of PRL receptor in ovarian and endometrial tumors as well as in endometrial hyperplasia, signifying the importance of PRL signaling in malignant and premalignant conditions. PRL mRNA was expressed in ovarian and endometrial tumors, indicating the presence of an autocrine loop. PRL potently induced proliferation in several ovarian and endometrial cancer cell lines. Binding of PRL to its receptor was followed by rapid phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, mitogen-activated protein kinase/ERK kinase 1, signal transducer and activator of transcription 3, CREB, ATF-2, and p53 and activation of 37 transcription factors in ovarian and endometrial carcinoma cells. PRL also activated Ras oncogene in these cells. When human immortalized normal ovarian epithelial cells were chronically exposed to PRL, a malignant transformation occurred manifested by the acquired ability of transformed cells to form clones, grow in soft agar, and form tumors in severe combined immunodeficient-beige mice. Transformation efficiency was diminished by a Ras inhibitor, providing proof that PRL-induced transformation uses the Ras pathway. In summary, we present findings that indicate an important role for PRL in ovarian and endometrial tumorigenesis. PRL may represent a risk factor for ovarian and endometrial cancers. [Cancer Res 2009;69(12):5226–33]

  J Liu , C Huan , M Chakraborty , H Zhang , D Lu , M. S Kuo , G Cao and X. C. Jiang
 

Rationale: Sphingomyelin synthase (SMS)2 contributes to de novo sphingomyelin (SM) biosynthesis and plasma membrane SM levels. SMS2 deficiency in macrophages diminishes nuclear factor B and mitogen-activated protein kinase activation induced by inflammatory stimuli.

Objective: The effects of SMS2 deficiency on the development of atherosclerosis are investigated.

Methods and Results: We measured cholesterol efflux from macrophages of wild-type (WT) and SMS2 knockout (KO) mice. We transplanted SMS2 KO mouse bone marrow into low-density lipoprotein (LDL) receptor (LDLr) knockout mice (SMS2–/–->LDLr–/–), creating a mouse model of SMS2 deficiency in the macrophages. We found that SMS2 deficiency caused significant induction of cholesterol efflux in vitro and in vivo. Moreover, we found that SMS2 KO mice had less interleukin-6 and tumor necrosis factor in the circulation before and after endotoxin stimulation, compared with controls. More importantly, after 3 months on a western-type diet, SMS2–/–->LDLr–/– mice showed decreased atherosclerotic lesions in the aortic arch, root (57%, P<0.001), and the entire aorta (42%, P<0.01), compared with WT->LDLr–/– mice. Analysis of plaque morphology revealed that SMS2–/–->LDLr–/– mice had significantly less necrotic core area (71%, P<0.001), less macrophage content (37%, P<0.01), and more collagen content (35%, P<0.05) in atherosclerotic lesions. We also found that SMS2–/–->LDLr–/– mice had significantly lower free cholesterol and cholesteryl ester levels in the brachiocephalic artery than WT->LDLr–/– mice (33 and 52%, P<0.01 and P<0.001, respectively).

Conclusions: SMS2 deficiency in the macrophages reduces atherosclerosis in mice. Macrophage SMS2 is thus a potential therapeutic target for treatment of this disease.

  P Wang , J Liu , Y Li , S Wu , J Luo , H Yang , R Subbiah , J Chatham , O Zhelyabovska and Q. Yang
 

Rationale: Peroxisome proliferator-activated receptors (PPARs) (, , and /β) are nuclear hormone receptors and ligand-activated transcription factors that serve as key determinants of myocardial fatty acid metabolism. Long-term cardiomyocyte-restricted PPAR deficiency in mice leads to depressed myocardial fatty acid oxidation, bioenergetics, and premature death with lipotoxic cardiomyopathy.

Objective: To explore the essential role of PPAR in the adult heart.

Methods and Results: We investigated the consequences of inducible short-term PPAR knockout in the adult mouse heart. In addition to a substantial transcriptional downregulation of lipid metabolic proteins, short-term PPAR knockout in the adult mouse heart attenuated cardiac expression of both Cu/Zn superoxide dismutase and manganese superoxide dismutase, leading to increased oxidative damage to the heart. Moreover, expression of key mitochondrial biogenesis determinants such as PPAR coactivator-1 were substantially decreased in the short-term PPAR deficient heart, concomitant with a decreased mitochondrial DNA copy number. Rates of palmitate and glucose oxidation were markedly depressed in cardiomyocytes of PPAR knockout hearts. Consequently, PPAR deficiency in the adult heart led to depressed cardiac performance and cardiac hypertrophy.

Conclusions: PPAR is an essential regulator of cardiac mitochondrial protection and biogenesis and PPAR activation can be a potential therapeutic target for cardiac disorders.

  A Moran , D Gu , D Zhao , P Coxson , Y. C Wang , C. S Chen , J Liu , J Cheng , K Bibbins Domingo , Y. M Shen , J He and L. Goldman
 

Background— The relative effects of individual and combined risk factor trends on future cardiovascular disease in China have not been quantified in detail.

Methods and Results— Future risk factor trends in China were projected based on prior trends. Cardiovascular disease (coronary heart disease and stroke) in adults ages 35 to 84 years was projected from 2010 to 2030 using the Coronary Heart Disease Policy Model–China, a Markov computer simulation model. With risk factor levels held constant, projected annual cardiovascular events increased by >50% between 2010 and 2030 based on population aging and growth alone. Projected trends in blood pressure, total cholesterol, diabetes (increases), and active smoking (decline) would increase annual cardiovascular disease events by an additional 23%, an increase of approximately 21.3 million cardiovascular events and 7.7 million cardiovascular deaths over 2010 to 2030. Aggressively reducing active smoking in Chinese men to 20% prevalence in 2020 and 10% prevalence in 2030 or reducing mean systolic blood pressure by 3.8 mm Hg in men and women would counteract adverse trends in other risk factors by preventing cardiovascular events and 2.9 to 5.7 million total deaths over 2 decades.

Conclusions— Aging and population growth will increase cardiovascular disease by more than a half over the coming 20 years, and projected unfavorable trends in blood pressure, total cholesterol, diabetes, and body mass index may accelerate the epidemic. National policy aimed at controlling blood pressure, smoking, and other risk factors would counteract the expected future cardiovascular disease epidemic in China.

  M. A Merlin , J Krimmel , J Liu and A. Marques Baptista
  Objectives

The objective of this study was to survey medical students for a measurable opinion or knowledge increase in prehospital care after a fourth-year clerkship in emergency medicine (EM). The goal of the mandatory prehospital care aspect of the clerkship was twofold: to diminish the prehospital knowledge gap in medical school by teaching students about prehospital protocols and disaster medicine and to increase student interest.

Methods

The study setting was within a university-based academic EM department with a prehospital system of 250 prehospital personnel. Data were collected from two similar questionnaires administered pre- and post-rotation to 49 fourth-year medical students. Statistical analyses were applied to collected data to quantify the changes of opinion and knowledge. Questions used a Likert five-point Scale.

Results

The data verified the improvement of students' knowledge in multiple areas of assessment. Greater than 35% opinion change (two points on the Likert Scale) was found in areas of prehospital care, 911 dispatch and education differences in prehospital providers (all p<0.0001; 95% CI 0.90 to 1.02, 0.66 to 0.90 and 0.66 to 0.90, respectively). Greater than 35% opinion change was also found in understanding triage (p=0.03; 95% CI 0.29 to 0.58) and general teaching of prehospital care, fellowship opportunities and use of a monitor/defibrillator (p<0.0001, p<0.0001 and p=0.04, respectively).

Conclusions

We found medical students developed a significantly improved understanding of prehospital care. Without extraordinary effort, academic emergency departments could easily include a significant experience and education within fourth-year EM clerkships.

  J Liu , E. S Park and M. Jo
 

Runt-related transcription factor 1 (RUNX1), a transcription factor, is transiently induced by the LH surge and regulates gene expression in periovulatory granulosa cells. Potential binding sites for RUNX are present in the 5'-flanking region of the Ptgs2 (prostaglandin-endoperoxide synthase 2) gene. Periovulatory Ptgs2 expression is essential for ovulation. In the present study, we investigated the role of RUNX1 in mediating the LH-induced expression of Ptgs2 in periovulatory granulosa cells. We first determined whether the suppression of Runx1 expression or activity affects Ptgs2 expression using cultured preovulatory granulosa cells isolated from immature rat ovaries primed with pregnant mare serum gonadotropin for 48 h. Knockdown of human chorionic gonadotropin-induced Runx1 expression by small interfering RNA or inhibition of endogenous RUNX activities by dominant-negative RUNX decreased human chorionic gonadotropin or agonist-stimulated Ptgs2 expression and transcriptional activity of Ptgs2 promoter reporter constructs. Results from chromatin immunoprecipitation assays revealed in vivo binding of endogenous RUNX1 to the Ptgs2 promoter region in rat periovulatory granulosa cells. Direct binding of RUNX1 to two RUNX-binding motifs in the Ptgs2 promoter region was confirmed by EMSA. The mutation of these two binding motifs resulted in decreased transcriptional activity of Ptgs2 promoter reporter constructs in preovulatory granulosa cells. Taken together, these findings provide experimental evidence that the LH-dependent induction of Ptgs2 expression results, in part, from RUNX1-mediated transactivation of the Ptgs2 promoter. The results of the present study assign potential significance for LH-induced RUNX1 in the ovulatory process via regulating Ptgs2 gene expression.

  R. L Milne , J Benitez , H Nevanlinna , T Heikkinen , K Aittomaki , C Blomqvist , J. I Arias , M. P Zamora , B Burwinkel , C. R Bartram , A Meindl , R. K Schmutzler , A Cox , I Brock , G Elliott , M. W. R Reed , M. C Southey , L Smith , A. B Spurdle , J. L Hopper , F. J Couch , J. E Olson , X Wang , Z Fredericksen , P Schurmann , M Bremer , P Hillemanns , T Dork , P Devilee , C. J van Asperen , R. A. E. M Tollenaar , C Seynaeve , P Hall , K Czene , J Liu , Y Li , S Ahmed , A. M Dunning , M Maranian , P. D. P Pharoah , G Chenevix Trench , J Beesley , kConFab Investigators , N. N Antonenkova , I. V Zalutsky , H Anton Culver , A Ziogas , H Brauch , C Justenhoven , Y. D Ko , S Haas , P. A Fasching , R Strick , A. B Ekici , M. W Beckmann , G. G Giles , G Severi , L Baglietto , D. R English , O Fletcher , N Johnson , I dos Santos Silva , J Peto , C Turnbull , S Hines , A Renwick , N Rahman , B. G Nordestgaard , S. E Bojesen , H Flyger , D Kang , K. Y Yoo , D. Y Noh , A Mannermaa , V Kataja , V. M Kosma , M Garcia Closas , S Chanock , J Lissowska , L. A Brinton , J Chang Claude , S Wang Gohrke , C. Y Shen , H. C Wang , J. C Yu , S. T Chen , M Bermisheva , T Nikolaeva , E Khusnutdinova , M. K Humphreys , J Morrison , R Platte , D. F Easton and on behalf of the Breast Cancer Association Consortium
  Background

A recent genome-wide association study identified single-nucleotide polymorphism (SNP) 2q35-rs13387042 as a marker of susceptibility to estrogen receptor (ER)–positive breast cancer. We attempted to confirm this association using the Breast Cancer Association Consortium.

Methods

2q35-rs13387042 SNP was genotyped for 31 510 women with invasive breast cancer, 1101 women with ductal carcinoma in situ, and 35 969 female control subjects from 25 studies. Odds ratios (ORs) were estimated by logistic regression, adjusted for study. Heterogeneity in odds ratios by each of age, ethnicity, and study was assessed by fitting interaction terms. Heterogeneity by each of invasiveness, family history, bilaterality, and hormone receptor status was assessed by subclassifying case patients and applying polytomous logistic regression. All statistical tests were two-sided.

Results

We found strong evidence of association between rs13387042 and breast cancer in white women of European origin (per-allele OR = 1.12, 95% confidence interval [CI] = 1.09 to 1.15; Ptrend = 1.0 x 10–19). The odds ratio was lower than that previously reported (P = .02) and did not vary by age or ethnicity (all P ≥ .2). However, it was higher when the analysis was restricted to case patients who were selected for a strong family history (P = .02). An association was observed for both ER-positive (OR = 1.14, 95% CI = 1.10 to 1.17; P = 10–15) and ER-negative disease (OR = 1.10, 95% CI = 1.04 to 1.15; P = .0003) and both progesterone receptor (PR)–positive (OR = 1.15, 95% CI = 1.11 to 1.19; P = 5 x 10–14) and PR-negative disease (OR = 1.10, 95% CI = 1.06 to 1.15; P = .00002).

Conclusion

The rs13387042 is associated with both ER-positive and ER-negative breast cancer in European women.

  A. V Peterson , K. A Kealey , S. L Mann , P. M Marek , E. J Ludman , J Liu and J. B. Bricker
  Background

The Hutchinson Study of High School Smoking randomized trial was designed to rigorously evaluate a proactive, personalized telephone counseling intervention for adolescent smoking cessation.

Methods

Fifty randomly selected Washington State high schools were randomized to the experimental or control condition. High school junior smokers were proactively identified (N = 2151). Trained counselors delivered the motivational interviewing plus cognitive behavioral skills training telephone intervention to smokers in experimental schools during their senior year of high school. Participants were followed up, with 88.8% participation, to outcome ascertainment more than 1 year after random assignment. The main outcome was 6-months prolonged abstinence from smoking. All statistical tests were two-sided.

Results

The intervention increased the percentage who achieved 6-month prolonged smoking abstinence among all smokers (21.8% in the experimental condition vs 17.7% in the control condition, difference = 4.0%, 95% confidence interval [CI] = –0.2 to 8.1, P = .06) and in particular among daily smokers (10.1% vs 5.9%, difference = 4.1%, 95% CI = 0.8 to 7.1, P = .02). There was also generally strong evidence of intervention impact for 3-month, 1-month, and 7-day abstinence and duration since last cigarette (P = .09, .015, .01, and .03, respectively). The intervention effect was strongest among male daily smokers and among female less-than-daily smokers.

Conclusions

Proactive identification and recruitment of adolescents via public high schools can produce a high level of intervention reach; a personalized motivational interviewing plus cognitive behavioral skills training counseling intervention delivered by counselor-initiated telephone calls is effective in increasing teen smoking cessation; and both daily and less-than-daily teen smokers participate in and benefit from telephone-based smoking cessation intervention.

  A Golden , J Liu and O. Cohen Fix
  Andy Golden, Jun Liu, and Orna Cohen-Fix

The nuclear envelope (NE) is a dynamic structure, undergoing periods of growth, breakdown and reassembly during the cell cycle. In yeast, altering lipid synthesis by inactivating the yeast homolog of lipin, a phosphatidic acid phosphohydrolase, leads to disorganization of the peripheral ER and abnormal nuclear shape. These results suggest that lipid metabolism contributes to NE dynamics; however, since yeast undergo closed mitosis, the relevance of these observations to higher eukaryotes is unclear. In mammals, lipin has been implicated in adipose tissue differentiation, insulin resistance, lipid storage and obesity, but the underlying cellular defects caused by altering lipin levels are not known. Here, we identify the Caenorhabditis elegans lipin homolog (LPIN-1) and examine its affect on NE dynamics. We find that downregulating LPIN-1 by RNAi results in the appearance of membrane sheets and other abnormal structures in the peripheral ER. Moreover, lpin-1 RNAi causes defects in NE breakdown, abnormal chromosome...

  K Wang , D Tang , M Wang , J Lu , H Yu , J Liu , B Qian , Z Gong , X Wang , J Chen , M Gu and Z. Cheng
  Kejian Wang, Ding Tang, Mo Wang, Jufei Lu, Hengxiu Yu, Jiafan Liu, Baoxiang Qian, Zhiyun Gong, Xin Wang, Jianmin Chen, Minghong Gu, and Zhukuan Cheng

MER3, a ZMM protein, is required for the formation of crossovers in Saccharomyces cerevisiae and Arabidopsis. Here, MER3, the first identified ZMM gene in a monocot, is characterized by map-based cloning in rice (Oryza sativa). The null mutation of MER3 results in complete sterility without any vegetative defects. Cytological analyses show that chiasma frequency is reduced dramatically in mer3 mutants and the remaining chiasmata distribute randomly among different pollen mother cells, implying possible coexistence of two kinds of crossover in rice. Immunocytological analyses reveal that MER3 only exists as foci in prophase I meiocytes. In addition, MER3 does not colocalize with PAIR2 at the beginning of prophase I, but locates on one end of PAIR2 fragments at later stages, whereas MER3 foci merely locate on one end of REC8 fragments when signals start to be seen in early prophase I. The normal loading of PAIR2 and REC8 in mer3...

  X Zhang , Q Chen , J Feng , J Hou , F Yang , J Liu , Q Jiang and C. Zhang
 

Nedd1 is a new member of the -tubulin ring complex (TuRC) and targets the TuRC to the centrosomes for microtubule nucleation and spindle assembly in mitosis. Although its role is known, its functional regulation mechanism remains unclear. Here we report that the function of Nedd1 is regulated by Cdk1 and Plk1. During mitosis, Nedd1 is firstly phosphorylated at T550 by Cdk1, which creates a binding site for the polo-box domain of Plk1. Then, Nedd1 is further phosphorylated by Plk1 at four sites: T382, S397, S637 and S426. The sequential phosphorylation of Nedd1 by Cdk1 and Plk1 promotes its interaction with -tubulin for targeting the TuRC to the centrosome and is important for spindle formation. Knockdown of Plk1 by RNAi decreases Nedd1 phosphorylation and attenuates Nedd1 accumulation at the spindle pole and subsequent -tubulin recruitment at the spindle pole for microtubule nucleation. Taken together, we propose that the sequential phosphorylation...

  J Liu , Z. C Feng and J. Wu
 

Premature rupture of membranes (PROM) is a common pregnancy complication and is associated with significant risks of fetal and neonatal morbidity and mortality. However, there is a lack of data concerning PROM in China. We investigated the incidence rate and analysed the influence of PROM on fetal and neonatal health. This used data from five specialist hospitals and one general hospital in mainland China. The total number of deliveries and the number of those complicated by PROM were recorded between January 2003 and December 2007. The time from initiation of PROM until delivery (latent period), the volume of amniotic fluid at delivery, fetal conditions and neonatal clinical conditions were recorded. The results suggest that the incidence of PROM was 19.53% and it could influence various aspects of the health of fetuses and neonates, including platelet parameters, erythrocyte parameters, neonatal jaundice and myocardial injury.

  J Liu and Z. C. Feng
 

To explore whether or not the umbilical blood levels of cytokines can be used to indicate the adverse outcomes of hypoxic-ischemic encephalopathy (HIE) patients. Umbilical artery blood and peripheral venous blood samples were collected on the 1st, 3rd and 7th days after birth to detect the levels of IL-1β, IL-8 and TNF-. Neurological examination and Denver developmental screening test (DDST-II) were performed at the 6 and 12 months evaluations to detect any neurodevelopmental abnormalities. The results showed: (i) the serum concentrations of IL-1β, IL-8 and TNF- in umbilical and peripheral blood were significantly higher in HIE patients than control groups; (ii) the umbilical blood concentrations of IL-1β exhibited the best positive correlation with HIE grades, when compared with IL-8 and TNF-; and (iii) abnormal neurological outcomes at 6 and 12 months of age were best predicted by umbilical levels of IL-1β. Thus, umbilical concentrations of IL-1β were associated with the grades and adverse outcomes of HIE.

  J Liu , C. D MacCalman , Y. l Wang and P. C. K. Leung
 

The potential roles of GnRH I and GnRH II have been assigned in promoting the invasive capacity of human trophoblasts by regulating matrix metalloproteinases-2 and -9, type I tissue inhibitor of matrix metalloproteinase, and urokinase plasminogen activator/plasminogen activator inhibitor protease systems during human placentation, and GnRH II has been shown to be more potent than GnRH I. However, the mechanisms for the differential effects of these two hormones remain unclear. In this study, we examined the invasion-promoting effects and the signaling pathways of GnRH I and GnRH II in human trophoblasts. The data revealed that both GnRH I and GnRH II were key autocrine and/or paracrine regulators in facilitating trophoblast invasion. The GnRH receptor antagonist (Antide) and specific small interfering RNA for GnRH receptor inhibited the regulatory effects of GnRH I, but not GnRH II, on trophoblast invasion. Both GnRH I and II activated protein kinase C, ERK1/2, and c-Jun N-terminal kinase to mediate their effects on trophoblast invasion, whereas only GnRH II elicited invasion-promoting action through transactivating the tyrosine kinase activity of epidermal growth factor receptor in trophoblasts. Our observations elucidate a ligand-dependent selective cross-communication between GnRH receptor and epidermal growth factor receptor signaling systems in human trophoblastic cell, and this would further our understanding on the differentially biological significance of these two forms of GnRH in extrapituitary tissues.

  J Ai , Y Wang , J. A Dar , J Liu , L Liu , J. B Nelson and Z. Wang
 

The development of castration-resistant prostate cancer (PCa) requires that under castration conditions, the androgen receptor (AR) remains active and thus nuclear. Heat shock protein 90 (Hsp90) plays a key role in androgen-induced and -independent nuclear localization and activation of AR. Histone deacetylase 6 (HDAC6) is implicated, but has not been proven, in regulating AR activity via modulating Hsp90 acetylation. Here, we report that knockdown of HDAC6 in C4-2 cells using short hairpin RNA impaired ligand-independent nuclear localization of endogenous AR and inhibited PSA expression and cell growth in the absence or presence of dihydrotestosterone (DHT). The dose-response curve of DHT-stimulated C4-2 colony formation was shifted by shHDAC6 such that approximately 10-fold higher concentration of DHT is required, indicating a requirement for HDAC6 in AR hypersensitivity. HDAC6 knockdown also inhibited C4-2 xenograft tumor establishment in castrated, but not in testes-intact, nude mice. Studies using HDAC6-deficient mouse embryonic fibroblasts cells showed that inhibition of AR nuclear localization by HDAC6 knockdown can be largely alleviated by expressing a deacetylation mimic Hsp90 mutant. Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation. Targeting HDAC6 alone or in combination with other therapeutic approaches is a promising new strategy for prevention and/or treatment of castration-resistant PCa.

  A. S Hancock , A Du , J Liu , M Miller and C. L. May
 

The major role of glucagon is to promote hepatic gluconeogenesis and glycogenolysis to raise blood glucose levels during hypoglycemic conditions. Several animal models have been established to examine the in vivo function of glucagon in the liver through attenuation of glucagon via glucagon receptor knockout animals and pharmacological interventions. To investigate the consequences of glucagon loss to hepatic glucose production and glucose homeostasis, we derived mice with a pancreas specific ablation of the -cell transcription factor, Arx, resulting in a complete loss of the glucagon-producing pancreatic -cell. Using this model, we found that glucagon is not required for the general health of mice but is essential for total hepatic glucose production. Our data clarifies the importance of glucagon during the regulation of fasting and postprandial glucose homeostasis.

  I Burstyn , N Kapur , C Shalapay , F Bamforth , T.C Wild , J Liu and D. LeGatt
  Introduction

Our main objective was to estimate smoking prevalence as well as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of self-reported smoking among pregnant women in Edmonton, Canada, at 15–16 weeks of gestation.

Methods

We used serum samples to assemble a cohort of pregnant women who underwent an optional second-trimester screening for chromosomal and developmental anomalies. We determined cotinine concentrations for 92 self-reported smokers (11% of the cohort) and for 285 self-reported nonsmoking mothers, using adapted urinary cotinine assay. Self-reports were collected at the time of delivery. In a validation study, serum cotinine was determined for known smokers and nonsmokers and used, within a Bayesian statistical framework, to define the distribution of cutoffs that differentiate true smokers from nonsmokers. This distribution of cutoffs was used to construct multiple two-by-two tables to obtain the distribution of sensitivity, specificity, PPV, NPV, and prevalence.

Results

Sensitivity was poor (M = 47.4%, SD = 17.3%), but specificity was nearly perfect (M = 94.9%, SD = 1.1%). PPV (M = 66.6%, SD = 11.7%) was smaller than NPV (M = 84.7%, SD = 14.3%). In our sample, the prevalence of true smoking at 15–16 weeks of gestation was described by a skewed distribution with a mean of 21.6% (SD = 13.8%) and a median of 16.6%.

Discussion

The strength of the present study includes blinding of subjects to the intention to test their sera for a biomarker of smoking. A limitation was the use of a nonrandom sample restricted to pregnancies that resulted in live births. We discuss data collection methods that would elicit more accurate smoking histories from pregnant women.

  J Liu , D. K Lieu , C. W Siu , J. D Fu , H. F Tse and R. A. Li
 

Cardiomyocytes (CMs) are nonregenerative. Self-renewable pluripotent human embryonic stem cells (hESCs) can differentiate into CMs for cell-based therapies. We recently reported that Ca2+ handling, crucial to excitation-contraction coupling of hESC-derived CMs (hESC-CMs), is functional but immature. Such immature properties as smaller cytosolic Ca2+ transient amplitudes, slower kinetics, and reduced Ca2+ content of sarcoplasmic reticulum (SR) can be attributed to the differential developmental expression profiles of specific Ca2+ handling and regulatory proteins in hESC-CMs and their adult counterparts. In particular, calsequestrin (CSQ), the most abundant, high-capacity but low-affinity, Ca2+-binding protein in the SR that is anchored to the ryanodine receptor, is robustly expressed in adult CMs but completely absent in hESC-CMs. Here we hypothesized that gene transfer of CSQ in hESC-CMs suffices to induce functional improvement of SR. Transduction of hESC-CMs by the recombinant adenovirus Ad-CMV-CSQ-IRES-GFP (Ad-CSQ) significantly increased the transient amplitude, upstroke velocity, and transient decay compared with the control Ad-CMV-GFP (Ad-GFP) and Ad-CMV-CSQ-IRES-GFP (Ad-CSQ, which mediated the expression of a nonfunctional, truncated version of CSQ) groups. Ad-CSQ increased the SR Ca2+ content but did not alter L-type Ca2+ current. Pharmacologically, untransduced wild-type, Ad-GFP-, Ad-CSQ-, and Ad-CSQ-transduced hESC-CMs behaved similarly. Whereas ryanodine significantly reduced the Ca2+ transient amplitude and slowed the upstroke, thapsigargin slowed the decay. Neither triadin nor junctin was affected. We conclude that CSQ expression in hESC-CMs facilitates Ca2+ handling maturation. Our results shed insights into the suitability of hESC-CMs for therapies and as certain heart disease models for drug screening.

  H. B Naik , M Beshire , B. M Walsh , J Liu and D. I. Soybel
 

Secretory compartments of neurons, endocrine cells, and exocrine glands are acidic and contain high levels of labile Zn2+. Previously, we reported evidence that acidity is regulated, in part, by the content of Zn2+ in the secretory [i.e., tubulovesicle (TV)] compartment of the acid-secreting gastric parietal cell. Here we report studies focusing on the mechanisms of Zn2+ transport by the TV compartment in the mammalian (rabbit) gastric parietal cell. Uptake of Zn2+ by isolated TV structures was monitored with a novel application of the fluorescent Zn2+ reporter N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ). Uptake was suppressed by removal of external ATP or blockade of H+-K+-ATPase that mediates luminal acid secretion. Uptake was diminished with dissipation of the proton gradient across the TV membrane, suggesting Zn2+/H+ antiport as the connection between Zn2+ uptake and acidity in the TV lumen. In isolated gastric glands loaded with the reporter fluozin-3, inhibition of H+-K+-ATPase arrested the flow of Zn2+ from the cytoplasm to the TV compartment and secretory stimulation with forskolin enhanced vectorial movement of cytoplasmic Zn2+ into the tubulovesicle/lumen (TV/L) compartment. Our findings suggest that Zn2+ accumulation in the TV/L compartment is physiologically coupled to secretion of acid. These findings offer novel insight into mechanisms regulating Zn2+ homeostasis in the gastric parietal cell and potentially other cells in which acidic subcellular compartments serve signature functional roles.

  Y Ge , A. L Wu , C Warnes , J Liu , C Zhang , H Kawasome , N Terada , M. D Boppart , C. J Schoenherr and J. Chen
 

Rapamycin-sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (mTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of mTOR kinase activity. However, the kinase-independent function of mTOR remains controversial, and no in vivo studies have examined those mTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of mTOR function, we have generated and characterized transgenic mice expressing two mutants of mTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR- and RR/KI-mTOR muscles, consistent with mTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of mTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, mTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively.

  T Vaithianathan , D Narayanan , M. T Asuncion Chin , L. H Jeyakumar , J Liu , S Fleischer , J. H Jaggar and A. M. Dopico
 

Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 ± 8, 334 ± 15, and 441 ± 27 pS in symmetric 300 mM Cs+ were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to ≤100 pS when Cs+ was replaced with Ca2+. The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NPo)-voltage curve was bell shaped, with NPo drastically decreasing when voltage was switched from –30 to –40 mV. NPo increased when intracellular calcium (Ca2+i) was raised within 0.1–100 µM to abruptly diminish with higher Ca2+i. Thus maximal activity occurred within the Ca2+i range found in rat cerebral artery myocytes under physiological conditions. NPo was reduced by ruthenium red (80 µM), increased monotonically by caffeine (0.1–5 mM) or ryanodine (0.05–5 µM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 µM), resistant to dantrolene (100 µM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.

  A. S Yu , B. A Hirayama , G Timbol , J Liu , E Basarah , V Kepe , N Satyamurthy , S. C Huang , E. M Wright and J. R. Barrio
 

This work provides evidence of previously unrecognized uptake of glucose via sodium-coupled glucose transporters (SGLTs) in specific regions of the brain. The current understanding of functional glucose utilization in brain is largely based on studies using positron emission tomography (PET) with the glucose tracer 2-deoxy-2-[F-18]fluoro-d-glucose (2-FDG). However, 2-FDG is only a good substrate for facilitated-glucose transporters (GLUTs), not for SGLTs. Thus, glucose accumulation measured by 2-FDG omits the role of SGLTs. We designed and synthesized two high-affinity tracers: one, -methyl-4-[F-18]fluoro-4-deoxy-d-glucopyranoside (Me-4FDG), is a highly specific SGLT substrate and not transported by GLUTs; the other one, 4-[F-18]fluoro-4-deoxy-d-glucose (4-FDG), is transported by both SGLTs and GLUTs and will pass through the blood brain barrier (BBB). In vitro Me-4FDG autoradiography was used to map the distribution of uptake by functional SGLTs in brain slices with a comparable result from in vitro 4-FDG autoradiography. Immunohistochemical assays showed that uptake was consistent with the distribution of SGLT protein. Ex vivo 4-FDG autoradiography showed that SGLTs in these areas are functionally active in the normal in vivo brain. The results establish that SGLTs are a normal part of the physiology of specific areas of the brain, including hippocampus, amygdala, hypothalamus, and cerebral cortices. 4-FDG PET imaging also established that this BBB-permeable SGLT tracer now offers a functional imaging approach in humans to assess regulation of SGLT activity in health and disease.

  J Liu , Y Song , B Tian , J Qian , Y Dong , B Liu and Z. Sun
 

It is well established that promyelocytic leukaemia nuclear bodies (PML NBs) play important roles in DNA damage responses (DDR). After irradiation, PML NBs dynamically recruit or release important proteins involved in cell-cycle regulation, DNA repair and apoptosis. As PML protein is the key molecule of PML NBs’ dynamic assembling, we aimed to characterize the PML-interacting proteins in 60Co-irradiated MCF-7 cells. A proteomic approach using CoIP, mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of 124 proteins that may associate with PML after irradiation. Bioinformatic analysis of the identified proteins showed that most of them were related to characterized PML functions, such as transcriptional regulation, cell-cycle regulation, cell-death regulation and response to stress. Four proteins, B23, MVP, G3BP1 and DHX9, were verified to co-localize with PML differentially before and after ionizing radiation (IR) treatment. The proteins identified in this study will significantly improve our understanding of the dynamic organization and multiple functions of PML NBs in DDR.

  C. S Rex , L. Y Chen , A Sharma , J Liu , A. H Babayan , C. M Gall and G. Lynch
 

The releasable factor adenosine blocks the formation of long-term potentiation (LTP). These experiments used this observation to uncover the synaptic processes that stabilize the potentiation effect. Brief adenosine infusion blocked stimulation-induced actin polymerization within dendritic spines along with LTP itself in control rat hippocampal slices but not in those pretreated with the actin filament stabilizer jasplakinolide. Adenosine also blocked activity-driven phosphorylation of synaptic cofilin but not of synaptic p21-activated kinase (PAK). A search for the upstream origins of these effects showed that adenosine suppressed RhoA activity but only modestly affected Rac and Cdc42. A RhoA kinase (ROCK) inhibitor reproduced adenosine's effects on cofilin phosphorylation, spine actin polymerization, and LTP, whereas a Rac inhibitor did not. However, inhibitors of Rac or PAK did prolong LTP's vulnerability to reversal by latrunculin, a toxin which blocks actin filament assembly. Thus, LTP induction initiates two synaptic signaling cascades: one (RhoA-ROCK-cofilin) leads to actin polymerization, whereas the other (Rac-PAK) stabilizes the newly formed filaments.

  Z Zheng , H Zhu , Q Wan , J Liu , Z Xiao , D. P Siderovski and Q. Du
 

Disrupting LGN’s function at lateral membrane domains displaces the axis of cell division in cyst-forming MDCK cells.

 
 
 
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