JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Grading of Peripheral Cytopenias Caused by Nonalcoholic Cirrhotic Portal Hypertension and Its Clinical Significance.
Cell Biochem. Biophys.
PUBLISHED: 11-08-2014
Show Abstract
Hide Abstract
This study investigates peripheral cytopenias in patients with splenomegaly caused by nonalcoholic cirrhotic portal hypertension. Data from 330 splenomegaly cases caused by nonalcoholic cirrhotic portal hypertension were collected and analyzed using univariate and multivariate analysis. The cytopenias were scored and graded according to the F value of the multiple linear regression equation. Based on the severity of thrombocytopenia, cytopenia was graded as mild, moderate, or severe, and determined by a score of <2 points, 2-3 points, and >3 points. 30 % of the patients had monolineage cytopenias, 35.8 % had bilineage cytopenias, and 34.2 % had trilineage cytopenias. All patients were treated surgically. In the univariate analysis, the severity of erythropenia was different in the surgical outcome when compared to the intra-group (P < 0.05). In the multivariate analysis, thrombocytopenia was different in the surgical outcomes when compared with leukopenia and erythropenia (P < 0.05). There was a significant difference in surgical outcomes between the three grades (mild, moderate, and severe) of cytopenia (P < 0.05). Peripheral cytopenias have a significant impact on the clinical outcomes. The more severe the cytopenias, the worse the surgical outcomes are. Thrombocytopenia is a major factor influencing surgical outcomes. The thrombocytopenia-based three-level grading of cytopenias provides a basis for analyzing individual cases, planning treatment, and assessing prognosis in clinical practice.
Related JoVE Video
Culturable heavy metal-resistant and plant growth promoting bacteria in V-Ti magnetite mine tailing soil from Panzhihua, China.
PLoS ONE
PUBLISHED: 09-04-2014
Show Abstract
Hide Abstract
To provide a basis for using indigenous bacteria for bioremediation of heavy metal contaminated soil, the heavy metal resistance and plant growth-promoting activity of 136 isolates from V-Ti magnetite mine tailing soil were systematically analyzed. Among the 13 identified bacterial genera, the most abundant genus was Bacillus (79 isolates) out of which 32 represented B. subtilis and 14 B. pumilus, followed by Rhizobium sp. (29 isolates) and Ochrobactrum intermedium (13 isolates). Altogether 93 isolates tolerated the highest concentration (1000 mg kg(-1)) of at least one of the six tested heavy metals. Five strains were tolerant against all the tested heavy metals, 71 strains tolerated 1,000 mg kg(-1) cadmium whereas only one strain tolerated 1,000 mg kg(-1) cobalt. Altogether 67% of the bacteria produced indoleacetic acid (IAA), a plant growth-promoting phytohormone. The concentration of IAA produced by 53 isolates was higher than 20 µg ml(-1). In total 21% of the bacteria produced siderophore (5.50-167.67 µg ml(-1)) with two Bacillus sp. producing more than 100 µg ml(-1). Eighteen isolates produced both IAA and siderophore. The results suggested that the indigenous bacteria in the soil have beneficial characteristics for remediating the contaminated mine tailing soil.
Related JoVE Video
Convergent transcription through microsatellite repeat tracts induces cell death.
Mol. Biol. Rep.
PUBLISHED: 05-24-2014
Show Abstract
Hide Abstract
Microsatellite sequences, composed of short tandem repeats and randomly distributed in human genome, can become unstable during various DNA metabolic processes. Expansions of CAG, GAA, CGG and CCTG repeats located in specific genes are responsible for several human disorders. It is known that a major percentage of human genes simultaneously express both sense and antisense transcripts. Recently, we reported that convergent transcription through a CAG95 tract in human cells leads to cell cycle arrest as well as robust apoptosis. In this study, we studied the effects of convergent transcription through other types of repeats, using cell lines that contain substrates with inducible sense and antisense transcription through CGG66, GAA102, or CCTG134 tracts. We found that convergent transcription through all these repeats inhibits cell growth and induces cell death, though more moderately than convergent transcription through a CAG tract. These results suggest that convergent transcription through various types of tandem repeats represent a novel type of stress to cells.
Related JoVE Video
Constitutive ERK1/2 activation contributes to production of double minute chromosomes in tumour cells.
J. Pathol.
PUBLISHED: 04-24-2014
Show Abstract
Hide Abstract
Double minute chromosomes (DMs) are extrachromosomal cytogenetic structures found in tumour cells. As hallmarks of gene amplification, DMs often carry oncogenes and drug-resistance genes and play important roles in malignant tumour progression and drug resistance. The mitogen-activated protein kinase (MAPK) signalling pathway is frequently dysregulated in human malignant tumours, which induces genomic instability, but it remains unclear whether a close relationship exists between MAPK signalling and DMs. In the present study, we focused on three major components of MAPK signalling, ERK1/2, JNK1/2/3 and p38, to investigate the relationship between MAPK and DM production in tumour cells. We found that the constitutive phosphorylation of ERK1/2, but not JNK1/2/3 and p38, was closely associated with DMs in tumour cells. Inhibition of ERK1/2 activation in DM-containing and ERK1/2 constitutively phosphorylated tumour cells was able to markedly decrease the number of DMs, as well as the degree of amplification and expression of DM-carried genes. The mechanism was found to be an increasing tendency of DM DNA to break, become enveloped into micronuclei (MNs) and excluded from the tumour cells during the S/G2 phases of the cell cycle, events that accompanied the reversion of malignant behaviour. Our study reveals a linkage between ERK1/2 activation and DM stability in tumour cells. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Related JoVE Video
[Individualized neurosurgical treatments of spastic cerebral palsy].
Zhonghua Yi Xue Za Zhi
PUBLISHED: 04-22-2014
Show Abstract
Hide Abstract
To explore the outcomes of individualized neurosurgical treatments of spastic cerebral palsy.
Related JoVE Video
Clinical study on the relationship between hematocytopenia and splenomegaly caused by cirrhotic portal hypertension.
Cell Biochem. Biophys.
PUBLISHED: 04-04-2014
Show Abstract
Hide Abstract
This clinical study was designed to evaluate the presence of hematocytopenia in patients with splenomegaly caused by non-alcoholic cirrhotic portal hypertension. For this purpose, we randomly selected 358 patients with splenomegaly caused by non-alcoholic cirrhotic portal hypertension and admitted to the clinical data in our hospital between January 1991 and June 2009. Among these 358 patients, 322 patients (90.0 %) showed hematocytopenia, including multi-hemocyte decrease in 206 patients (i.e., 89 patients with a decrease in white blood cell count (WBC) + red blood cell count (RBC) + platelets count (PLT)); 52 patients with WBC + PLT decrease; 29 patients with RBC + PLT decrease; and 36 patients with WBC + RBC decrease) and single-hemocyte decrease in 116 patients (i.e., 31 cases with single PLT decrease; 29 cases with single WBC decrease; and 56 patients with single RBC decrease). After splenectomy, 36 patients (10.0 %) with hematocytopenia presented a statistical improvement of blood cell to normal level (P < 0.05), while 32 patients did not have any change as compared to pre-operative one (P > 0.05). It has to be noted that 4 patients did not received any surgery. Hematocytopenia was not detected in all the patients with splenomegaly caused by cirrhotic portal hypertension, thus it is probably a complication of splenomegaly but not an inevitable manifestation. It was concluded that splenectomy could be an effective treatment for splenomegaly associated with hematocytopenia, but patients without hematocytopenia could choose a non-surgical alternative treatment.
Related JoVE Video
Neuroprotective effect of ischemic preconditioning in focal cerebral infarction: relationship with upregulation of vascular endothelial growth factor.
Neural Regen Res
PUBLISHED: 04-01-2014
Show Abstract
Hide Abstract
Neuroprotection by ischemic preconditioning has been confirmed by many studies, but the precise mechanism remains unclear. In the present study, we performed cerebral ischemic preconditioning in rats by simulating a transient ischemic attack twice (each a 20-minute occlusion of the middle cerebral artery) before inducing focal cerebral infarction (2 hour occlusion-reperfusion in the same artery). We also explored the mechanism underlying the neuroprotective effect of ischemic preconditioning. Seven days after occlusion-reperfusion, tetrazolium chloride staining and immunohistochemistry revealed that the infarct volume was significantly smaller in the group that underwent preconditioning than in the model group. Furthermore, vascular endothelial growth factor immunoreactivity was considerably greater in the hippocampal CA3 region of preconditioned rats than model rats. Our results suggest that the protective effects of ischemic preconditioning on focal cerebral infarction are associated with upregulation of vascular endothelial growth factor.
Related JoVE Video
Hyperglycemia regulates TXNIP/TRX/ROS axis via p38 MAPK and ERK pathways in pancreatic cancer.
Curr Cancer Drug Targets
PUBLISHED: 03-28-2014
Show Abstract
Hide Abstract
Approximately 85% of pancreatic cancer patients suffer from glucose intolerance or even diabetes because high glucose levels can contribute to oxidative stress which promotes tumor development. As one of the reactive oxygen species (ROS)-regulating factors, thioredoxin-interacting protein (TXNIP), is involved in the maintenance of thioredoxin (TRX)-mediated redox regulation. In this study, we demonstrated that high glucose levels increased the expression of TXNIP in time- and concentration-dependent manners and modulated the activity of TRX and ROS production in pancreatic cancer cells, BxPC-3 and Panc-1. We also found that glucose activated both p38 MAPK and ERK pathways and inhibitors of these pathways impaired the TXNIP/TRX/ROS axis. Knockdown of TXNIP restored TRX activity and decreased ROS production under high glucose conditions. Moreover, we observed that the integrated optical density (IOD) of TXNIP staining as well as the protein and mRNA expression levels of TXNIP were higher in the tumor tissues of pancreatic cancer patients with diabetes. Taken together, these results indicate that hyperglycemia-induced TXNIP expression is involved in diabetes-mediated oxidative stress in pancreatic cancer via p38 MAPK and ERK pathways.
Related JoVE Video
GPC1 regulated by miR-96-5p, rather than miR-182-5p, in inhibition of pancreatic carcinoma cell proliferation.
Int J Mol Sci
PUBLISHED: 03-09-2014
Show Abstract
Hide Abstract
To determine the relationships between miR-96-5p/-182-5p and GPC1 in pancreatic cancer (PC), we conducted the population and in vitro studies. We followed 38 pancreatic cancer patients, measured and compared the expression of miR-96-5p/-182-5p, GPC1, characteristics and patients' survival time of different miR-96-5p/-182-5p expression levels in PC tissues. In an in vitro study, we investigated the proliferation, cycle and apotosis in cells transfected with mimics/inhibitors of the two miRNAs, and determine their effects on GPC1 by dual-luciferase assay. In the follow-up study, we found that the expressions of miR-96-5p/-182-5p were lower/higher in PC tissues; patients with lower/higher levels of miR-96-5p/-182-5p suffered poorer characteristics and decreased survival time. In the in vitro study, the expressions of miR-96-5p/-182-5p were different in cells. Proliferation of cells transfected with miR-96-5p mimics/inhibitors was lower/higher in Panc-1/BxPC-3; when transfected with miR-182-5p mimics/inhibitors, proliferation of cells were higher/lower in AsPC-1/Panc-1. In a cell cycle study, panc-1 cells transfected with miR-96-5p mimics was arrested at G0/G1; BxPC-3 cells transfected with miR-96-5p inhibitors showed a significantly decrease at G0/G1; AsPC-1 cells transfected with miR-182-5p mimics was arrested at S; Panc-1 cells transfected with miR-182-5p inhibitors showed a decrease at S. MiR-96-5p mimics increased the apoptosis rate in Panc-1 cells, and its inhibitors decreased the apoptosis rate in BxPC-3. Dual luciferase assay revealed that GPC1 was regulated by miR-96-5p, not -182-5p. We found that miR-96-5p/-182-5p as good markers for PC; miR-96-5p, rather than -182-5p, inhibits GPC1 to suppress proliferation of PC cells.
Related JoVE Video
miRNA-940 reduction contributes to human Tetralogy of Fallot development.
J. Cell. Mol. Med.
PUBLISHED: 02-13-2014
Show Abstract
Hide Abstract
Tetralogy of Fallot (TOF) is a complex congenital heart defect and the microRNAs regulation in TOF development is largely unknown. Herein, we explored the role of miRNAs in TOF. Among 75 dysregulated miRNAs identified from human heart tissues, miRNA-940 was the most down-regulated one. Interestingly, miRNA-940 was most highly expressed in normal human right ventricular out-flow tract comparing to other heart chambers. As TOF is caused by altered proliferation, migration and/or differentiation of the progenitor cells of the secondary heart field, we isolated Sca-1(+) human cardiomyocyte progenitor cells (hCMPC) for miRNA-940 function analysis. miRNA-940 reduction significantly promoted hCMPCs proliferation and inhibited hCMPCs migration. We found that JARID2 is an endogenous target regulated by miRNA-940. Functional analyses showed that JARID2 also affected hCMPCs proliferation and migration. Thus, decreased miRNA-940 affects the proliferation and migration of the progenitor cells of the secondary heart field by targeting JARID2 and potentially leads to TOF development.
Related JoVE Video
PINK1 and its familial Parkinson's disease-associated mutation regulate brain vascular endothelial inflammation.
J. Mol. Neurosci.
PUBLISHED: 01-03-2014
Show Abstract
Hide Abstract
Parkinson's disease (PD) is a debilitating disorder that affects movement. Inflammation-mediated endothelial dysfunction has been found to be involved in neurodegenerative diseases, including PD. More than 40 PTEN-induced putative kinase 1 (PINK1) mutations have been found in PD patients. The effects of PINK1 in vascular inflammation are as yet unknown. In this study, our findings revealed that PINK1 can be increased by the inflammatory cytokine tumor necrosis factor-? in primary human brain microvascular endothelial cells (HBMECs). We found that wild-type PINK1 prevents expression of the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1), thus inhibiting the attachment of monocytes to brain endothelial cells. However, PINK1G309D, the loss-of-function mutation associated with early-onset familial PD, promotes expression of VCAM-1 and exacerbates attachment of monocytes to brain endothelial cells. Mechanism studies revealed that overexpression of wild-type PINK1 inhibits the VCAM-1 promoter by inhibiting the transcriptional activity of interferon regulatory factor 1 (IRF-1). However, PINK1G309D promotes the VCAM-1 promoter by increasing the transcriptional activity of IRF-1.
Related JoVE Video
Evaluation of the diagnostic performance of magnetic resonance spectroscopy in brain tumors: a systematic review and meta-analysis.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
The aim of this study was to determine the suitability of magnetic resonance spectroscopy (MRS) for screening brain tumors, based on a systematic review and meta-analysis of published data on the diagnostic performance of MRS.
Related JoVE Video
The Wnt antagonist sFRP1 as a favorable prognosticator in human biliary tract carcinoma.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
Inactivation of Secreted Frizzled-Related Protein-1 (SFRP1) and overexpression of ?-catenin play important roles in the development and progression of a wide range of malignancies. We sought to determine whether the expression of SFRP1 and ?-catenin correlates with clinicopathologic parameters in human biliary tract cancer (BTC) and to evaluate the potential roles of these proteins as prognostic indicators. The expression of SFRP1 and ?-catenin in 78 patients with BTC and 36 control patients as investigated by immunohistochemistry. A wide variety of statistical parameters were assessed to determine the association between these proteins and the occurrence, clinical features, and overall survival rate in BTC.SFRP1 and ?-catenin had an inverse correlation (r?=?-0.636, P<0.0001) as assessed by Spearman rank analysis, with 52 (66.7%) of the BTC samples negative for SFRP1 expression and 53 (68.0%) positive for ?-catenin expression. Expression of each protein was associated with the histological type and lymph node invasion of BTC. A significantly poorer overall survival rate was observed for patients with low SFRP1 expression (P<0.0001) or high ?-catenin expression (P?=?0.007). SFRP1 expression (P<0.0001), ?-catenin expression (P<0.01) and histological type (P<0.01) were correlated with overall survival rate as assessed by univariate analysis; while multivariate analysis suggested that SFRP1 (hazard ratio, 10.514; 95% confidence intervals, 2.381-39.048; P<0.0001) may serve as an independent prognostic factor for BTC. Collectively, these results demonstrate that SFRP1 is a favorable prognostic factor for human BTC and that its expression inversely correlates with that of ?-catenin.
Related JoVE Video
MiR-34a targets GAS1 to promote cell proliferation and inhibit apoptosis in papillary thyroid carcinoma via PI3K/Akt/Bad pathway.
Biochem. Biophys. Res. Commun.
PUBLISHED: 10-24-2013
Show Abstract
Hide Abstract
MicroRNAs (miRNAs) are fundamental regulators of cell proliferation, differentiation, and apoptosis, and are implicated in tumorigenesis of many cancers. MiR-34a is best known as a tumor suppressor through repression of growth factors and oncogenes. Growth arrest specific1 (GAS1) protein is a tumor suppressor that inhibits cancer cell proliferation and induces apoptosis through inhibition of RET receptor tyrosine kinase. Both miR-34a and GAS1 are frequently down-regulated in various tumors. However, it has been reported that while GAS1 is down-regulated in papillary thyroid carcinoma (PTC), miR-34a is up-regulated in this specific type of cancer, although their potential roles in PTC tumorigenesis have not been examined to date. A computational search revealed that miR-34a putatively binds to the 3-UTR of GAS1 gene. In the present study, we confirmed previous findings that miR-34a is up-regulated and GAS1 down-regulated in PTC tissues. Further studies indicated that GAS1 is directly targeted by miR-34a. Overexpression of miR-34a promoted PTC cell proliferation and colony formation and inhibited apoptosis, whereas knockdown of miR-34a showed the opposite effects. Silencing of GAS1 had similar growth-promoting effects as overexpression of miR-34a. Furthermore, miR-34a overexpression led to activation of PI3K/Akt/Bad signaling pathway in PTC cells, and depletion of Akt reversed the pro-growth, anti-apoptotic effects of miR-34a. Taken together, our results demonstrate that miR-34a regulates GAS1 expression to promote proliferation and suppress apoptosis in PTC cells via PI3K/Akt/Bad pathway. MiR-34a functions as an oncogene in PTC.
Related JoVE Video
Induction of recombination between diverged sequences in a mammalian genome by a double-strand break.
Cell. Mol. Life Sci.
PUBLISHED: 09-18-2013
Show Abstract
Hide Abstract
To investigate whether mammalian cells can carry out recombinational double-strand break (DSB) repair between highly diverged sequences, mouse fibroblasts were transfected with DNA substrates that contained a "recipient" thymidine kinase (tk) gene disrupted by the recognition site for endonuclease I-SceI. Substrates also contained a linked "donor" tk gene sequence. Following DSB induction by I-SceI, selection for tk-expressing clones allowed recovery of repair events occurring by nonhomologous end-joining or recombination with the donor sequence. Although recombinational repair was most efficient when donor and recipient shared near-perfect homology, we recovered recombination events between recipient and donor sequences displaying 20 % nucleotide mismatch. Recombination between such imperfectly matched ("homeologous") sequences occurred at a frequency of 1.7 × 10(-7) events per cell and constituted 3 % of the DSB repair events recovered with the pair of homeologous sequences. Additional experiments were done with a substrate containing a donor sequence comprised of a region sharing high homology with the recipient and an adjacent region homeologous to the recipient. Recombinational DSB repair tracts initiating within high homology propagated into homeology in 11 of 112 repair events. These collective results contrasted with our earlier work in which spontaneous recombination (not intentionally induced by a DSB) between homeologous sequences occurred at an undetectable frequency of less than 10(-9) events per cell, and in which events initiating within high homology propagated into adjoining homeology in one of 81 events examined. Our current work suggests that homology requirements for recombination are effectively relaxed in proximity to a DSB in a mammalian genome.
Related JoVE Video
Requirement for integrin-linked kinase in neural crest migration and differentiation and outflow tract morphogenesis.
BMC Biol.
PUBLISHED: 09-10-2013
Show Abstract
Hide Abstract
Neural crest defects lead to congenital heart disease involving outflow tract malformation. Integrin-linked-kinase (ILK) plays important roles in multiple cellular processes and embryogenesis. ILK is expressed in the neural crest, but its role in neural crest and outflow tract morphogenesis remains unknown.
Related JoVE Video
HCN4 dynamically marks the first heart field and conduction system precursors.
Circ. Res.
PUBLISHED: 06-06-2013
Show Abstract
Hide Abstract
To date, there has been no specific marker of the first heart field to facilitate understanding of contributions of the first heart field to cardiac lineages. Cardiac arrhythmia is a leading cause of death, often resulting from abnormalities in the cardiac conduction system (CCS). Understanding origins and identifying markers of CCS lineages are essential steps toward modeling diseases of the CCS and for development of biological pacemakers.
Related JoVE Video
Expression of Isl1 during mouse development.
Gene Expr. Patterns
PUBLISHED: 04-29-2013
Show Abstract
Hide Abstract
The LIM-homeodomain transcription factor Isl1 plays essential roles in cell proliferation, differentiation and survival during embryogenesis. To better visualize Isl1 expression and provide insight into the role of Isl1 during development, we generated an Isl1 nuclear LacZ (nLacZ) knockin mouse line. We have analyzed Isl1nlacZ expression during development by Xgal staining, and compared expression of Isl1nlacZ with endogenous Isl1 by coimmunostaining with antibodies to Isl1 and ?-galactosidase. Results demonstrated that during development, Isl1 nLacZ is expressed in a pattern that recapitulates endogenous Isl1 protein expression. Consistent with previous in situ and immunohistochemistry data, we observed Isl1nlacZ expression in multiple tissues and cell types, including the central and peripheral nervous system, neural retina, inner ear, pharyngeal mesoderm and endoderm and their derivatives (craniofacial structures, thymus, thyroid gland and trachea), cardiovascular system (cardiac outflow tract, carotid arteries, umbilical vessels, sinoatrial node and atrial septum), gastrointestinal system (oral epithelium, stomach, pancreas, mesentery) and hindlimb.
Related JoVE Video
The LIM-Homeodomain transcription factor Islet-1 is required for the development of sympathetic neurons and adrenal chromaffin cells.
Dev. Biol.
PUBLISHED: 04-04-2013
Show Abstract
Hide Abstract
Islet-1 is a LIM-Homeodomain transcription factor with important functions for the development of distinct neuronal and non-neuronal cell populations. We show here that Islet-1 acts genetically downstream of Phox2B in cells of the sympathoadrenal cell lineage and that the development of sympathetic neurons and chromaffin cells is impaired in mouse embryos with a conditional deletion of Islet-1 controlled by the wnt1 promotor. Islet-1 is not essential for the initial differentiation of sympathoadrenal cells, as indicated by the correct expression of pan-neuronal and catecholaminergic subtype specific genes in primary sympathetic ganglia of Islet-1 deficient mouse embryos. However, our data indicate that the subsequent survival of sympathetic neuron precursors and their differentiation towards TrkA expressing neurons depends on Islet-1 function. In contrast to spinal sensory neurons, sympathetic neurons of Islet-1 deficient mice did not display ectopic expression of genes normally present in the CNS. In Islet-1 deficient mouse embryos the numbers of chromaffin cells were only mildly reduced, in contrast to that of sympathetic neurons, but the initiation of the adrenaline synthesizing enzyme PNMT was abrogated and the expression level of chromogranin A was diminished. Microarray analysis revealed that developing chromaffin cells of Islet-1 deficient mice displayed normal expression levels of TH, DBH and the transcription factors Phox2B, Mash-1, Hand2, Gata3 and Insm1, but the expression levels of the transcription factors Gata2 and Hand1, and AP-2ß were significantly reduced. Together our data indicate that Islet-1 is not essentially required for the initial differentiation of sympathoadrenal cells, but has an important function for the correct subsequent development of sympathetic neurons and chromaffin cells.
Related JoVE Video
Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries.
Cell Res.
PUBLISHED: 02-06-2013
Show Abstract
Hide Abstract
Coronary arteries bring blood flow to the heart muscle. Understanding the developmental program of the coronary arteries provides insights into the treatment of coronary artery diseases. Multiple sources have been described as contributing to coronary arteries including the proepicardium, sinus venosus (SV), and endocardium. However, the developmental origins of coronary vessels are still under intense study. We have produced a new genetic tool for studying coronary development, an AplnCreER mouse line, which expresses an inducible Cre recombinase specifically in developing coronary vessels. Quantitative analysis of coronary development and timed induction of AplnCreER fate tracing showed that the progenies of subepicardial endothelial cells (ECs) both invade the compact myocardium to form coronary arteries and remain on the surface to produce veins. We found that these subepicardial ECs are the major sources of intramyocardial coronary vessels in the developing heart. In vitro explant assays indicate that the majority of these subepicardial ECs arise from endocardium of the SV and atrium, but not from ventricular endocardium. Clonal analysis of Apln-positive cells indicates that a single subepicardial EC contributes equally to both coronary arteries and veins. Collectively, these data suggested that subepicardial ECs are the major source of intramyocardial coronary arteries in the ventricle wall, and that coronary arteries and veins have a common origin in the developing heart.
Related JoVE Video
Repair of chromosomal double-strand breaks by precise ligation in human cells.
DNA Repair (Amst.)
PUBLISHED: 01-03-2013
Show Abstract
Hide Abstract
Double-strand breaks (DSBs), a common type of DNA lesion, occur daily in human cells as a result of both endogenous and exogenous damaging agents. DSBs are repaired in two general ways: by the homology-dependent, error-free pathways of homologous recombination (HR) and by the homology-independent, error-prone pathways of nonhomologous end-joining (NHEJ), with NHEJ predominating in most cells. DSBs with compatible ends can be re-joined in vitro with DNA ligase alone, which raises the question of whether such DSBs require the more elaborate machinery of NHEJ to be repaired in cells. Here we report that chromosomal DSBs with compatible ends introduced by the rare-cutting endonuclease, ISceI, are repaired by precise ligation nearly 100% of the time in human cells. Precise ligation depends on the classical NHEJ components Ku70, XRCC4, and DNA ligase IV, since siRNA knockdowns of these factors significantly reduced the efficiency of precise ligation. Interestingly, knockdown of the tumor suppressors p53 or BRCA1 showed similar effects as the knockdowns of NHEJ factors. In contrast, knockdown of components involved in alternative NHEJ, mismatch repair, nucleotide excision repair, and single-strand break repair did not reduce precise ligation. In summary, our results demonstrate that DSBs in human cells are efficiently repaired by precise ligation, which requires classical NHEJ components and is enhanced by p53 and BRCA1.
Related JoVE Video
Immunological response in H22 transplanted mice undergoing Aconitum coreanum polysaccharide treatment.
Int. J. Biol. Macromol.
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
In this study, a polysaccharide (ACP-a1), with a molecular weight of 3.2×10(5)Da, was successfully purified and identified from the roots of Aconitum coreanum (Lèvl.) Rapaics. Gas chromatography (GC) analysis indicated that ACP-a1 was mainly composed of ?-d-mannose and ?-d-glucose in a molar ratio of 1.2:3.5. The effects of ACP-a1 on the tumor growth and immune function were assessed in hepatoma H22 bearing mice. Results showed that ACP-a1 significantly inhibited the growth of hepatoma H22 transplanted in mice and prolonged the survival time of H22 tumor-bearing mice. Besides, the body weight, peripheral white blood cells (WBC), thymus index and spleen index of H22 tumor-bearing were also improved after ACP-a1 treatment. Furthermore, ACP-a1 could promote the secretion of serum cytokines in H22 tumor-bearing mice, such as IL-2, TNF-? and IFN-?. Taken together, our results indicate that ACP-a1 inhibits tumor growth in vivo at least partly via improving immune responses of host organism, and seems to be safe and effective as a novel agent with immunomodulatory activity for the use of anti-tumor therapy.
Related JoVE Video
Xpa deficiency reduces CAG trinucleotide repeat instability in neuronal tissues in a mouse model of SCA1.
Hum. Mol. Genet.
PUBLISHED: 09-15-2011
Show Abstract
Hide Abstract
Expansion of trinucleotide repeats (TNRs) is responsible for a number of human neurodegenerative disorders. The molecular mechanisms that underlie TNR instability in humans are not clear. Based on results from model systems, several mechanisms for instability have been proposed, all of which focus on the ability of TNRs to form alternative structures during normal DNA transactions, including replication, DNA repair and transcription. These abnormal structures are thought to trigger changes in TNR length. We have previously shown that transcription-induced TNR instability in cultured human cells depends on several genes known to be involved in transcription-coupled nucleotide excision repair (NER). We hypothesized that NER normally functions to destabilize expanded TNRs. To test this hypothesis, we bred an Xpa null allele, which eliminates NER, into the TNR mouse model for spinocerebellar ataxia type 1 (SCA1), which carries an expanded CAG repeat tract at the endogenous mouse Sca1 locus. We find that Xpa deficiency does not substantially affect TNR instability in either the male or female germline; however, it dramatically reduces CAG repeat instability in neuronal tissues-striatum, hippocampus and cerebral cortex-but does not alter CAG instability in kidney or liver. The tissue-specific effect of Xpa deficiency represents a novel finding; it suggests that tissue-to-tissue variation in CAG repeat instability arises, in part, by different underlying mechanisms. These results validate our original findings in cultured human cells and suggest that transcription may induce NER-dependent TNR instability in neuronal tissues in humans.
Related JoVE Video
Discovery of a pharmacologically active antagonist of the two-pore-domain potassium channel K2P9.1 (TASK-3).
ChemMedChem
PUBLISHED: 07-18-2011
Show Abstract
Hide Abstract
TWIK-related acid-sensitive K(+) (K(2P) 9.1, TASK-3) ion channels have the capacity to regulate the activity of neuronal pathways by influencing the resting membrane potential of neurons on which they are expressed. The central nervous system (CNS) expression of these channels suggests potential roles in neurologic disorders, and it is believed that the development of TASK-3 antagonists could lead to the therapeutic treatment of a number of neurological conditions. While a therapeutic potential for TASK-3 channel modulation exists, there are only a few documented examples of potent and selective small-molecule channel blockers. Herein, we describe the discovery and lead optimization efforts for a novel series of TASK-3 channel antagonists based on a 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine high-throughput screening lead from which a subseries of potent and selective inhibitors were identified. One compound was profiled in detail with respect to its physical properties and demonstrated pharmacological target engagement as indicated by its ability to modulate sleep architecture in rodent electroencephalogram (EEG) telemetry models.
Related JoVE Video
Topoisomerase 1 and single-strand break repair modulate transcription-induced CAG repeat contraction in human cells.
Mol. Cell. Biol.
PUBLISHED: 05-31-2011
Show Abstract
Hide Abstract
Expanded trinucleotide repeats are responsible for a number of neurodegenerative diseases, such as Huntington disease and myotonic dystrophy type 1. The mechanisms that underlie repeat instability in the germ line and in the somatic tissues of human patients are undefined. Using a selection assay based on contraction of CAG repeat tracts in human cells, we screened the Prestwick chemical library in a moderately high-throughput assay and identified 18 novel inducers of repeat contraction. A subset of these compounds targeted pathways involved in the management of DNA supercoiling associated with transcription. Further analyses using both small molecule inhibitors and small interfering RNA (siRNA)-mediated knockdowns demonstrated the involvement of topoisomerase 1 (TOP1), tyrosyl-DNA phosphodiesterase 1 (TDP1), and single-strand break repair (SSBR) in modulating transcription-dependent CAG repeat contractions. The TOP1-TDP1-SSBR pathway normally functions to suppress repeat instability, since interfering with it stimulated repeat contractions. We further showed that the increase in repeat contractions when the TOP1-TDP1-SSBR pathway is compromised arises via transcription-coupled nucleotide excision repair, a previously identified contributor to transcription-induced repeat instability. These studies broaden the scope of pathways involved in transcription-induced CAG repeat instability and begin to define their interrelationships.
Related JoVE Video
Isl1 is required for multiple aspects of motor neuron development.
Mol. Cell. Neurosci.
PUBLISHED: 03-25-2011
Show Abstract
Hide Abstract
The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in multiple organs and plays essential roles during embryogenesis. Isl1 is required for the survival and specification of spinal cord motor neurons. Due to early embryonic lethality and loss of motor neurons, the role of Isl1 in other aspects of motor neuron development remains unclear. In this study, we generated Isl1 mutant mouse lines expressing graded doses of Isl1. Our study has revealed essential roles of Isl1 in multiple aspects of motor neuron development, including motor neuron cell body localization, motor column formation and axon growth. In addition, Isl1 is required for survival of cranial ganglia neurons.
Related JoVE Video
MicroRNA-134 as a potential plasma biomarker for the diagnosis of acute pulmonary embolism.
J Transl Med
PUBLISHED: 03-05-2011
Show Abstract
Hide Abstract
Acute pulmonary embolism (APE) remains a diagnostic challenge due to a variable clinical presentation and the lack of a reliable screening tool. MicroRNAs (miRNAs) regulate gene expression in a wide range of pathophysiologic processes. Circulating miRNAs are emerging biomarkers in heart failure, type 2 diabetes and other disease states; however, using plasma miRNAs as biomarkers for the diagnosis of APE is still unknown.
Related JoVE Video
MicroRNA expression signature in atrial fibrillation with mitral stenosis.
Physiol. Genomics
PUBLISHED: 02-15-2011
Show Abstract
Hide Abstract
The aim of this study was to investigate the microRNA (miRNA) signature in atrial fibrillation (AF) with mitral stenosis (MS). miRNA arrays were used to evaluate the expression signature of the right atrial appendages of healthy individuals (n=9), patients with MS and AF (n=9) and patients with MS without AF (n=4). The results were validated with qRT-PCR analysis. GOmir was used to predict the potential miRNA targets and to analyze their functions. DIANA-mirPath was used to incorporate the miRNAs into pathways. miRNA arrays revealed that 136 and 96 miRNAs were expressed at different levels in MS patients with AF and in MS patients without AF, respectively, compared with healthy controls. More importantly, 28 miRNAs were expressed differently in the MS patients with AF compared with the MS patients without AF; of these miRNAs, miR-1202 was the most dysregulated. The unsupervised hierarchical clustering analysis based on the 28 differently expressed miRNAs showed that the heat map of miRNA expression categorized two well-defined clusters that corresponded to MS with AF and MS without AF. The qRT-PCR results correlated well with the microarray data. Bioinformatic analysis indicated the potential miRNA targets and molecular pathways. This study shows that there is a distinct miRNA expression signature in AF with MS. The findings may be useful for the development of therapeutic interventions that are based on rational target selection in these patients.
Related JoVE Video
Transcription-induced DNA toxicity at trinucleotide repeats: double bubble is trouble.
Cell Cycle
PUBLISHED: 02-15-2011
Show Abstract
Hide Abstract
Trinucleotide repeats (TNR) are a blessing and a curse. In coding regions, where they are enriched, short repeats offer the potential for continuous, rapid length variation with linked incremental changes in the activity of the encoded protein, a valuable source of variation for evolution. But at the upper end of these benign and beneficial lengths, trinucleotide repeats become very unstable, with a dangerous bias toward continual expansion, which can lead to neurological diseases in humans. The mechanisms of expansion are varied and the links to disease are complex. Where they have been delineated, however, they have often revealed unexpected, fundamental aspects of the underlying cell biology. Nowhere is this more apparent than in recent studies, which indicate that expanded CAG repeats can form toxic sites in the genome, which can, upon interaction with normal components of DNA metabolism, trigger cell death. Here we discuss the phenomenon of TNR-induced DNA toxicity, with special emphasis on the role of transcription. Transcription-induced DNA toxicity may have profound biological consequences, with particular relevance to repeat-associated neurodegenerative diseases.
Related JoVE Video
Prolyl hydroxylase 2: a novel regulator of ?2 -adrenoceptor internalization.
J. Cell. Mol. Med.
PUBLISHED: 01-25-2011
Show Abstract
Hide Abstract
Adrenergic receptor (AR)-mediated signalling is modulated by oxygen levels. Prolyl hydroxylases (PHDs) are crucial for intracellular oxygen sensing and organism survival. However, it remains to be clarified whether or how PHDs are involved in the regulation of ?(2) -adrenoceptor (?(2) -AR) signalling. Here we show that PHD2 can modulate the rate of ?(2) -AR internalization through interactions with ?-arrestin 2. PHD2 hydroxylates ?-arrestin 2 at the proline (Pro)(176), Pro(179) and Pro(181) sites, which retards the recruitment of ?-arrestin 2 to the plasma membrane and inhibits subsequent co-internalization with ?(2) -AR into the cytosol. ?(2) -AR internalization is critical to control the temporal and spatial aspects of ?(2) -AR signalling. Identifying novel regulators of ?(2) -AR internalization will enable us to develop new strategies to manipulate receptor signalling and provide potential targets for drug development in the prevention and treatment of diseases associated with ?(2) -AR signalling dysregulation.
Related JoVE Video
Prolyl hydroxylase 3 interacts with Bcl-2 to regulate doxorubicin-induced apoptosis in H9c2 cells.
Biochem. Biophys. Res. Commun.
PUBLISHED: 08-24-2010
Show Abstract
Hide Abstract
Prolyl hydroxylases (PHDs) are dioxygenases that use oxygen as a co-substrate to hydroxylate proline residues. Three PHD isoforms (PHD1, PHD2 and PHD3) have been identified in mammalian cells. PHD3 expression is upregulated in some cardiac diseases such as cardiomyopathy, myocardial ischemia-reperfusion injury and congestive heart failure, all of which are associated with apoptosis. However, the role of PHDs in cardiomyocyte apoptosis remains unknown. Here, we have found that exposure of embryonic rat heart-derived H9c2 cells to doxorubicin (DOX) induced cell apoptosis as evaluated by caspase-3/7 activity, mitochondrial membrane potential (??m) and cell viability, and that this apoptosis was linked to PHD3 upregulation. PHD inhibition or PHD3 silencing substantially ameliorated DOX-induced apoptosis, but PHD1 or PHD2 knockdown did not significantly influence apoptosis. Furthermore, immunoprecipitation experiments showed that PHD3 upregulation reduced the formation of the Bax-Bcl-2 complex, inhibiting the anti-apoptotic effect of Bcl-2. Thus, PHD3 upregulation may be partially responsible for DOX-induced cardiomyocyte apoptosis via its interaction with Bcl-2. Inhibition of PHD3 is likely to be cardioprotective against apoptosis in some heart disorders.
Related JoVE Video
Convergent transcription through a long CAG tract destabilizes repeats and induces apoptosis.
Mol. Cell. Biol.
PUBLISHED: 07-20-2010
Show Abstract
Hide Abstract
Short repetitive sequences are common in the human genome, and many fall within transcription units. We have previously shown that transcription through CAG repeat tracts destabilizes them in a way that depends on transcription-coupled nucleotide excision repair and mismatch repair. Recent observations that antisense transcription accompanies sense transcription in many human genes led us to test the effects of antisense transcription on triplet repeat instability in human cells. Here, we report that simultaneous sense and antisense transcription (convergent transcription) initiated from two inducible promoters flanking a CAG95 tract in a nonessential gene enhances repeat instability synergistically, arrests the cell cycle, and causes massive cell death via apoptosis. Using chemical inhibitors and small interfering RNA (siRNA) knockdowns, we identified the ATR (ataxia-telangiectasia mutated [ATM] and Rad3 related) signaling pathway as a key mediator of this cellular response. RNA polymerase II, replication protein A (RPA), and components of the ATR signaling pathway accumulate at convergently transcribed repeat tracts, accompanied by phosphorylation of ATR, CHK1, and p53. Cell death depends on simultaneous sense and antisense transcription and is proportional to their relative levels, it requires the presence of the repeat tract, and it occurs in both proliferating and nonproliferating cells. Convergent transcription through a CAG repeat represents a novel mechanism for triggering a cellular stress response, one that is initiated by events at a single locus in the genome and resembles the response to DNA damage.
Related JoVE Video
Gastrin-releasing peptide promotes the growth of HepG2 cells via EGFR-independent ERK1/2 activation.
Oncol. Rep.
PUBLISHED: 07-03-2010
Show Abstract
Hide Abstract
Gastrin-releasing peptide (GRP) plays an important role in regulating tumor growth and migration. However, little is known about its role in human hepatocellular carcinoma (HCC) cells. This study explored the effect of GRP on the growth of HCC HepG2 cells and the underlying mechanisms. Expression of GRP and its cognate receptor (GRPR) were detected by immunocytochemisty, reverse transcription-PCR and Western blotting and compared between two human HCC cell lines (HepG2 and MHCC97H) and a normal hepatic cell line (HL-7702). The effects of GRP on cell proliferation and signaling pathways were examined by Western blotting, MTT assay and flow cytometry. Both GRP and GRPR were overexpressed in HepG2 and MHCC97H cells. GRP activated MAPK/ERK1/2 in HepG2 cells, leading to enhanced proliferation, reduced apoptosis and accelerated cell cycle progression. The effect of GRP on ERK1/2 was effectively attenuated by the GRPR antagonist PD176252 or MEK inhibitor U0126, but not by the TNF-alpha protease inhibitor TAPI-1 or the EGFR tyrosine kinase inhibitor PD153035. The effect of GRP on the growth of HepG2 cells was significantly attenuated by PD176252 or U0126. GRP serves as a mitogen for HepG2 and MHCC97H cells. GRP promotes the growth of HepG2 cells through interaction with GRPR co-expressed in tumor cells, and subsequently activates MAPK/ERK1/2 via EGFR-independent mechanisms.
Related JoVE Video
beta2- but not beta1-adrenoceptor activation modulates intracellular oxygen availability.
J. Physiol. (Lond.)
PUBLISHED: 06-14-2010
Show Abstract
Hide Abstract
beta-Adrenoceptors (beta-ARs) play a critical role in the regulation of cardiovascular function. Intracellular oxygen homeostasis is crucial for the survival of cardiomyocytes. However, it is still unclear whether beta-AR activation can modulate intracellular oxygen. Here we used mitochondrial and cytosolic target Renilla luciferase to detect intracellular oxygen concentration. Pharmacological experiments revealed that beta2-AR activation specifically regulates intracellular oxygen in cardiomyocytes and COS7 cells. This effect was abrogated by inhibitory G protein (Gi) inhibition, endothelial nitric oxide synthase (eNOS) blockade, and NO scavenging, implicating that the beta2-AR-Gi-eNOS pathway is involved in this regulation. beta2-AR activation increased the AMP/ATP ratio, AMPK activity, ROS production and prolyl hydroxylase activity. These effects also contribute to the regulation of beta2-AR signalling, thus providing an additional layer of complexity to enforce the specificity of beta1-AR and beta2-AR signalling. Collectively, the study provides novel insight into the modulation of oxygen homeostasis, broadens the scope of beta2-AR function, and may have crucial implications for beta2-AR signalling regulation.
Related JoVE Video
Hsp90 modulates CAG repeat instability in human cells.
Cell Stress Chaperones
PUBLISHED: 03-02-2010
Show Abstract
Hide Abstract
The Hsp90 molecular chaperone has been implicated as a contributor to evolution in several organisms by revealing cryptic variation that can yield dramatic phenotypes when the chaperone is diverted from its normal functions by environmental stress. In addition, as a cancer drug target, Hsp90 inhibition has been documented to sensitize cells to DNA-damaging agents, suggesting a function for Hsp90 in DNA repair. Here we explore the potential role of Hsp90 in modulating the stability of nucleotide repeats, which in a number of species, including humans, exert subtle and quantitative consequences for protein function, morphological and behavioral traits, and disease. We report that impairment of Hsp90 in human cells induces contractions of CAG repeat tracks by tenfold. Inhibition of the recombinase Rad51, a downstream target of Hsp90, induces a comparable increase in repeat instability, suggesting that Hsp90-enabled homologous recombination normally functions to stabilize CAG repeat tracts. By contrast, Hsp90 inhibition does not increase the rate of gene-inactivating point mutations. The capacity of Hsp90 to modulate repeat-tract lengths suggests that the chaperone, in addition to exposing cryptic variation, might facilitate the expression of new phenotypes through induction of novel genetic variation.
Related JoVE Video
Gastrin-releasing peptide links stressor to cancer progression.
J. Cancer Res. Clin. Oncol.
PUBLISHED: 01-13-2010
Show Abstract
Hide Abstract
Gastrin-releasing peptide (GRP) plays an important role in cancer growth and metastasis; however, the mechanisms of how GRP affects cancer progression are not well understood. Recent studies revealed that chronic stress is a major risk factor for cancer progression, and this effect may be mediated by GRP. In this review, we will discuss the mechanisms and implications of GRP linking stressor to cancer progression.
Related JoVE Video
R loops stimulate genetic instability of CTG.CAG repeats.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 12-22-2009
Show Abstract
Hide Abstract
Transcription stimulates the genetic instability of trinucleotide repeat sequences. However, the mechanisms leading to transcription-dependent repeat length variation are unclear. We demonstrate, using biochemical and genetic approaches, that the formation of stable RNA.DNA hybrids enhances the instability of CTG.CAG repeat tracts. In vitro transcribed CG-rich repeating sequences, unlike AT-rich repeats and nonrepeating sequences, form stable, ribonuclease A-resistant structures. These RNA.DNA hybrids are eliminated by ribonuclease H treatment. Mutation in the rnhA1 gene that decreases the activity of ribonuclease HI stimulates the instability of CTG.CAG repeats in E. coli. Importantly, the effect of ribonuclease HI depletion on repeat instability requires active transcription. We also showed that transcription-dependent CTG.CAG repeat instability in human cells is stimulated by siRNA knockdown of RNase H1 and H2. In addition, we used bisulfite modification, which detects single-stranded DNA, to demonstrate that the nontemplate DNA strand at transcribed CTG.CAG repeats remains partially single-stranded in human genomic DNA, thus indicating that it is displaced by an RNA.DNA hybrid. These studies demonstrate that persistent hybrids between the nascent RNA transcript and the template DNA strand at CTG.CAG tracts promote instability of DNA trinucleotide repeats.
Related JoVE Video
Particularly interesting cysteine- and histidine-rich protein in cardiac development and remodeling.
J. Investig. Med.
PUBLISHED: 12-03-2009
Show Abstract
Hide Abstract
Integrin-mediated cell-extracellular matrix interaction plays key roles in tissue morphogenesis and integrity. The Lin11-Isl-1-Mec-3 (LIM) domain-only particularly interesting cysteine- and histidine-rich (PINCH) protein functions as an adaptor essential for the assembly and function of the focal adhesion complex that links integrin signaling to the cytoskeleton and other intracellular signaling pathways and regulates diverse cellular processes such as cell adhesion, migration, growth, differentiation, and survival. Recent biochemical and genetic studies have greatly advanced our knowledge surrounding the molecular interactions and functions of each component of the focal adhesion complex and revealed a requirement for PINCH in early embryogenesis, in morphogenesis of the neural crest and cardiac outflow, and in myocardial growth and remodeling. In this review article, we will provide an overview of the current knowledge of the molecular interactions of PINCH with other components of focal adhesions, highlighting recent discoveries of the in vivo role of PINCH and discuss its potential implication for human heart disease.
Related JoVE Video
Islet-to-LMO stoichiometries control the function of transcription complexes that specify motor neuron and V2a interneuron identity.
Development
PUBLISHED: 08-12-2009
Show Abstract
Hide Abstract
LIM transcription factors bind to nuclear LIM interactor (Ldb/NLI/Clim) in specific ratios to form higher-order complexes that regulate gene expression. Here we examined how the dosage of LIM homeodomain proteins Isl1 and Isl2 and LIM-only protein Lmo4 influences the assembly and function of complexes involved in the generation of spinal motor neurons (MNs) and V2a interneurons (INs). Reducing the levels of Islet proteins using a graded series of mutations favored V2a IN differentiation at the expense of MN formation. Although LIM-only proteins (LMOs) are predicted to antagonize the function of Islet proteins, we found that the presence or absence of Lmo4 had little influence on MN or V2a IN specification. We did find, however, that the loss of MNs resulting from reduced Islet levels was rescued by eliminating Lmo4, unmasking a functional interaction between these proteins. Our findings demonstrate that MN and V2a IN fates are specified by distinct complexes that are sensitive to the relative stoichiometries of the constituent factors and we present a model to explain how LIM domain proteins modulate these complexes and, thereby, this binary-cell-fate decision.
Related JoVE Video
Targeted ablation of PINCH1 and PINCH2 from murine myocardium results in dilated cardiomyopathy and early postnatal lethality.
Circulation
PUBLISHED: 08-03-2009
Show Abstract
Hide Abstract
PINCH proteins are 5 LIM domain-only adaptor proteins that function as key components of the integrin signaling pathway and play crucial roles in multiple cellular processes. Two PINCH proteins, PINCH1 and PINCH2, have been described in mammals and share high homology. Both PINCH1 and PINCH2 are ubiquitously expressed in most tissues and organs, including myocardium. Cardiac-specific PINCH1 knockout or global PINCH2 knockout mice exhibit no basal cardiac phenotype, which may reflect a redundant role for these 2 PINCH proteins in myocardium. A potential role for PINCH proteins in myocardium remains unknown.
Related JoVE Video
Crop diversity for yield increase.
PLoS ONE
PUBLISHED: 05-30-2009
Show Abstract
Hide Abstract
Traditional farming practices suggest that cultivation of a mixture of crop species in the same field through temporal and spatial management may be advantageous in boosting yields and preventing disease, but evidence from large-scale field testing is limited. Increasing crop diversity through intercropping addresses the problem of increasing land utilization and crop productivity. In collaboration with farmers and extension personnel, we tested intercropping of tobacco, maize, sugarcane, potato, wheat and broad bean--either by relay cropping or by mixing crop species based on differences in their heights, and practiced these patterns on 15,302 hectares in ten counties in Yunnan Province, China. The results of observation plots within these areas showed that some combinations increased crop yields for the same season between 33.2 and 84.7% and reached a land equivalent ratio (LER) of between 1.31 and 1.84. This approach can be easily applied in developing countries, which is crucial in face of dwindling arable land and increasing food demand.
Related JoVE Video
Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-29-2009
Show Abstract
Hide Abstract
Expanded triplet repeats have been identified as the genetic basis for a growing number of neurological and skeletal disorders. To examine the contribution of double-strand break repair to CAG x CTG repeat instability in mammalian systems, we developed zinc finger nucleases (ZFNs) that recognize and cleave CAG repeat sequences. Engineered ZFNs use a tandem array of zinc fingers, fused to the FokI DNA cleavage domain, to direct double-strand breaks (DSBs) in a site-specific manner. We first determined that the ZFNs cleave CAG repeats in vitro. Then, using our previously described tissue culture assay for identifying modifiers of CAG repeat instability, we found that transfection of ZFN-expression vectors induced up to a 15-fold increase in changes to the CAG repeat in human and rodent cell lines, and that longer repeats were much more sensitive to cleavage than shorter ones. Analysis of individual colonies arising after treatment revealed a spectrum of events consistent with ZFN-induced DSBs and dominated by repeat contractions. We also found that expressing a dominant-negative form of RAD51 in combination with a ZFN, dramatically reduced the effect of the nuclease, suggesting that DSB-induced repeat instability is mediated, in part, through homology directed repair. These studies identify a ZFN as a useful reagent for characterizing the effects of DSBs on CAG repeats in cells.
Related JoVE Video
Transcription destabilizes triplet repeats.
Mol. Carcinog.
PUBLISHED: 04-25-2009
Show Abstract
Hide Abstract
Triplet repeat expansion is the molecular basis for several human diseases. Intensive studies using systems in bacteria, yeast, flies, mammalian cells, and mice have provided important insights into the molecular processes that are responsible for mediating repeat instability. The age-dependent, ongoing repeat instability in somatic tissues, especially in terminally differentiated neurons, strongly suggests a robust role for pathways that are independent of DNA replication. Several genetic studies have indicated that transcription can play a critical role in repeat instability, potentially providing a basis for the instability observed in neurons. Transcription-induced repeat instability can be modulated by several DNA repair proteins, including those involved in mismatch repair (MMR) and transcription-coupled nucleotide excision repair (TC-NER). Though the mechanism is unclear, it is likely that transcription facilitates the formation of repeat-specific secondary structures, which act as intermediates to trigger DNA repair, eventually leading to changes in the length of the repeat tract. In addition, other processes associated with transcription can also modulate repeat instability, as shown in a variety of different systems. Overall, the mechanisms underlying repeat instability in humans are unexpectedly complicated. Because repeat-disease genes are widely expressed, transcription undoubtedly contributes to the repeat instability observed in many diseases, but it may be especially important in nondividing cells. Transcription-induced instability is likely to involve an extensive interplay not only of the core transcription machinery and DNA repair proteins, but also of proteins involved in chromatin remodeling, regulation of supercoiling, and removal of stalled RNA polymerases, as well as local DNA sequence effects.
Related JoVE Video
A catalytic resonance fluorometry method for determination of hydroquinone and its applications.
Anal Sci
PUBLISHED: 04-11-2009
Show Abstract
Hide Abstract
Pyronin Y (PY) has a strong resonance fluorescence in sulfuric acid medium. The characteristics of the resonance fluorescence spectra and the factors affecting the spectra were studied. A catalytic resonance fluorometry method for the determination of hydroquinone was proposed based on the catalytic effect of hydroquinone on the oxidation of PY by potassium bromate. The oxidation of PY resulted in the decrease of the resonance fluorescence intensity. The influences of several variables on the sensitivity were studied. At the optimized conditions, the decrease of the resonance fluorescence intensity was in proportion to the concentration of hydroquinone in the range of 4.42-1.60 x 10(3) microg l(-1), and the detection limit was 1.46 microg l(-1). The proposed method was applied successfully for the determination of trace hydroquinone in environment samples. The relative standard deviation was less than 3.90% and the recoveries were in the range of 95.2-104.0%.
Related JoVE Video
Mdm2 deficiency suppresses MYCN-Driven neuroblastoma tumorigenesis in vivo.
Neoplasia
PUBLISHED: 03-11-2009
Show Abstract
Hide Abstract
Neuroblastoma is derived from neural crest precursor components of the peripheral sympathetic nervous system and accounts for more than 15% of all pediatric cancer deaths. A clearer understanding of the molecular basis of neuroblastoma is required for novel therapeutic approaches to improve morbidity and mortality. Neuroblastoma is uniformly p53 wild type at diagnosis and must overcome p53-mediated tumor suppression during pathogenesis. Amplification of the MYCN oncogene correlates with the most clinically aggressive form of the cancer, and MDM2, a primary inhibitor of the p53 tumor suppressor, is a direct transcriptional target of, and positively regulated by, both MYCN and MYCC. We hypothesize that MDM2 contributes to MYCN-driven tumorigenesis helping to ameliorate p53-dependent apoptotic oncogenic stress during tumor initiation and progression. To study the interaction of MYCN and MDM2, we generated an Mdm2 haploinsufficient transgenic animal model of neuroblastoma. In Mdm2(+/-)MYCN transgenics, tumor latency and animal survival are remarkably extended, whereas tumor incidence and growth are reduced. Analysis of the Mdm2/p53 pathway reveals remarkable p53 stabilization counter-balanced by epigenetic silencing of the p19(Arf) gene in the Mdm2 haploinsufficient tumors. In human neuroblastoma xenograft models, conditional small interfering RNA-mediated knockdown of MDM2 in cells expressing wild-type p53 dramatically suppresses tumor growth in a p53-dependent manner. In summary, we provided evidence for a crucial role for direct inhibition of p53 by MDM2 and suppression of the p19(ARF)/p53 axis in neuroblastoma tumorigenesis, supporting the development of therapies targeting these pathways.
Related JoVE Video
The molecular resonance fluorescence method for determination of arsenic in hair samples.
Spectrochim Acta A Mol Biomol Spectrosc
PUBLISHED: 01-14-2009
Show Abstract
Hide Abstract
A molecular resonance fluorescence quenching method for the determination of arsenic was proposed. The method is based on the quenching effect of As (V) on the molecular resonance fluorescence of Rhodamine B (RhB) in sulfuric acid medium and in the presence of molybdate and PVA. The influences of acidity, chemical and manifold variables on the sensitivity were studied. At the optimized conditions, the linear range for the determination of arsenic was 2.0x10(-3)-0.12microgml(-1) with the detection limit of 1.20x10(-9)gml(-1). The relative standard deviations for the eleven replicate determinations of 0.006microgml(-1) and 0.060microgml(-1) of arsenic were 1.12% and 1.61%, respectively. The proposed method was applied successfully for the determination of trace arsenic in hair samples with the recoveries of 94.8-104.6%.
Related JoVE Video
Diverse effects of individual mismatch repair components on transcription-induced CAG repeat instability in human cells.
DNA Repair (Amst.)
PUBLISHED: 01-05-2009
Show Abstract
Hide Abstract
Several neurodegerative diseases are caused by expansion of a trinucleotide repeat tract in a critical gene. The mechanism of repeat instability is not yet defined, but in mice it requires MutSbeta, a complex of MSH2 and MSH3. We showed previously that transcription through a CAG repeat tract induces repeat instability in human cells via a pathway that requires the mismatch repair (MMR) components, MSH2 and MSH3, and the entire transcription-coupled nucleotide excision repair pathway [Y. Lin, V. Dion, J.H. Wilson, Transcription promotes contraction of CAG repeat tracts in human cells, Nat. Struct. Mol. Biol. 13 (2006) 179-180; Y. Lin, J.H. Wilson, Transcription-induced CAG repeat contraction in human cells is mediated in part by transcription-coupled nucleotide excision repair, Mol. Cell Biol. 27 (2007) 6209-6217]. Here, we examine the role of downstream MMR processing components on transcription-induced CAG instability, using our selection assay for repeat contraction. In contrast to knockdowns of MSH2 or MSH3, which reduce repeat contractions, we show that siRNA-mediated depletion of MLH1 or PMS2 increases contraction frequency. Knockdown of DNMT1, which has been identified as an MMR factor in genetic studies, also elevates the frequency of contraction. Simultaneous knockdowns of MLH1 or DNMT1 along with MSH2, XPA, or BRCA1, whose individual knockdowns each decrease CAG contraction, yield intermediate frequencies. In sharp contrast, double knockdown of MLH1 and DNMT1 additively increases the frequency of CAG contraction. These results show that MMR components can alter repeat stability in diverse ways, either enhancing or suppressing CAG contraction, and they provide insight into the influence of MMR components on transcription-induced CAG repeat instability.
Related JoVE Video
[Rapid determination of pesticide multiresidues in vegetables and fruits by accelerated solvent extraction coupled with online gel permeation chromatography-gas chromatography-mass spectrometry].
Se Pu
Show Abstract
Hide Abstract
A novel method was developed for the rapid determination of 22 representative pesticide residues in vegetables and fruits based on accelerated solvent extraction (ASE) coupled with online gel permeation chromatography-gas chromatography-mass spectrometry (GPC-GC-MS). The sample was extracted by accelerated solvent extraction with dichloromethane-acetone (1:1, v/v) and purified with a carbon/NH2 column, evaporated to dryness by nitrogen, then dissolved in cyclohexane-acetone (7:3, v/v), and finally identified and quantified by GPC-GC-MS system in selected ion monitoring (SIM) mode. The results showed that the linearities of the 22 pesticides were good in their linear ranges. The limits of detection (S/N = 3) were 0.3-1.8 microg/kg. The limits of quantification (S/N = 10) ranged from 1-6 microg/kg. The recoveries for all at three spiked levels in Chinese cabbages and apples ranged from 70.5% to 107.5% with the relative standard deviations (RSDs) of 2.1%-8.7%. The proposed method is accurate, sensitive and highly efficient in the extraction, and can be used for the quick determination of the pesticide multiresidues in vegetables and fruits.
Related JoVE Video
The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions.
Nat. Genet.
Show Abstract
Hide Abstract
Watermelon, Citrullus lanatus, is an important cucurbit crop grown throughout the world. Here we report a high-quality draft genome sequence of the east Asia watermelon cultivar 97103 (2n = 2× = 22) containing 23,440 predicted protein-coding genes. Comparative genomics analysis provided an evolutionary scenario for the origin of the 11 watermelon chromosomes derived from a 7-chromosome paleohexaploid eudicot ancestor. Resequencing of 20 watermelon accessions representing three different C. lanatus subspecies produced numerous haplotypes and identified the extent of genetic diversity and population structure of watermelon germplasm. Genomic regions that were preferentially selected during domestication were identified. Many disease-resistance genes were also found to be lost during domestication. In addition, integrative genomic and transcriptomic analyses yielded important insights into aspects of phloem-based vascular signaling in common between watermelon and cucumber and identified genes crucial to valuable fruit-quality traits, including sugar accumulation and citrulline metabolism.
Related JoVE Video
Nucleotide excision repair, mismatch repair, and R-loops modulate convergent transcription-induced cell death and repeat instability.
PLoS ONE
Show Abstract
Hide Abstract
Expansion of CAG•CTG tracts located in specific genes is responsible for 13 human neurodegenerative disorders, the pathogenic mechanisms of which are not yet well defined. These disease genes are ubiquitously expressed in human tissues, and transcription has been identified as one of the major pathways destabilizing the repeats. Transcription-induced repeat instability depends on transcription-coupled nucleotide excision repair (TC-NER), the mismatch repair (MMR) recognition component MSH2/MSH3, and RNA/DNA hybrids (R-loops). Recently, we reported that simultaneous sense and antisense transcription-convergent transcription-through a CAG repeat not only promotes repeat instability, but also induces a cell stress response, which arrests the cell cycle and eventually leads to massive cell death via apoptosis. Here, we use siRNA knockdowns to investigate whether NER, MMR, and R-loops also modulate convergent-transcription-induced cell death and repeat instability. We find that siRNA-mediated depletion of TC-NER components increases convergent transcription-induced cell death, as does the simultaneous depletion of RNase H1 and RNase H2A. In contrast, depletion of MSH2 decreases cell death. These results identify TC-NER, MMR recognition, and R-loops as modulators of convergent transcription-induced cell death and shed light on the molecular mechanism involved. We also find that the TC-NER pathway, MSH2, and R-loops modulate convergent transcription-induced repeat instability. These observations link the mechanisms of convergent transcription-induced repeat instability and convergent transcription-induced cell death, suggesting that a common structure may trigger both outcomes.
Related JoVE Video
MicroRNA-204 is required for differentiation of human-derived cardiomyocyte progenitor cells.
J. Mol. Cell. Cardiol.
Show Abstract
Hide Abstract
Human cardiomyocyte progenitor cells (hCMPCs) are cardiac progenitor cells that are unique for their efficient differentiation into beating cardiomyocytes without requiring co-culture with neonatal cardiomyocytes. hCMPCs have shown great potential in preserving the function of infarcted mouse myocardium. MiRNA-204 has been reported to be up-regulated in differentiated hCMPCs, however, its biological significance is unclear. In this study, hCMPC proliferation, viability, apoptosis and necrosis were determined using the ELISA Kit (colorimetric BrdU detection), Cell Counting Kit-8, and Annexin V and propidium iodide staining, respectively. MiRNA-204 inhibition promoted hCMPC proliferation without affecting cell viability and the level of apoptosis and necrosis, indicating that miRNA-204 might be required for hCMPC differentiation. Quantitative reverse transcriptase-polymerase chain reactions were used to detect the expression profile of cardiac genes, including MEF2C, GATA-4, Nkx-2.5, TropT, ?MHC, and cActin. Cardiac ?-actin staining was used to quantify the degree of differentiation. MiRNA-204 inhibition significantly down-regulated TropT, ?MHC, and cActin and reduced differentiation by 47.81% after 2 weeks of differentiation induction. Interestingly, miRNA-204 mimics (30 nM) did not promote hCMPC proliferation and differentiation. The bioinformatic tool GOmir identified the activating transcription factor 2 (ATF-2) as a potential target, which was confirmed by Western blot and a luciferase reporter assay. ATF-2 overexpression promoted hCMPC proliferation, further demonstrating the role played by ATF-2 as a target gene of miRNA-204. Therefore, miRNA-204 is required for hCMPC differentiation and ATF-2 is a target gene of miRNA-204 in hCMPCs. This study indicates that miRNA-204 is among the regulators that drive hCMPC proliferation and differentiation, and miRNA-204 might be used to influence cell fate.
Related JoVE Video
The transcription factor encyclopedia.
Dimas Yusuf, Stefanie L Butland, Magdalena I Swanson, Eugene Bolotin, Amy Ticoll, Warren A Cheung, Xiao Yu Cindy Zhang, Christopher T D Dickman, Debra L Fulton, Jonathan S Lim, Jake M Schnabl, Oscar H P Ramos, Mireille Vasseur-Cognet, Charles N de Leeuw, Elizabeth M Simpson, Gerhart U Ryffel, Eric W-F Lam, Ralf Kist, Miranda S C Wilson, Raquel Marco-Ferreres, Jan J Brosens, Leonardo L Beccari, Paola Bovolenta, Bérénice A Benayoun, Lara J Monteiro, Helma D C Schwenen, Lars Grontved, Elizabeth Wederell, Susanne Mandrup, Reiner A Veitia, Harini Chakravarthy, Pamela A Hoodless, M Michela Mancarelli, Bruce E Torbett, Alison H Banham, Sekhar P Reddy, Rebecca L Cullum, Michaela Liedtke, Mario P Tschan, Michelle Vaz, Angie Rizzino, Mariastella Zannini, Seth Frietze, Peggy J Farnham, Astrid Eijkelenboom, Philip J Brown, David Laperriere, Dominique Leprince, Tiziana de Cristofaro, Kelly L Prince, Marrit Putker, Luis del Peso, Gieri Camenisch, Roland H Wenger, Michal Mikula, Marieke Rozendaal, Sylvie Mader, Jerzy Ostrowski, Simon J Rhodes, Capucine Van Rechem, Gaylor Boulay, Sam W Z Olechnowicz, Mary B Breslin, Michael S Lan, Kyster K Nanan, Michael Wegner, Juan Hou, Rachel D Mullen, Stephanie C Colvin, Peter John Noy, Carol F Webb, Matthew E Witek, Scott Ferrell, Juliet M Daniel, Jason Park, Scott A Waldman, Daniel J Peet, Michael Taggart, Padma-Sheela Jayaraman, Julien J Karrich, Bianca Blom, Farhad Vesuna, Henriette O'Geen, Yunfu Sun, Richard M Gronostajski, Mark W Woodcroft, Margaret R Hough, Edwin Chen, G Nicholas Europe-Finner, Magdalena Karolczak-Bayatti, Jarrod Bailey, Oliver Hankinson, Venu Raman, David P LeBrun, Shyam Biswal, Christopher J Harvey, Jason P DeBruyne, John B Hogenesch, Robert F Hevner, Christophe Héligon, Xin M Luo, Marissa Cathleen Blank, Kathleen Joyce Millen, David S Sharlin, Douglas Forrest, Karin Dahlman-Wright, Chunyan Zhao, Yuriko Mishima, Satrajit Sinha, Rumela Chakrabarti, Elodie Portales-Casamar, Frances M Sladek, Philip H Bradley, Wyeth W Wasserman.
Genome Biol.
Show Abstract
Hide Abstract
Here we present the Transcription Factor Encyclopedia (TFe), a new web-based compendium of mini review articles on transcription factors (TFs) that is founded on the principles of open access and collaboration. Our consortium of over 100 researchers has collectively contributed over 130 mini review articles on pertinent human, mouse and rat TFs. Notable features of the TFe website include a high-quality PDF generator and web API for programmatic data retrieval. TFe aims to rapidly educate scientists about the TFs they encounter through the delivery of succinct summaries written and vetted by experts in the field. TFe is available at http://www.cisreg.ca/tfe.
Related JoVE Video
Cyclin-dependent kinase 4 is a novel target in micoRNA-195-mediated cell cycle arrest in bladder cancer cells.
FEBS Lett.
Show Abstract
Hide Abstract
miRNAs are a class of small-noncoding RNAs capable of negatively regulating gene expression. Here, we found that miR-195 is down-regulated in human bladder cancer tissue versus normal adjacent tissue. To better characterize the role of miR-195 in bladder cancer, we conducted gain of function analysis by transfecting bladder cancer cell line T24 with chemically synthesized miR-195 mimic. We identified CDK4, an early G1 cell cycle regulator, as a novel target of miR-195. Selective over-expression of miR-195 could induce G1-phase arrest in T24 cells, and subsequently inhibit T24 cell growth. These findings indicate that miR-195 could be a potential tumor suppressor in bladder cancer.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.