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1.  Comparison of hemodynamic changes between phenylephrine and combined phenylephrine and glycopyrrolate groups after spinal anesthesia for cesarean delivery 
Hypotension remains a common clinical problem of spinal anesthesia for cesarean delivery and phenylephrine is used as a vasopressor to address this. However, phenylephrine reduces maternal cardiac output (CO) due to reflex bradycardia. Glycopyrrolate is safe for the fetus, and increases heart rate (HR). Using a noninvasive measure of CO, we compared maternal hemodynamic changes between the phenylephrine only group (group P) and the phenylephrine plus glycopyrrolate group (group PG).
In this randomized study, 60 women scheduled for elective cesarean delivery were allocated to group P (n = 30) or group PG (n = 30). In both groups, phenylephrine was infused at 50 µg/min. This infusions stopped if systolic blood pressure (SBP) was higher than the baseline value, and phenylephrine 100 µg was injected if SBP was lower than 80% of the baseline value from spinal anesthesia to delivery. In group PG, glycopyrrolate 0.2 mg was injected intravenously after spinal anesthesia. Hemodynamic parameters, such as SBP, heart rate (HR), stroke volume index (SVI), cardiac index (CI) were measured before and until 15 min after spinal anesthesia.
There were no significant differences in SBP and SVI compared to the baseline value in each group and between the two groups. HR and CI reduced significantly from 8 min to 15 min in group P compared to the baseline value as well as group PG for each time-point. However, HR and CI were maintained in group PG.
The use of glycopyrrolate added to phenylephrine infusion to prevent hypotension by spinal anesthesia for cesarean delivery was effective in maintaining HR and CI.
PMCID: PMC3272527  PMID: 22323952
Cesarean section; Glycopyrrolate; Hypotension; Phenylephrine; Spinal anesthesia
2.  Multiple redundant sequence elements within the fission yeast ura4 replication origin enhancer 
Some origins in eukaryotic chromosomes fire more frequently than others. In the fission yeast, Schizosaccharomyces pombe, the relative firing frequencies of the three origins clustered 4-8 kbp upstream of the ura4 gene are controlled by a replication enhancer - an element that stimulates nearby origins in a relatively position-and orientation-independent fashion. The important sequence motifs within this enhancer were not previously localized.
Systematic deletion of consecutive segments of ~50, ~100 or ~150 bp within the enhancer and its adjacent core origin (ars3002) revealed that several of the ~50-bp stretches within the enhancer contribute to its function in partially redundant fashion. Other stretches within the enhancer are inhibitory. Some of the stretches within the enhancer proved to be redundant with sequences within core ars3002. Consequently the collection of sequences important for core origin function was found to depend on whether the core origin is assayed in the presence or absence of the enhancer. Some of the important sequences in the core origin and enhancer co-localize with short runs of adenines or thymines, which may serve as binding sites for the fission yeast Origin Recognition Complex (ORC). Others co-localize with matches to consensus sequences commonly found in fission yeast replication origins.
The enhancer within the ura4 origin cluster in fission yeast contains multiple sequence motifs. Many of these stimulate origin function in partially redundant fashion. Some of them resemble motifs also found in core origins. The next step is to identify the proteins that bind to these stimulatory sequences.
PMCID: PMC29090  PMID: 11178109
3.  Genome-wide Transcriptome Profiling of Homologous Recombination DNA Repair 
Nature communications  2014;5:3361.
Homologous recombination (HR) repair deficiency predisposes to cancer development, but also sensitizes cancer cells to DNA-damage-inducing therapeutics. Here we identify an HR-defect (HRD) gene signature, which can be used to functionally assess HR repair status without interrogating individual genetic alterations in cells. By using this HRD gene signature as a functional network analysis tool, we discover that simultaneous loss of two major tumor suppressors BRCA1 and PTEN extensively rewire the HR repair-deficient phenotype, which is found in cells with defects in either BRCA1 or PTEN alone. Moreover, the HRD gene signature serves as an effective drug discovery platform to identify agents targeting HR repair as potential chemo/radio-sensitizers. More importantly, this HRD gene signature is able to predict clinical outcomes across multiple cancer lineages. Our findings, therefore, provide a molecular profile of HR repair to assess its status at a functional network level, which can provide both biological insights and have clinical implications in cancer.
PMCID: PMC4017859  PMID: 24553445
4.  65-gene–based risk score classifier predicts overall survival in hepatocellular carcinoma 
Hepatology (Baltimore, Md.)  2012;55(5):1443-1452.
Clinical application of the prognostic gene expression signature has been delayed due to the large number of genes and complexity of prediction algorithms. In current study, we aim to develop an easy-to-use risk score with a limited number of genes that can robustly predict prognosis of patients with HCC. The risk score was developed by using Cox coefficient values of 65 genes in the training set (n=139) and its robustness was validated in test sets (n=292). The risk score was a highly significant predictor of overall survival (OS) in the first test cohort (P = 5.6 × 10-5, n = 100) and the second test cohort (P = 5.0 × 10-5, n = 192). In multivariate analysis, the risk score was significant risk factor among clinical variables examined together (hazard ratio [HR], 1.36; 95% confidential interval [CI], 1.13-1.64; P = 0.001 for OS).
The risk score classifier we have developed can identify two clinically distinct HCC subtypes at early and late stage of the disease in a simple and highly reproducible manner across multiple data sets.
PMCID: PMC4060518  PMID: 22105560
Hepatocellular Carcinoma; Gene expression signature; Microarrays; Prognostic biomarkers
5.  Glioma pathogenesis-related protein 1 induces prostate cancer cell death through Hsc70-mediated suppression of AURKA and TPX2 
Molecular oncology  2012;7(3):484-496.
In this study we report that expression of glioma pathogenesis-related protein 1 (GLIPR1) regulated numerous apoptotic, cell cycle, and spindle/centrosome assembly-related genes, including AURKA and TPX2, and induced apoptosis and/or mitotic catastrophe (MC) in prostate cancer (PCa) cells, including p53-mutated/deleted, androgen-insensitive metastatic PCa cells. Mechanistically, GLIPR1 interacts with heat shock cognate protein 70 (Hsc70); this interaction is associated with SP1 and c-Myb destabilization and suppression of SP1-and c-Myb-mediated AURKA and TPX2 transcription. Inhibition of AURKA and TPX2 using siRNA mimicked enforced GLIPR1 expression in the induction of apoptosis and MC. Recombinant GLIPR1-ΔTM protein inhibited AURKA and TPX2 expression, induced apoptosis and MC, and suppressed orthotopic xenograft tumor growth. Our results define a novel GLIPR1-regulated signaling pathway that controls apoptosis and/or mitotic catastrophe in PCa cells and establishes the potential of this pathway for targeted therapies.
PMCID: PMC3907183  PMID: 23333597
Glioma pathogenesis-related; protein 1 (GLIPR1); Hsc70; c-Myb; AURKA; TPX2; Prostate cancer
6.  GLIPR1 Suppresses Prostate Cancer Development through Targeted Oncoprotein Destruction 
Cancer research  2011;71(24):10.1158/0008-5472.CAN-11-1714.
Downregulation of the proapoptotic p53 target gene GLIPR1 occurs frequently in prostate cancer (PCa), but the functional meaning of this event is obscure. Here we report the discovery of functional relationship between GLIPR1 and c-Myc in PCa where c-Myc is often upregulated. We found that the expression of GLIPR1 and c-Myc were inversely correlated in human PCa. Restoration of GLIPR1 expression in PCa cells downregulated c-myc levels, inhibiting cell cycle progression. Downregulation was linked to a reduction in β-catenin/TCF4-mediated transcription of the c-myc gene, which were caused by GLIPR1-mediated redistribution of casein kinase 1α (CK1α) from the Golgi apparatus to the cytoplasm where CK1α could phosphorylate β-catenin and mediate its destruction. In parallel, GLIPR1 also promoted c-Myc protein ubiquitination and degradation by glycogen synthase kinase-3α- and/or CK1α-mediated c-Myc phosphorylation. Notably, genetic ablation of the mouse homolog of Glipr1 cooperated with c-myc overexpression to induce prostatic intraepithelial neoplasia (PIN) and PCa. Together, our findings provide evidence for CK1α-mediated destruction of c-Myc and identify c-Myc S252 as a crucial CK1α phosphorylation site for c-Myc degradation. Further, they reveal parallel mechanisms of c-myc downregulation by GLIPR1 that when ablated in the prostate are sufficient to drive c-Myc expression and malignant development.
PMCID: PMC3813470  PMID: 22025562
GLIPR1; c-Myc; CK1α; oncoprotein destruction; prostate cancer
7.  Silencing of p130Cas in Ovarian Carcinoma: A Novel Mechanism for Tumor Cell Death 
We investigated the clinical and biological significance of p130cas, an important cell signaling molecule, in ovarian carcinoma.
Expression of p130cas in ovarian tumors, as assessed by immunohistochemistry, was associated with tumor characteristics and patient survival. The effects of p130cas gene silencing with small interfering RNAs incorporated into neutral nanoliposomes (siRNA-DOPC), alone and in combination with docetaxel, on in vivo tumor growth and on tumor cell proliferation (proliferating cell nuclear antigen) and apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling) were examined in mice bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) or taxane-resistant (HeyA8-MDR) ovarian tumors (n = 10 per group). To determine the specific mechanisms by which p130cas gene silencing abrogates tumor growth, we measured cell viability (MTT assay), apoptosis (fluorescence-activated cell sorting), autophagy (immunoblotting, fluorescence, and transmission electron microscopy), and cell signaling (immunoblotting) in vitro. All statistical tests were two-sided.
Of 91 ovarian cancer specimens, 70 (76%) had high p130cas expression; and 21 (24%) had low p130cas expression. High p130cas expression was associated with advanced tumor stage (P < .001) and higher residual disease (>1 cm) following primary cytoreduction surgery (P = .007) and inversely associated with overall survival and progression-free survival (median overall survival: high p130cas expression vs low expression, 2.14 vs 9.1 years, difference = 6.96 years, 95% confidence interval = 1.69 to 9.48 years, P < .001; median progression-free survival: high p130cas expression vs low expression, 1.04 vs 2.13 years, difference = 1.09 years, 95% confidence interval = 0.47 to 2.60 years, P = .01). In mice bearing orthotopically implanted HeyA8 or SKOV3ip1 ovarian tumors, treatment with p130cas siRNA-DOPC in combination with docetaxel chemotherapy resulted in the greatest reduction in tumor growth compared with control siRNA therapy (92%–95% reduction in tumor growth; P < .001 for all). Compared with control siRNA therapy, p130cas siRNA-DOPC reduced SKOV3ip1 cell proliferation (31% reduction, P < .001) and increased apoptosis (143% increase, P < .001) in vivo. Increased tumor cell apoptosis may have persisted despite pan-caspase inhibition by the induction of autophagy and related signaling pathways.
Increased p130cas expression is associated with poor clinical outcome in human ovarian carcinoma, and p130cas gene silencing decreases tumor growth through stimulation of apoptotic and autophagic cell death.
PMCID: PMC3206039  PMID: 21957230
8.  Convergence of major physiological stimuli for renin release on the Gs-alpha/cyclic adenosine monophosphate signaling pathway 
Control of the renin system by physiological mechanisms such as the baroreceptor or the macula densa (MD) is characterized by asymmetry in that the capacity for renin secretion and expression to increase is much larger than the magnitude of the inhibitory response. The large stimulatory reserve of the renin–angiotensin system may be one of the causes for the remarkable salt-conserving power of the mammalian kidney. Physiological stimulation of renin secretion and expression relies on the activation of regulatory pathways that converge on the cyclic adenosine monophosphate/protein kinase A (cAMP/ PKA) pathway. Mice with selective Gs-alpha (Gsα) deficiency in juxtaglomerular granular cells show a marked reduction of basal renin secretion, and an almost complete unresponsiveness of renin release to furosemide, hydralazine, or isoproterenol. Cyclooxygenase-2 generating prostaglandin E2 (PGE2) and prostacyclin (PGI2) in MD and thick ascending limb cells is one of the main effector systems utilizing Gsα-coupled receptors to stimulate the renin–angiotensin system. In addition, β-adrenergic receptors are critical for the expression of high basal levels of renin and for its release response to lowering blood pressure or MD sodium chloride concentration. Nitric oxide generated by nitric oxide synthases in the MD and in endothelial cells enhances cAMP-dependent signaling by stabilizing cAMP through cyclic guanosine monophosphate-dependent inhibition of phosphodiesterase 3. The stimulation of renin secretion by drugs that inhibit angiotensin II formation or action results from the convergent activation of cAMP probably through indirect augmentation of the activity of PGE2 and PGI2 receptors, β-adrenergic receptors, and nitric oxide.
PMCID: PMC3482793  PMID: 22124804
Cyclooxygenase; Nitric oxide synthase; Phosphodiesterase; Macula densa; Baroreceptor; ACE inhibition
9.  Impaired Glucose Tolerance in the Absence of Adenosine A1 Receptor Signaling 
Diabetes  2011;60(10):2578-2587.
The role of adenosine (ADO) in the regulation of glucose homeostasis is not clear. In the current study, we used A1-ADO receptor (A1AR)-deficient mice to investigate the role of ADO/A1AR signaling for glucose homeostasis.
After weaning, A1AR−/− and wild-type mice received either a standard diet (12 kcal% fat) or high-fat diet (HFD; 45 kcal% fat). Body weight, fasting plasma glucose, plasma insulin, and intraperitoneal glucose tolerance tests were performed in 8-week-old mice and again after 12–20 weeks of subsequent observation. Body composition was quantified by magnetic resonance imaging and epididymal fat-pad weights. Glucose metabolism was investigated by hyperinsulinemic-euglycemic clamp studies. To describe pathophysiological mechanisms, adipokines and Akt phosphorylation were measured.
A1AR−/− mice were significantly heavier than wild-type mice because of an increased fat mass. Fasting plasma glucose and insulin were significantly higher in A1AR−/− mice after weaning and remained higher in adulthood. An intraperitoneal glucose challenge disclosed a significantly slower glucose clearance in A1AR−/− mice. An HFD enhanced this phenotype in A1AR−/− mice and unmasked a dysfunctional insulin secretory mechanism. Insulin sensitivity was significantly impaired in A1AR−/− mice on the standard diet shortly after weaning. Clamp studies detected a significant decrease of net glucose uptake in A1AR−/− mice and a reduced glucose uptake in muscle and white adipose tissue. Effects were not triggered by leptin deficiency but involved a decreased Akt phosphorylation.
ADO/A1AR signaling contributes importantly to insulin-controlled glucose homeostasis and insulin sensitivity in C57BL/6 mice and is involved in the metabolic regulation of adipose tissue.
PMCID: PMC3178298  PMID: 21831968
10.  Prognostic gene expression signature associated with two molecularly distinct subtypes of colorectal cancer 
Gut  2011;61(9):1291-1298.
Despite continual efforts to develop prognostic and predictive models of colorectal cancer by using clinicopathological and genetic parameters, a clinical test that can discriminate between patients with good or poor outcome after treatment has not been established. Thus, the authors aim to uncover subtypes of colorectal cancer that have distinct biological characteristics associated with prognosis and identify potential biomarkers that best reflect the biological and clinical characteristics of subtypes.
Unsupervised hierarchical clustering analysis was applied to gene expression data from 177 patients with colorectal cancer to determine a prognostic gene expression signature. Validation of the signature was sought in two independent patient groups. The association between the signature and prognosis of patients was assessed by Kaplan–Meier plots, log-rank tests and the Cox model.
The authors identified a gene signature that was associated with overall survival and disease-free survival in 177 patients and validated in two independent cohorts of 213 patients. In multivariate analysis, the signature was an independent risk factor (HR 3.08; 95% CI 1.33 to 7.14; p=0.008 for overall survival). Subset analysis of patients with AJCC (American Joint Committee on Cancer) stage III cancer revealed that the signature can also identify the patients who have better outcome with adjuvant chemotherapy (CTX). Adjuvant chemotherapy significantly affected disease-free survival in patients in subtype B (3-year rate, 71.2% (CTX) vs 41.9% (no CTX); p=0.004). However, such benefit of adjuvant chemotherapy was not significant for patients in subtype A.
The gene signature is an independent predictor of response to chemotherapy and clinical outcome in patients with colorectal cancer.
PMCID: PMC3419333  PMID: 21997556
11.  Gene expression signature–based prognostic risk score in gastric cancer 
Despite continual efforts to develop a prognostic model of gastric cancer by using clinical and pathological parameters, a clinical test that can discriminate patients with good outcomes from those with poor outcomes after gastric cancer surgery has not been established. We aim to develop practical biomarker-based risk score that can predict relapse of gastric cancer after surgical treatment.
Experimental Design
Using microarray technologies, we generated and analyzed gene expression profiling data from 65 gastric cancer patients to identify biomarker genes associated with relapse. The association of expression patterns of identified genes with relapse and overall survival was validated in independent gastric cancer patients.
We uncovered two subgroups of gastric cancer that were strongly associated with the prognosis. For the easy translation of our findings into practice, we developed a scoring system based on the expression of six genes that predicted the likelihood of relapse after curative resection. In multivariate analysis, the risk score was an independent predictor of relapse in a cohort of 96 patients. We were able to validate the robustness of the 6-gene signature in an additional independent cohort.
The risk score derived from the 6-gene set successfully prognosticated the relapse of gastric cancer patients after gastrectomy.
PMCID: PMC3078023  PMID: 21447720
12.  Reconstruction of nuclear receptor network reveals that NR2E3 is a novel upstream regulator of ESR1 in breast cancer 
EMBO Molecular Medicine  2012;4(1):52-67.
ESR1 is one of the most important transcription factors and therapeutic targets in breast cancer. By applying systems-level re-analysis of publicly available gene expression data, we uncovered a potential regulator of ESR1. We demonstrated that orphan nuclear receptor NR2E3 regulates ESR1 via direct binding to the ESR1 promoter with concomitant recruitment of PIAS3 to the promoter in breast cancer cells, and is essential for physiological cellular activity of ESR1 in estrogen receptor (ER)-positive breast cancer cells. Moreover, expression of NR2E3 was significantly associated with recurrence-free survival and a favourable response to tamoxifen treatment in women with ER-positive breast cancer. Our results provide mechanistic insights on the regulation of ESR1 by NR2E3 and the clinical relevance of NR2E3 in breast cancer.
PMCID: PMC3376834  PMID: 22174013
breast cancer; genomics; microarray; NR2E3; nuclear receptor
13.  Cadherin-11 increases migration and invasion of prostate cancer cells and enhances their interaction with osteoblasts 
Cancer research  2010;70(11):4580-4589.
Cell adhesion molecules have been implicated in the colonization of cancer cells to distant organs. Prostate cancer (PCa) has a propensity to metastasize to bone and cadherin-11, which is an osteoblast cadherin aberrantly expressed in PCa cells derived from bone metastases, has been shown to play a role in the metastasis of PCa cells to bone. However, the mechanism by which cadherin-11 is involved in this process is not known. Here, we show that expression of cadherin-11 in cadherin-11-negative C4-2B4 cells increases their spreading and intercalation into an osteoblast layer, and also stimulates C4-2B4 cell migration and invasiveness. Downregulation of cadherin-11 in cadherin-11-expressing metastatic PC3 cells decreases cell motility and invasiveness. Further, both the juxtamembrane (JMD) and β-catenin binding domains (CBS) in the cytoplasmic tail of cadherin-11 are required for cell migration and invasion, but not spreading. Gene array analyses showed that several invasion related genes, including MMP-7 and MMP-15, are upregulated in cadherin-11, but not in cad11-ΔJMD or cad11-ΔCBS, expressing C4-2B4 cells. These observations suggest that cadherin-11 not only provides a physical link between PCa cells and osteoblasts but also increases PCa cell motility and invasiveness that may facilitate the metastatic colonization of PCa cells in bone.
PMCID: PMC2923552  PMID: 20484040
cadherin-11; prostate cancer; osteoblast; adhesion; bone metastasis
14.  Prognostic Biomarkers for Esophageal Adenocarcinoma Identified by Analysis of Tumor Transcriptome 
PLoS ONE  2010;5(11):e15074.
Despite many attempts to establish pre-treatment prognostic markers to understand the clinical biology of esophageal adenocarcinoma (EAC), validated clinical biomarkers or parameters remain elusive. We generated and analyzed tumor transcriptome to develop a practical biomarker prognostic signature in EAC.
Methodology/Principal Findings
Untreated esophageal endoscopic biopsy specimens were obtained from 64 patients undergoing surgery and chemoradiation. Using DNA microarray technology, genome-wide gene expression profiling was performed on 75 untreated cancer specimens from 64 EAC patients. By applying various statistical and informatical methods to gene expression data, we discovered distinct subgroups of EAC with differences in overall gene expression patterns and identified potential biomarkers significantly associated with prognosis. The candidate marker genes were further explored in formalin-fixed, paraffin-embedded tissues from an independent cohort (52 patients) using quantitative RT-PCR to measure gene expression. We identified two genes whose expression was associated with overall survival in 52 EAC patients and the combined 2-gene expression signature was independently associated with poor outcome (P<0.024) in the multivariate Cox hazard regression analysis.
Our findings suggest that the molecular gene expression signatures are associated with prognosis of EAC patients and can be assessed prior to any therapy. This signature could provide important improvement for the management of EAC patients.
PMCID: PMC2994829  PMID: 21152079
15.  Modulatory Effect of Rice Bran and Phytic Acid on Glucose Metabolism in High Fat-Fed C57BL/6N Mice 
The effect of dietary feeding of rice bran and phytic acid on the glucose metabolism in high fat-fed C57BL/6N mice fed was investigated. The mice were given with either a high fat diet only (HF group) or a high fat diet supplemented with rice bran (HF-RB group) or phytic acid (HF-PA group) for 7 weeks. The control mice (NC group) received a normal diet. At the end of the experimental period, the HF group exhibited substantially higher blood glucose level than the NC group. However, the HF-RB and HF-PA groups showed a marked decrease in the blood glucose level relative to HF mice. Furthermore, significantly higher glucokinase (GK) activity and lower phosphoenolpyruvate carboxykinase (PEPCK) activity were observed in HF-RB and HF-PA mice compared with that of the NC and HF ones. It was also found that the glucose-6-phosphatase (G6pase) activity and hepatic glycogen concentration were considerably higher in HF-RB and HF-PA groups, respectively, than that of the HF mice. These findings demonstrate that both rice bran and phytic acid could reduce the risk of high fat diet-induced hyperglycemia via regulation of hepatic glucose-regulating enzyme activities.
PMCID: PMC2901758  PMID: 20664725
rice bran; phytic acid; glucose metabolism; diabetes; high fat-fed mice
16.  Magnetic resonance imaging of urea transporter knockout mice reveals two distinct types of renal pelvic abnormality 
Kidney international  2008;74(9):1202-1208.
Many transgenic and knockout mouse models with increased urine flow have been noted to have structural abnormalities of the renal pelvis and renal inner medulla. Here, we describe an approach for in vivo study of such abnormalities in mice using high resolution contrast enhanced T1-weighted magnetic resonance imaging (MRI). The studies were carried out in mice in which the UT-A isoform 1 and 3 urea transporters had been deleted (UT-A1/3-/- mice). The experiments revealed three distinct variations in the appearance of the renal pelvis in these mice: 1) normal kidneys with no accumulation of contrast agent in the renal pelvis; 2) frank right-sided unilateral hydronephrosis with marked atrophy of the renal medulla, seen relatively infrequently; and 3) a renal pelvic reflux pattern characterized by the presence of contrast agent in the renal pelvis surrounding the renal inner medulla, with no substantial atrophy of the renal medulla, seen in most UT-A1/3-/- mice with advancing age. The reflux pattern was also found in aquaporin-1 knockout mice. UT-A1/3-/- mice also manifested increased mean arterial pressure. Feeding the UT-A1/3-/- mice a low protein diet did not prevent the demonstrated abnormalities of the renal pelvis. These studies demonstrate the feasibility of real time imaging of renal pelvic structure in genetically manipulated mice, providing a tool for non-destructive, temporal studies of kidney structure.
PMCID: PMC2614379  PMID: 18854850
kidney; hydronephrosis; renal pelvic reflux; blood pressure
17.  Bone marrow-derived cells do not repair endothelium in a mouse model of chronic endothelial cell dysfunction 
Cardiovascular Research  2009;84(2):317-325.
Bone marrow (BM)-derived endothelial progenitor cells (EPCs) in the circulation replace damaged vascular endothelium. We assessed the hypothesis that a BM transplant from healthy animals would restore normal arterial endothelium and prevent hypertension in young endothelial nitric oxide synthase-deficient (eNOS−/−) mice.
Methods and results
Radiation or busulfan-induced BM ablation in eNOS−/− mice on day 6, day 14, or day 28 was followed by a BM transplant consisting of enhanced green fluorescent protein positive (EGFP+) cells from C57BL/6J mice. Peripheral blood cell chimerism was always greater than 85% at 4 months after BM transplant. Molecular assays of heart, kidney, and liver revealed low-level chimerism in all treatment groups, consistent with residual circulating EGFP+ blood cells. When aorta, coronary, renal, hepatic, and splenic arteries in BM-transplanted eNOS−/− mice were examined by confocal microscopy, there were no EGFP- or eNOS-positive endothelial cells detected in these vessels in any of the treatment groups. Likewise, telemetry did not detect any reduction in blood pressure. Thus, no differences were observed in our measurements using several different treatment protocols.
We found no evidence for BM-derived EPC renewal of endothelium in this eNOS-deficient mouse model of a chronic vascular disease or in wild-type mice during postnatal growth. Hence, renewal of chronic dysfunctional endothelium and endothelial homeostasis may be dependent on resident vascular progenitor cells.
PMCID: PMC2761200  PMID: 19578071
Stem cells; Bone marrow transplantation; Endothelial NOS deficiency; Hypertension
18.  AP214, an analogue of α-melanocyte-stimulating hormone, ameliorates sepsis-induced acute kidney injury and mortality 
Kidney international  2008;73(11):1266-1274.
Sepsis remains a serious problem in critically ill patients with the mortality increasing to over half when there is attendant acute kidney injury. α-Melanocyte-stimulating hormone is a potent anti-inflammatory cytokine that inhibits many forms of inflammation including that with acute kidney injury. We tested whether a new α-melanocyte-stimulating hormone analogue (AP214), which has increased binding affinity to melanocortin receptors, improves sepsis-induced kidney injury and mortality using a cecal ligation and puncture mouse model. In the lethal cecal ligation-puncture model of sepsis, severe hypotension and bradycardia resulted and AP214 attenuated acute kidney injury of the lethal model with a bell-shaped dose-response curve. An optimum AP214 dose reduced acute kidney injury even when it was administered 6 hr after surgery and it significantly improved blood pressure and heart rate. AP214 reduced serum TNF-α and IL-10 levels with a bell-shaped dose-response curve. Additionally; NF-κB activation in the kidney and spleen, and splenocyte apoptosis were decreased by the treatment. AP214 significantly improved survival in both lethal and sublethal models. We have shown that AP214 improves hemodynamic failure, acute kidney injury, mortality and splenocyte apoptosis attenuating pro- and anti-inflammatory actions due to sepsis.
PMCID: PMC2398767  PMID: 18354376
19.  Persistence of Circadian Variation in Arterial Blood Pressure in β1/β2-Adrenergic Receptor-Deficient Mice 
The β-adrenergic pathway has been considered one important effector of circadian variation in arterial pressure. Experiments were performed in β1/β2-adrenergic receptor-deficient mice (β1/β2ADR-/-) to assess whether this pathway is required for circadian variation in mean arterial pressure (MAP) and to determine the impact of its loss on the response to changes in dietary salt. Twenty-four hour recordings of MAP, heart rate (HR) and locomotor activity were made in conscious 16-17 week old mice (wild-type, WT, n = 7, β1/β2ADR-/- n=10) by telemetry. Both WT and β1/β2ADR-/- mice demonstrated robust circadian variation in MAP and HR, although 24 hour mean MAP was 10% lower (102.02 ± 1.81 vs. 92.11 ± 2.62 mm Hg) in β1/β2 ADR-/- than WT, HR was 16% lower and day-night differences reduced. Both WT and β1/β2ADR-/- adapted to changed salt intake without changed MAP. However, the β1/β2ADR-/- mice demonstrated a striking reduction in locomotor activity in light and dark phases of the day. In WT, MAP was markedly affected by locomotor activity, resulting in bimodal distributions in both light and dark. When MAP was analyzed using only intervals without locomotor activity, bimodality and circadian differences were reduced, and there was no significant difference between the two genotypes. The results indicate that there is no direct effect or role for the β-adrenergic system in circadian variation of arterial pressure in mice, aside from the indirect consequences of altered locomotor activity. Our results also confirm that locomotor activity contributes strongly to circadian variation in BP in mice.
PMCID: PMC2386676  PMID: 18305025
Salt intake; sympathetic nervous system
20.  Absence of BLM leads to accumulation of chromosomal DNA breaks during both unperturbed and disrupted S phases 
The Journal of Cell Biology  2004;165(6):801-812.
Bloom's syndrome (BS), a disorder associated with genomic instability and cancer predisposition, results from defects in the Bloom's helicase (BLM) protein. In BS cells, chromosomal abnormalities such as sister chromatid exchanges occur at highly elevated rates. Using Xenopus egg extracts, we have studied Xenopus BLM (Xblm) during both unperturbed and disrupted DNA replication cycles. Xblm binds to replicating chromatin and becomes highly phosphorylated in the presence of DNA replication blocks. This phosphorylation depends on Xenopus ATR (Xatr) and Xenopus Rad17 (Xrad17), but not Claspin. Xblm and Xenopus topoisomerase IIIα (Xtop3α) interact in a regulated manner and associate with replicating chromatin interdependently. Immunodepletion of Xblm from egg extracts results in accumulation of chromosomal DNA breaks during both normal and perturbed DNA replication cycles. Disruption of the interaction between Xblm and Xtop3α has similar effects. The occurrence of DNA damage in the absence of Xblm, even without any exogenous insult to the DNA, may help to explain the genesis of chromosomal defects in BS cells.
PMCID: PMC2172405  PMID: 15197177
RecQ helicase; ATR; Rad17; DNA replication; DNA damage
21.  Multiple Orientation-Dependent, Synergistically Interacting, Similar Domains in the Ribosomal DNA Replication Origin of the Fission Yeast, Schizosaccharomyces pombe 
Molecular and Cellular Biology  1998;18(12):7294-7303.
Previous investigations have shown that the fission yeast, Schizosaccharomyces pombe, has DNA replication origins (500 to 1500 bp) that are larger than those in the budding yeast, Saccharomyces cerevisiae (100 to 150 bp). Deletion and linker substitution analyses of two fission yeast origins revealed that they contain multiple important regions with AT-rich asymmetric (abundant A residues in one strand and T residues in the complementary strand) sequence motifs. In this work we present the characterization of a third fission yeast replication origin, ars3001, which is relatively small (∼570 bp) and responsible for replication of ribosomal DNA. Like previously studied fission yeast origins, ars3001 contains multiple important regions. The three most important of these regions resemble each other in several ways: each region is essential for origin function and is at least partially orientation dependent, each region contains similar clusters of A+T-rich asymmetric sequences, and the regions can partially substitute for each other. These observations suggest that ars3001 function requires synergistic interactions between domains binding similar proteins. It is likely that this requirement extends to other fission yeast origins, explaining why such origins are larger than those of budding yeast.
PMCID: PMC109311  PMID: 9819416

Results 1-21 (21)