Biomedical subjects

Y Yang

Publications and source records attributed to Y Yang.

At least 19 recordsLinked to original sources

PubMed2012

MRI roadmap-guided transendocardial delivery of exon-skipping recombinant adeno-associated virus restores dystrophin expression in a canine model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) cardiomyopathy patients currently have no therapeutic options. We evaluated catheter-based transendocardial delivery of a recombinant adeno-associated virus (rAAV) expressing a small nuclear U7 RNA (U7smOPT) complementary to specific cis-acting splicing signals. Eliminating specific exons restores the open reading frame resulting in translation of truncated dystrophin protein. To test this approach in a clinically relevant DMD model, golden retriever muscular dystrophy (GRMD) dogs received serotype 6 rAAV-U7smOPT via the intracoronary or transendocardial route. Transendocardial injections were administered with an injection-tipped catheter and fluoroscopic guidance using X-ray fused with magnetic resonance imaging (XFM) roadmaps. Three months after treatment, tissues were analyzed for DNA, RNA, dystrophin protein, and histology. Whereas intracoronary delivery did not result in effective transduction, transendocardial injections, XFM guidance, enabled 30±10 non-overlapping injections per animal. Vector DNA was detectable in all samples tested and ranged from <1 to >3000 vector genome copies per cell. RNA analysis, western blot analysis, and immunohistology demonstrated extensive expression of skipped RNA and dystrophin protein in the treated myocardium. Left ventricular function remained unchanged over a 3-month follow-up. These results demonstrated that effective transendocardial delivery of rAAV-U7smOPT was achieved using XFM. This approach restores an open reading frame for dystrophin in affected dogs and has potential clinical utility.

Animals↗

PubMed2000

Three-amino acid extension loop homeodomain proteins Meis2 and TGIF differentially regulate transcription.

Three-amino acid extension loop (TALE) homeobox proteins are highly conserved transcription regulators. We report that two members of this family, Meis2 and TGIF, which frequently have overlapping consensus binding sites on complementary DNA strands in opposite orientations, can function competitively. For example, in the D(1A) gene, which encodes the predominant dopamine receptor in the striatum, Meis2 and TGIF bind to the activator sequence ACT (-1174 to -1154) and regulate transcription differentially in a cell type-specific manner. Among the five cloned splice variants of Meis2, isoforms Meis2a-d activate the D(1A) promoter in most cell types tested, whereas TGIF competes with Meis2 binding to DNA and represses Meis2-induced transcription activation. Consequently, Meis2 cannot activate the D(1A) promoter in a cell that has abundant TGIF expression. The Meis2 message is highly co-localized with the D(1A) message in adult striatal neurons, whereas TGIF is barely detectable in the adult brain. Our observations provide in vitro and in vivo evidence that Meis2 and TGIF differentially regulate their target genes. Thus, the delicate ratio between Meis2 and TGIF expression in a given cell type determines the cell-specific expression of the D(1A) gene. We also found that splice variant Meis2e, which has a truncated homeodomain, cannot bind to the D(1A) ACT sequence or activate transcription. However, Meis2e is an effective dominant negative regulator by blocking Meis2d-induced transcription activation. Thus, truncated homeoproteins with no DNA binding domains can have important regulatory functions.

Alternative Splicing↗

PubMed2000

Survival and histological evaluation of therapeutic window of post-ischemia treatment with magnesium sulfate in embolic stroke model of rat.

The neuroprotective window of magnesium for up to 8 h after ischemic insult was evaluated in a clinically relevant model of focal cerebral ischemia subjected to embolization of the right middle cerebral artery (MCA) with an autologous thrombus. The animals were treated with either vehicle or 5% magnesium sulfate (90 mg/kg) intravenously at 2, or 6, or 8 h after ischemic insult. Survival rate significantly increased only in animals treated with magnesium sulfate in treatment groups at 2 h after MCA embolization but improvement in neurological outcome was observed in all medicated groups. Compared with animals in control group, post-ischemia treatment with magnesium sulfate significantly reduced brain infarct volume (P<0.01) except the 8 h group. These data indicate that the therapeutic window for neuroprotection of magnesium can be extended up to 6 h in the focal cerebral ischemia model of this study. The long window of opportunity for effective dosing may be explained with the proposed multiple mechanisms of actions for magnesium.

Animals↗

PubMed2000

Surface and interface beta-chain residues synergistically affect hemoglobin assembly.

Homo- and heterotetramer formations of beta112 variants (beta(112Cys-->Asp), beta(112Cys-->Ser), beta(112Cys-->Thr), and beta(112Cys-->Val)) of hemoglobin were characterized in the presence and absence of beta(16Gly-->Asp) in vitro. In all cases an alteration in overall surface charge (beta(16Gly-->Asp)) decreased the beta(4) homotetramer stability (association constants as determined by gel-permeation chromatography) albeit to differing extents. In contrast, competition experiments of hemoglobin subunits showed that heterotetramer formation was promoted by this substitution. Order of increase in tetramer formation by the additional negative surface charge in the beta112 variants was as follows: Hb betaG16D, C112D > Hb betaG16D, C112S > Hb betaG16D > Hb G16D, C112T > Hb betaG16D, C112V. Thus, the overall surface charge of the beta chain and its contribution to electrostatic interaction in these instances appear to act in synergy with alpha(1)beta(1) interface residues to affect the assembly of hemoglobin molecules.

Amino Acid Substitution↗

PubMed2000

An epidermal plakin that integrates actin and microtubule networks at cellular junctions.

Plakins are cytoskeletal linker proteins initially thought to interact exclusively with intermediate filaments (IFs), but recently were found to associate additionally with actin and microtubule networks. Here, we report on ACF7, a mammalian orthologue of the Drosophila kakapo plakin genetically involved in epidermal-muscle adhesion and neuromuscular junctions. While ACF7/kakapo is divergent from other plakins in its IF-binding domain, it has at least one actin (K(d) = 0.35 microM) and one microtubule (K(d) approximately 6 microM) binding domain. Similar to its fly counterpart, ACF7 is expressed in the epidermis. In well spread epidermal keratinocytes, ACF7 discontinuously decorates the cytoskeleton at the cell periphery, including microtubules (MTs) and actin filaments (AFs) that are aligned in parallel converging at focal contacts. Upon calcium induction of intercellular adhesion, ACF7 and the cytoskeleton reorganize at cell-cell borders but with different kinetics from adherens junctions and desmosomes. Treatments with cytoskeletal depolymerizing drugs reveal that ACF7's cytoskeletal association is dependent upon the microtubule network, but ACF7 also appears to stabilize actin at sites where microtubules and microfilaments meet. We posit that ACF7 may function in microtubule dynamics to facilitate actin-microtubule interactions at the cell periphery and to couple the microtubule network to cellular junctions. These attributes provide a clear explanation for the kakapo mutant phenotype in flies.

Actin Cytoskeleton↗

PubMed2000

Purification, identification, and characterization of an osmotic response element binding protein.

Kidney cells, especially the epithelial cells lining the collecting tubules in the inner medulla, are constantly exposed to concentrated urine. They are protected from the osmotic effect of high levels of sodium ion and urea by accumulating compatible osmolytes such as sorbitol, betaine, and myo-inositol. These osmolytes are involved in maintaining cell volume and electrolyte contents because they do not perturb the protein structure and function over a wide range of concentrations. Sorbitol is produced via the reduction of glucose by aldose reductase (AR), while betaine and myo-inositol are transported into the cells through specific transporters. Under hyperosmotic stress, transcriptions of genes encoding these proteins are highly induced. The induction of transcription was found to be mediated through the osmotic response elements (OREs) located in the 5' flanking sequences of these genes. We had earlier identified the OREs in human AR gene. In this study we purified and identified the osmotic response element binding protein (OREBP). OREBP is a transcription factor of approximately 200 kDa in size, characterized by a Rel-like DNA binding domain and a glutamine-rich transactivation domain. Dominant negative OREBP significantly diminished hyperosmotic AR gene induction. Immunohistochemical analysis showed that this transcription factor is rapidly translocated into the nucleus upon hyperosmotic stress.

Aldehyde Reductase↗

PubMed2000

Quantitative effects of functional bundle branch block in patients with atrioventricular reentrant tachycardia.

Changes in the retrograde conduction time (ventriculoatrial [VA]) interval during functional bundle branch block (BBB) have been used to separate septal from free wall accessory pathways (APs), but different values of the VA interval prolongation (deltaVA) have been described in different reports. A total of 95 patients with single nondecremental APs who developed BBB during atrioventricular reentrant tachycardia were studied. Free wall APs were found in 60 patients, and 35 had septal APs. For patients with free wall APs, complete and incomplete BBB ipsilateral to the atrial insertion site of APs were observed in 39 of 60 patients (65%) and 31 of 60 patients (52%), respectively. For patients who had both complete (QRS > or = 120 ms) and incomplete (QRS <120 ms) BBB during atrioventricular reentrant tachycardia, deltaVA for patients with complete BBB was significantly greater than in those with incomplete BBB, 59 +/- 19 ms versus 30 +/- 11 ms, p <0.001. For patients with septal APs and complete and incomplete BBB during tachycardia, the mean deltaVA for those with complete BBB was 31 +/- 20 ms and was significantly longer than in patients with incomplete BBB (14 +/- 6 ms), p <0.001. There was no significant difference in deltaVA between those with free wall APs and incomplete BBB compared with those with septal APs and complete BBB. The criteria of QRS > or = 120 ms associated with deltaVA > or =40 ms served to best separate free wall from septal APs with a sensitivity of 88% and a specificity of 89%. Left anterior fascicular block was associated with marked lengthening of deltaVA for those with left free wall APs, whereas a left posterior fascicular block pattern resulted in a marked increase in the deltaVA for patients with posteroseptal APs. In the absence of fascicular block patterns, a deltaVA > or =40 ms provides strong evidence of a free wall AP, with a sensitivity of 95% and a specificity of 100%. The left posterior fascicle appears to provide predominant innervation of the posterior septum.

Adolescent↗

PubMed2000

Ligand-independent down-regulation of IFN-gamma receptor 1 following TCR engagement.

Activated T lymphocytes modulate the level of many molecules on their cell surface, including cytokine receptors. This regulation of cytokine receptor expression affects the ability of T cells to respond to cytokines and thus influences the outcome of an immune response. The receptor for IFN-gamma, a proinflammatory cytokine, consists of two copies of a ligand binding chain (IFN-gammaR1) as well as two copies of a second chain (IFN-gammaR2) required for signal transduction. The expression of IFN-gammaR2 is down-regulated at the mRNA level on CD4+ T cells when they differentiate into the Th1, but not the Th2, phenotype. This down-regulation has been demonstrated to depend on the ligand, IFN-gamma, which is produced by Th1 but not Th2 T cells. The regulation of the cell-surface expression of IFN-gamma receptors during primary T cell activation has not been reported. Naive and differentiated T lymphocytes express IFN-gammaR1 at the mRNA level and as a cell-surface protein. In this study, we present evidence that cell-surface expression of IFN-gammaR1 is transiently down-regulated on the surface of naive CD4+ T cells shortly after TCR engagement. Furthermore, this down-regulation is not mediated by the ligand, IFN-gamma, but results from TCR engagement and can be inhibited by cyclosporin A.

Animals↗

PubMed2000

Interactions between regulatory proteins that bind to the nicotinic receptor beta4 subunit gene promoter.

The genes encoding the alpha3, alpha5 and beta4 subunits of nicotinic acetylcholine receptors are tightly clustered within the genome. As these three subunits constitute the predominant acetylcholine receptor subtype expressed in the peripheral nervous system, their genomic proximity suggests a regulatory mechanism ensuring their coordinate expression. We previously identified two transcriptional regulatory elements within the beta4 promoter. One of these elements, a CT box, interacts with the regulatory factors heterogeneous nuclear ribonucleoprotein K and Puralpha. Another element, a CA box, interacts with Sp1 and Sp3. The binding site for a fifth factor, Sox10, overlaps the CT and CA boxes. As the CT and CA boxes are adjacent, we postulated that the proteins that bind to the elements interact. Here we report that the CT box-binding factors interact with each other as do the CA box-binding factors. However, there are no direct associations between the two pairs of proteins. Interestingly though, Sox10 directly interacts with all four proteins, suggesting a central role in beta4 gene expression for this member of the Sox family of regulatory factors.

Cyclic AMP Response Element-Binding Protein↗

PubMed2000

On-line coupling of subcritical water extraction with high-performance liquid chromatography via solid-phase trapping.

Although ambient water is very polar and cannot dissolve many organic species, water at elevated temperatures behaves like a polar organic solvent. Thus, subcritical water has been proven to be an effective extraction fluid for several classes of organic compounds. While solvent trapping was used to collect the extracted analytes in most of previous subcritical water extractions, sorbent trapping has also been developed for subcritical water extraction. In this study, an on-line system for subcritical water extraction and high-performance liquid chromatography (HPLC) was built and tested. A sorbent trap was used as the interface between subcritical water extraction and HPLC. Several shut-off valves have been utilized to switch the system from one mode to another (e.g., from the extraction mode to HPLC mode). The coupling technique of subcritical water extraction and HPLC eliminates the liquid-liquid extraction used in solvent trapping subcritical water extraction and provides higher sensitivity. Compared to the off-line system reported in an earlier work, the operation of this on-line system is even easier. Some peak broadening occurred after the coupling the water extraction with HPLC for the analytes studied. The performance of this on-line system was evaluated by the extraction and determination of caffeine, nitrotoluenes, polychlorinated biphenyls, chlorophenols and anilines.

Aniline Compounds↗

PubMed2000

Hematopoietic stem cell quiescence maintained by p21cip1/waf1.

Relative quiescence is a defining characteristic of hematopoietic stem cells, while their progeny have dramatic proliferative ability and inexorably move toward terminal differentiation. The quiescence of stem cells has been conjectured to be of critical biologic importance in protecting the stem cell compartment, which we directly assessed using mice engineered to be deficient in the G1 checkpoint regulator, cyclin-dependent kinase inhibitor, p21cip1/waf1 (p21). In the absence of p21, hematopoietic stem cell proliferation and absolute number were increased under normal homeostatic conditions. Exposing the animals to cell cycle-specific myelotoxic injury resulted in premature death due to hematopoietic cell depletion. Further, self-renewal of primitive cells was impaired in serially transplanted bone marrow from p21-/- mice, leading to hematopoietic failure. Therefore, p21 is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion. Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death.

Animals↗

PubMed2000

Enhanced neuroprotection and reduced hemorrhagic incidence in focal cerebral ischemia of rat by low dose combination therapy of urokinase and topiramate.

Thrombolysis is increasingly being used in treating acute ischemic stroke but it is also accompanied with a serious complication of cerebral hemorrhage in a dose-dependent fashion. As a lower dose may result in decreased effectiveness, we tested the efficacy of combining a neuroprotective agent, topiramate (TPM), with lower doses of intra-arterial urokinase in an embolic stroke model. Focal ischemia was produced by introduction of an autogenous thrombus into the right middle cerebral artery. Urokinase was infused via the ipsilateral internal carotid artery and neuroprotective agent, TPM, was administrated intra-peritoneally 2 h following ischemic insult. The animals were assigned to five groups: (1) control group (n=6); (2) urokinase 5000 units/kg (n=8); (3) urokinase at 2500 units/kg (n=8); (4) topiramate at 20 mg/kg (n=8); (5) urokinase at 2500 units/kg and topiramate at 20 mg/kg (n=8). Neurobehavioral outcome and the degree of brain infarct volume were assessed at 24 h. Three animals in the group treated by high dose urokinase developed intracranial hemorrhage but none in other groups. Animals in all medication-groups showed significant improvement in neurobehavioral score. Post-ischemia treatment with urokinase or TPM alone significantly attenuated brain infarct volume (low-dose urokinase, 39.1+/-13.0%, p<0.05; high-dose, 18.4+/-8.5%, p<0.001; TPM, 20. 1+/-11.2%, p<0.001) when compared to the control (54.2+/-9.04%). Addition of TPM to low dose urokinase achieved better neuroprotection (8.2+/-6.0%) than any single-drug-treated groups. Our data suggests that combination of low dose urokinase with a neuroprotective agent may benefit ischemic stroke treatment by improving neurologic recovery, attenuating infarction size, and reducing the risk of cerebral hemorrhage.

Animals↗

PubMed2000

TIP30 has an intrinsic kinase activity required for up-regulation of a subset of apoptotic genes.

CC3 is a metastasis suppressor that inhibits metastasis of the variant small cell lung carcinoma (v-SCLC) by predisposing cells to apoptosis. The same protein was also reported as a cellular cofactor, TIP30, which stimulates HIV-1 Tat-activated transcription by interacting with both Tat and RNA polymerase II. We report here that TIP30/CC3 is a novel serine/threonine kinase. It phosphorylates the heptapeptide repeats of the C-terminal domain (CTD) of the largest RNA polymerase II subunit in a Tat-dependent manner. Amino acid substitutions in the putative ATP binding motif that abolish the TIP30 kinase activity also inhibit the ability of TIP30 to enhance Tat-activated transcription or to sensitize NIH 3T3 and v-SCLC cells to apoptosis. Furthermore, ectopic expression of TIP30/CC3 in v-SCLC cells induces expression of a number of genes that include the apoptosis-related genes Bad and Siva, as well as metastasis suppressor NM23-H2. These data demonstrate a molecular mechanism for TIP30/CC3 function and suggest a novel pathway for regulating apoptosis.

Acetyltransferases↗

PubMed2000

Enhancement of DNA vaccine potency by linkage of antigen gene to an HSP70 gene.

Nucleic acid vaccines represent an attractive approach to generating antigen-specific immunity because of their stability and simplicity of delivery. However, there is still a need to increase the potency of DNA vaccines. Using human papillomavirus type 16 E7 as a model antigen, we evaluated the effect of linkage to Mycobacterium tuberculosis heat shock protein 70 (HSP70) on the potency of antigen-specific immunity generated by naked DNA vaccines. We found that vaccines containing E7-HSP70 fusion genes increased the frequency of E7-specific CD8+ T cells by at least 30-fold relative to vaccines containing the wild-type E7 gene. More importantly, this fusion converted a less effective vaccine into one with significant potency against established E7-expressing tumors. Surprisingly, E7-HSP70 fusion vaccines exclusively targeted CD8+ T cells; immunological and antitumor effects were completely CD4-independent. These results indicate that fusion of HSP70 to an antigen gene may greatly enhance the potency of DNA vaccines via CD8-dependent pathways.

Animals↗

PubMed2000

Toxaphene is antiestrogenic in a human breast-cancer cell assay.

Toxaphene is a complex mixture of chlorinated bornanes, bornenes, and bornadienes and was a heavily used insecticide in the United States until its use was restricted in 1982. There are conflicting reports regarding the potential for toxaphene to induce estrogenic responses in human and nonhuman animals. Due to the public concern over environmental estrogens, the estrogenicity of toxaphene was examined in a human breast-cancer cell assay, the MCF-7 focus assay, which is based on in vitro postconfluent cell proliferation and tissue restructuring. In this assay, 0.1-1 nM 17beta-estradiol (E2) produces maximum postconfluent proliferation and formation of multicellular nodules or foci. Toxaphene was also tested for its ability (1) to bind the estrogen receptor (ER) in a competitive binding assay using recombinant human ERalpha (rhER) and in a whole-cell competitive ER binding assay, and (2) to alter the catabolism of E2 in MCF-7 cell cultures. Results from the MCF-7 focus assay showed: (1) Toxaphene alone was not estrogenic between the concentrations of 0.5 nM and 10 microM, (2) toxaphene in binary combinations with chlordane, dieldrin, or endosulfan (alpha or beta) was not estrogenic, and (3) toxaphene was weakly antiestrogenic (it reduced the number of foci induced by 0.1 nM and 0.01 nM E2). Results from the competitive binding assays showed that (1) toxaphene alone did not bind rhER or ER in MCF-7 cells, and (2) toxaphene in binary combinations with other pesticides did not bind rhER. Results from the growth assay and radiometric analysis of E2 catabolism showed that (1) toxaphene did not alter the growth rate of MCF-7 cell cultures over 13 d, and (2) toxaphene did not alter the catabolism of E2. In conclusion, results from the MCF-7 focus assay demonstrate that toxaphene is weakly antiestrogenic rather than estrogenic.

Binding, Competitive↗

PubMed2000

Isolating aging mutants: a novel method yields three strains of the nematode Caenorhabditis elegans with extended life spans.

We designed a novel procedure for the isolation of mutant strains with significantly increased life spans in the nematode Caenorhabditis elegans. This procedure involves using heat-shock to screen a large number of animals and isolate a few which are more resistant to heat-shock stress. From the heat-shock-resistant animals, three mutant strains, HG25, HG96, and HG246, all exhibiting increased life span, were isolated. One mutant strain (HG246) develops more slowly than the wild-type strain, N2. Two mutant strains, HG96 and HG246, exhibit lower fertility than the wild-type. Each of the three mutant strains has a normal appearance. Their locomotive behavior also appears normal; only HG246 shows slightly slower movement. Their feeding behavior appears normal, and the males of HG25 and HG96 show normal mating behavior. However, the males of HG246, either are defective in their mating ability or their sperm are defective. The results indicate that heat-shock can be used as a means to facilitate the isolation of mutants which have longer life expectancy.

Aging↗

PubMed2000

Enteroviral capsid protein VP1 is present in myocardial tissues from some patients with myocarditis or dilated cardiomyopathy.

BACKGROUND: There are still discrepancies in the association of enterovirus and myocardial disease, partially due to lack of data on the detection of virus antigens in tissues. It is desirable to localize enteroviral antigens so as to establish a link between the two and to study mechanisms of virus persistence. METHODS AND RESULTS: Nineteen fixed explanted or postmortem myocardial samples were obtained from patients with myocarditis or dilated cardiomyopathy (DCM). Control samples were collected from 11 subjects who had died accidentally or of noncardiovascular disease. Viral antigen was detected by an improved immunohistochemical technique using an enterovirus group-specific antibody to viral capsid protein VP1. Nine of 11 myocarditis cases (81.8%) and 6 of 8 DCM cases (75%) were positive. Signals were localized in the cytoplasm of myocytes. Intense immunostaining was observed in acute myocarditis, whereas VP1 was detected in scattered myocytes in chronic myocarditis or DCM. Enteroviral RNA was detected in 6 of 11 myocarditis samples (54.5%) and 3 of 8 DCM samples (37.5%) by the reverse transcription-nested polymerase chain reaction, correlating with antigen detection (kappa=0.6+/-0.21). Neither viral antigen nor RNA was detected in any controls. CONCLUSIONS: Our findings demonstrate a direct link between enterovirus infection and some myocarditis or DCM cases. The pattern of VP1 detection may correlate with disease stage and severity. The data suggest that viral protein synthesis may be involved in persistent enterovirus infection in the pathogenesis of DCM.

Adolescent↗

PubMed2000

APOBEC-1 dependent cytidine to uridine editing of apolipoprotein B RNA in yeast.

Cytidine to uridine editing of apolipoprotein B (apoB) mRNA requires the cytidine deaminase APOBEC-1 as well as a tripartite sequence motif flanking a target cytidine in apoB mRNA and an undefined number of auxiliary proteins that mediate RNA recognition and determine site-specific editing. Yeast engineered to express APOBEC-1 and apoB mRNA supported editing under conditions of late log phase growth and stationary phase. The cis -acting sequence requirements and the intracellular distribution of APOBEC-1 in yeast were similar to those described in mammalian cells. These findings suggest that auxiliary protein functions necessary for the assembly of editing complexes, or 'editosomes', are expressed in yeast and that the distribution of editing activity is to the cell nucleus.

APOBEC-1 Deaminase↗