Biomedical subjects

D Duan

Publications and source records attributed to D Duan.

At least 37 records · Page 2Linked to original sources

PubMed2000

Molecular distribution of volume-regulated chloride channels (ClC-2 and ClC-3) in cardiac tissues.

The molecular identification of cardiac chloride channels has provided probes to investigate their distribution and abundance in heart. In this study, the molecular expression and distribution of volume-regulated chloride channels ClC-2 and ClC-3 in cardiac tissues were analyzed and quantified. Total RNA was isolated from atria and ventricles of several species (dog, guinea pig, and rat) and subjected to a quantitative RT-PCR strategy. ClC-2 and ClC-3 mRNA expression were calculated relative to beta-actin expression within these same tissues. The transcriptional levels of ClC-3 mRNA were between 1.8 and 10.2% of beta-actin expression in atria and between 3.4 and 8.6% of beta-actin in ventricles (n = 3 for each tissue). The levels of ClC-2 in both atria and ventricles were significantly less than those measured for ClC-3 (n = 3; P < 0.05). ClC-2 mRNA levels were between 0.04-0.08% and 0.03-0.18% of beta-actin expression in atria and ventricles, respectively (n = 3 for each tissue). Immunoblots of atrial and ventricular wall protein extracts demonstrated ClC-2- and ClC-3-specific immunoreactivity at 97 and 85 kDa, respectively. Immunohistochemical localization in guinea pig cardiac muscle demonstrates a ubiquitous distribution of ClC-2 and ClC-3 channels in the atrial and ventricular wall. Confocal analysis detected colocalization of ClC-2 and ClC-3 in sarcolemmal membranes and distinct ClC-3 immunoreactivity in cytoplasmic regions. The molecular expression of ClC-2 and ClC-3 in cardiac tissue is consistent with the proposed role of these chloride channels in the regulation of cardiac cell volume and the modulation of cardiac electrical activity.

Actins↗

PubMed2000

Anion transport in heart.

Anion transport proteins in mammalian cells participate in a wide variety of cell and intracellular organelle functions, including regulation of electrical activity, pH, volume, and the transport of osmolites and metabolites, and may even play a role in the control of immunological responses, cell migration, cell proliferation, and differentiation. Although significant progress over the past decade has been achieved in understanding electrogenic and electroneutral anion transport proteins in sarcolemmal and intracellular membranes, information on the molecular nature and physiological significance of many of these proteins, especially in the heart, is incomplete. Functional and molecular studies presently suggest that four primary types of sarcolemmal anion channels are expressed in cardiac cells: channels regulated by protein kinase A (PKA), protein kinase C, and purinergic receptors (I(Cl.PKA)); channels regulated by changes in cell volume (I(Cl.vol)); channels activated by intracellular Ca(2+) (I(Cl.Ca)); and inwardly rectifying anion channels (I(Cl.ir)). In most animal species, I(Cl.PKA) is due to expression of a cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator Cl(-) channel. New molecular candidates responsible for I(Cl.vol), I(Cl.Ca), and I(Cl.ir) (ClC-3, CLCA1, and ClC-2, respectively) have recently been identified and are presently being evaluated. Two isoforms of the band 3 anion exchange protein, originally characterized in erythrocytes, are responsible for Cl(-)/HCO(3)(-) exchange, and at least two members of a large vertebrate family of electroneutral cotransporters (ENCC1 and ENCC3) are responsible for Na(+)-dependent Cl(-) cotransport in heart. A 223-amino acid protein in the outer mitochondrial membrane of most eukaryotic cells comprises a voltage-dependent anion channel. The molecular entities responsible for other types of electroneutral anion exchange or Cl(-) conductances in intracellular membranes of the sarcoplasmic reticulum or nucleus are unknown. Evidence of cardiac expression of up to five additional members of the ClC gene family suggest a rich new variety of molecular candidates that may underlie existing or novel Cl(-) channel subtypes in sarcolemmal and intracellular membranes. The application of modern molecular biological and genetic approaches to the study of anion transport proteins during the next decade holds exciting promise for eventually revealing the actual physiological, pathophysiological, and clinical significance of these unique transport processes in cardiac and other mammalian cells.

Animals↗

PubMed2000

Endosomal processing limits gene transfer to polarized airway epithelia by adeno-associated virus.

The restriction of viral receptors and coreceptors to the basolateral surface of airway epithelial cells has been blamed for the inefficient transfer of viral vectors to the apical surface of this tissue. We now report, however, that differentiated human airway epithelia internalize rAAV type-2 virus efficiently from their apical surfaces, despite the absence of known adeno-associated virus-2 (AAV-2) receptors or coreceptors at these sites. The dramatically lower transduction efficiency of rAAV infection from the apical surface of airway cells appears to result instead from differences in endosomal processing and nuclear trafficking of apically or basolaterally internalized virions. AAV capsid proteins are ubiquitinated after endocytosis, and gene transfer can be significantly enhanced by proteasome or ubiquitin ligase inhibitors. Tripeptide proteasome inhibitors increased persistent rAAV gene delivery from the apical surface >200-fold, to a level nearly equivalent to that achieved with basolateral infection. In vivo application of proteasome inhibitor in mouse lung augmented rAAV gene transfer from undetectable levels to a mean of 10.4 +/- 1.6% of the epithelial cells in large bronchioles. Proteasome inhibitors also increased rAAV-2-mediated gene transfer to the liver tenfold, but they did not affect transduction of skeletal or cardiac muscle. These findings suggest that tissue-specific ubiquitination of viral capsid proteins interferes with rAAV-2 transduction and provides new approaches to circumvent this barrier for gene therapy of diseases such as cystic fibrosis.

Animals↗

PubMed2000

[Expression of proteolytic enzymes of metastatic tumor cells and the effects of some influencing factors].

OBJECTIVE: To elucidate the types of proteolytic enzymes the dendritic cell sarcoma (DCS) cell expressed in vitro and the effects of modulating factors. METHODS: The proteolytic spots of DCS cells were examined, with ten different proteinase inhibitors, various antibodies and matrix. RESULTS: Aprotinin, EDTA-Na2 and pepstatin could inhibit the proteolysis of DCS cells respectively. Anti-ubiquitin antibody and anti-DCS antibody (1:200-1:100) showed no obvious inhibitory effects. DCS cells spread out fairly well but with no proteolysis. CONCLUSIONS: Under the conditions of our experiments the DCS cells had serine proteinase, metalloproteinase and aspartate proteinase activities. Antiubiquitin antibodies and anti-DCS antibodies showed no obvious influence on the proteolysis of DCS cells. DCS cells displayed different proteolysis status on different matrix.

Aspartic Acid Endopeptidases↗

PubMed1999

Purinoceptor-coupled Cl- channels in mouse heart: a novel, alternative pathway for CFTR regulation.

1. P2-purinoceptors couple extracellular ATP to the activation of a Cl- current (ICl,ATP) in heart. We studied the molecular mechanism and intracellular signalling pathways of ICl,ATP activation in mouse heart. 2. Extracellular adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS; 100 microM) activated ICl,ATP in both atrial and ventricular myocytes. A specific PKC inhibitor, bisindolylmaleimide blocked the effect of ATPgammaS while a PKC activator, phorbol 12, 13-dibutyrate (PDBu) activated a current with identical properties to ICl,ATP. Maximal activation of ICl,ATP by ATPgammaS or PDBu occluded further modulation by the other agonist, suggesting that they may activate the same population of Cl- channels. 3. Isoprenaline increased ICl,ATP pre-activated by ATPgammaS or PDBu, while isoprenaline or forskolin alone failed to activate any Cl- current in these myocytes. Adenosine 3',5'-cyclic monophosphothionate, a PKA inhibitor, prevented ATPgammaS or PDBu activation of ICl,ATP. Thus, ICl,ATP is regulated by dual intracellular phosphorylation pathways involving both PKA and PKC in a synergistic manner similar to cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels. 4. Glibenclamide (50 microM) significantly blocked ICl,ATP activated by ATPgammaS or by the CFTR channel activator, levamisole. 5. The slope conductance of the unitary ICl,ATP in cell-attached patches was 11.8 +/- 0.3 pS, resembling the known properties of CFTR Cl- channels in cardiac myocytes. 6. The reverse transcription polymerase chain reaction and Northern blot analysis revealed CFTR mRNA expression in mouse heart. 7. We conclude that ICl,ATP in mouse heart is due to activation of CFTR Cl- channels through a novel intracellular signalling pathway involving purinergic activation of PKC and PKA.

Adenosine Triphosphate↗

PubMed1999

Structural analysis of adeno-associated virus transduction circular intermediates.

Recombinant adeno-associated virus (rAAV) has recently been demonstrated to form circular intermediates following transduction in muscle tissue and cell lines. Although restriction enzyme and Southern blot analysis has revealed a consistent monomer and multimer head-to-tail conformation, detailed structural sequence analysis has been lacking due to the high secondary structure of the ITR arrays. To gain further insight into potential mechanisms by which AAV circular genomes are formed from linear single-stranded viral DNA, we have performed chemical sequencing of ITR arrays within seven circular intermediates independently isolated from primary fibroblasts and Hela cells. Results from these studies demonstrated several types of circular intermediates with mosaic ITR elements flanked by two D sequences. The most predominant form consisted of a structure similar to that of previously generated AAV double-D plasmids, with one complete ITR flanked by two D-region elements. However, intermediately deleted ITR arrays with more than one complete ITR were also seen. Based on this structural information, we have proposed a model for formation of AAV circular intermediates by recombination/ligation between ITR ends of panhandle single-stranded AAV genomes.

Base Sequence↗

PubMed1999

Two independent molecular pathways for recombinant adeno-associated virus genome conversion occur after UV-C and E4orf6 augmentation of transduction.

Numerous environmental influences have been demonstrated to enhance recombinant adeno-associated virus (rAAV) transduction. Such findings are the foundation of developing new and innovative strategies to improve the efficiency of rAAV as a gene therapy vector. Several of these environmental factors included genotoxic stresses such as UV and y irradiation as well as certain adenoviral gene products such as E4orf6. The mechanisms by which these environmental stimuli increase rAAV transduction are only partially understood but have been suggested to involve both endocytosis and uptake of virus to the nucleus, as well as conversion of single-stranded DNA viral genomes to double-stranded expressible forms. Two molecular intermediates of rAAV genomes, which have been demonstrated to correlate with transgene expression and/or the persistence of rAAV, include both replication form (Rf) monomers and dimers as well as circular intermediates. In the present study, we demonstrate that augmentation of rAAV transduction by UV irradiation and the adenoviral protein E4orf6 correlates with distinct increases in either circular or replication form intermediates, respectively. UV irradiation of primary fibroblasts at 15 J/m2 resulted in a 15-fold induction of head-to-tail circular intermediates, with minimal induction of replication form rAAV genomes. In contrast, E4orf6-augmented rAAV transduction was correlated with the formation of replication form intermediates, with no alteration in the abundance of circular intermediates. These findings demonstrate that rAAV transduction can occur through two independent molecular pathways that convert single-stranded AAV genomes to expressible forms of DNA.

Adenovirus E4 Proteins↗

PubMed1999

A serine residue in ClC-3 links phosphorylation-dephosphorylation to chloride channel regulation by cell volume.

In many mammalian cells, ClC-3 volume-regulated chloride channels maintain a variety of normal cellular functions during osmotic perturbation. The molecular mechanisms of channel regulation by cell volume, however, are unknown. Since a number of recent studies point to the involvement of protein phosphorylation/dephosphorylation in the control of volume-regulated ionic transport systems, we studied the relationship between channel phosphorylation and volume regulation of ClC-3 channels using site-directed mutagenesis and patch-clamp techniques. In native cardiac cells and when overexpressed in NIH/3T3 cells, ClC-3 channels were opened by cell swelling or inhibition of endogenous PKC, but closed by PKC activation, phosphatase inhibition, or elevation of intracellular Ca2+. Site-specific mutational studies indicate that a serine residue (serine51) within a consensus PKC-phosphorylation site in the intracellular amino terminus of the ClC-3 channel protein represents an important volume sensor of the channel. These results provide direct molecular and pharmacological evidence indicating that channel phosphorylation/dephosphorylation plays a crucial role in the regulation of volume sensitivity of recombinant ClC-3 channels and their native counterpart, ICl.vol.

Animals↗

PubMed1999

Formation of adeno-associated virus circular genomes is differentially regulated by adenovirus E4 ORF6 and E2a gene expression.

A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5' viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rfm) and dimer (Rfd) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.

Adenovirus E2 Proteins↗

PubMed1999

Dynamin is required for recombinant adeno-associated virus type 2 infection.

Recombinant adeno-associated virus (rAAV) vectors for gene therapy of inherited disorders have demonstrated considerable potential for molecular medicine. Recent identification of the viral receptor and coreceptors for AAV type 2 (AAV-2) has begun to explain why certain organs may demonstrate higher efficiencies of gene transfer with this vector. However, the mechanisms by which AAV-2 enters cells remain unknown. In the present report, we have examined whether the endocytic pathways of rAAV-2 are dependent on dynamin, a GTPase protein involved in clathrin-mediated internalization of receptors and their ligands from the plasma membrane. Using a recombinant adenovirus expressing a dominant-inhibitory form of dynamin I (K44A), we have demonstrated that rAAV-2 infection is partially dependent on dynamin function. Overexpression of mutant dynamin I significantly inhibited AAV-2 internalization and gene delivery, but not viral binding. Furthermore, colocalization of rAAV and transferrin in the same endosomal compartment provides additional evidence that clathrin-coated pits are the predominant pathway for endocytosis of AAV-2 in HeLa cells.

Cell Line, Transformed↗

PubMed1999

Submucosal gland development in the airway is controlled by lymphoid enhancer binding factor 1 (LEF1).

Previous studies have demonstrated that transcription of the lymphoid enhancer binding factor 1 (Lef1) gene is upregulated in submucosal gland progenitor cells just prior to gland bud formation in the developing ferret trachea. In the current report, several animal models were utilized to functionally investigate the role of LEF1 in initiating and supporting gland development in the airway. Studies on Lef1-deficient mice and antisense oligonucleotides in a ferret xenograft model demonstrate that LEF1 is functionally required for submucosal gland formation in the nasal and tracheal mucosa. To determine whether LEF1 expression was sufficient for the induction of airway submucosal glands, two additional model systems were utilized. In the first, recombinant adeno-associated virus was used to overexpress the human LEF1 gene in a human bronchial xenograft model of regenerative gland development in the adult airway. In a second model, the LEF1 gene was ectopically overexpressed under the direction of the proximal airway-specific CC10 promoter in transgenic mice. In both of these models, morphometric analyses revealed no increase in the number or size of airway submucosal glands, indicating that ectopic LEF1 expression alone is insufficient to induce submucosal gland development. In summary, these studies demonstrate that LEF1 expression is required, but in and of itself is insufficient, for the initiation and continued morphogenesis of submucosal glands in the airway.

Adult↗

PubMed1999

[Supraglottic horizontal partial laryngectomy].

OBJECTIVE: To evaluate the supraglottic horizontal partial laryngectomy in the treatment of laryngeal cancers. METHODS: Some researches and modifications are performed on the standard supraglottic horzontal partial laryngectomy: the wounded area in the laryngeal cavity is covered with the lateral membrane of thyroid cartilage; the laryngeal cavity is closed as its anterior wall by folding and sewing up the sternohyoid muscles. From 1983 to 1993, 92 patients (80 men and 12 women) with quamous cell carcinoma of larynx were treated with supraglottic horizontal partial laryngectomy. The patients age ranged from 39 to 72 years; 72% aged from 50 to 59. There were 72 cases (78.3%) in the stages III and IV. RESULTS: The 3 and 5 year survival rates of the patients were 75% and 72% respectively. Most cases have acquired good speech and swallow function with 2-4 weeks; 84.8% (78 patients) were extubated. CONCLUSION: The authors believed that partial laryngectomy was safe and beneficial to some patients with laryngeal cancers.

Adult↗

PubMed1999

[Conservative surgery for glottic cancer with vocal cord fixation (T3)].

OBJECTIVE: To study the method of reconstruction of laryngeal function by unilateral bi-pedicled and bi-muscular flaps for glottic cancer of T3 category. METHODS: From 1984 to 1992, the unilateral bi-pedicled and bi-muscular flaps were used in 68 patients with T3 glottic cancer for repairing the surgical defect, enlarging the piriform recess on the affected side and closing the anterior wall of the laryngeal cavity after vertical laryngectomy. RESULTS: The 5-year survival rate was 73.5% (50/68). The decannulation rate was 91.2% (62/68). sixty-three patients had satisfactory phonation (92.6%). All patients resumed mouth-food-taking within 7 to 20 days after operation. CONCLUSION: The unilateral bi-pedicled and bi-muscular flaps are appropriate for reconstruction of surgical defect in T3 glottic cancer.

Adult↗

PubMed1998

Polarity influences the efficiency of recombinant adenoassociated virus infection in differentiated airway epithelia.

To better understand mechanisms that limit rAAV transduction in the lung, we have evaluated several unique features of rAAV infection in polarized primary airway epithelial cultures. rAAV was found to transduce the basolateral surface of airway epithelia 200-fold more efficiently than the apical membrane. These differences in membrane infection correlated with the abundance of apical heparan sulfate proteoglycan (AAV-2 receptor) and virus binding. UV irradiation augmented rAAV transduction greater than 20-fold, only when virus was applied to the apical membrane. Ultrastructural analysis of UV-irradiated primary cultures demonstrated significant changes in microvilli architecture following exposure to 25 J/m2 UV. Although virus binding and the abundance of heparan sulfate proteoglycan were not increased at the apical membrane following UV irradiation, increased receptor-independent endocytosis of fluorescent beads was seen at the apical membrane following UV irradiation. We hypothesize that endocytotic processes associated with apical membrane-specific pathways of viral entry, and/or processing of virus to the nucleus, may be altered following UV irradiation. Interestingly, UV irradiation had an inhibitory effect on rAAV transduction from the basolateral membrane, which correlated with a decrease in the abundance of heparan sulfate proteoglycan at the basal membrane. In summary, these findings suggest that independent pathways of viral transduction may occur in the apical and basolateral compartments of polarized airway epithelia.

Bronchi↗

PubMed1998

Functional and molecular expression of volume-regulated chloride channels in canine vascular smooth muscle cells.

1. We examined the possibility of functional and molecular expression of volume-regulated Cl- channels in vascular smooth muscle using the whole-cell patch-clamp technique and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on cells from canine pulmonary and renal arteries. 2. Decreasing external osmolarity induced cell swelling, which was accompanied by activation of Cl--dependent outward-rectifying membrane currents with an anion permeability sequence of SCN- > I- > Br- > Cl- > aspartate-. These currents were sensitive to block by DIDS, extracellular ATP and the antioestrogen compound tamoxifen. 3. Experiments were performed to determine whether the molecular form of the volume-regulated chloride channel (ClC-3) is expressed in pulmonary and renal arteries. Quantitative RT-PCR confirmed expression of ClC-3 in both types of smooth muscle. ClC-3 expression was 76.4 % of beta-actin in renal artery and 48.0 % of beta-actin in pulmonary artery. 4. We conclude that volume-regulated Cl- channels are expressed in vascular smooth muscle cells and exhibit functional properties similar to those found in other types of cells, presumably contributing to the regulation of cell volume, electrical activity and, possibly, myogenic tone.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1998

Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue.

Adeno-associated viral (AAV) vectors have demonstrated great utility for long-term gene expression in muscle tissue. However, the mechanisms by which recombinant AAV (rAAV) genomes persist in muscle tissue remain unclear. Using a recombinant shuttle vector, we have demonstrated that circularized rAAV intermediates impart episomal persistence to rAAV genomes in muscle tissue. The majority of circular intermediates had a consistent head-to-tail configuration consisting of monomer genomes which slowly converted to large multimers of >12 kbp by 80 days postinfection. Importantly, long-term transgene expression was associated with prolonged (80-day) episomal persistence of these circular intermediates. Structural features of these circular intermediates responsible for increased persistence included a DNA element encompassing two viral inverted terminal repeats (ITRs) in a head-to-tail orientation, which confers a 10-fold increase in the stability of DNA following incorporation into plasmid-based vectors and transfection into HeLa cells. These studies suggest that certain structural characteristics of AAV circular intermediates may explain long-term episomal persistence with this vector. Such information may also aid in the development of nonviral gene delivery systems with increased efficiency.

Animals↗

PubMed1998

Lef1 transcription factor expression defines airway progenitor cell targets for in utero gene therapy of submucosal gland in cystic fibrosis.

Cystic fibrosis (CF) has emerged as a paradigm disorder for assessing the utility of gene therapy in the treatment of genetic diseases. It is hypothesized that submucosal glands may play an important role in the pathophysiology of CF lung disease. However, this region poses several significant obstacles for gene therapy due to its inaccessibility from the lumen of adult proximal airways. In utero gene therapy to correct submucosal gland dysfunction in CF provides an attractive alternative strategy to target gland progenitor cells prior to gland formation and morphogenesis. Such approaches will require the use of integrating vectors capable of transducing expanding stem-cell/progenitor-cell populations in the lung. We described a newborn-ferret model of the proximal airway which was used to evaluate the phenotypic characteristics of submucosal gland progenitors and to test gene therapy strategies for targeting these cell types. To this end, we have isolated the ferret cDNA to the lymphoid enhancing factor 1 (Lef1) and have demonstrated that its mRNA expression specifically defines a subset of surface airway epithelial progenitor cells involved in the formation of primordial submucosal gland buds and subsequent gland morphogenesis. Such findings suggest that the transcriptional switch regulating activation of Lef1 expression defines the phenotype of early submucosal gland progenitor cells. In an effort to prove the principle of gene targeting to this progenitor-cell population, we evaluated the efficiency of recombinant retroviral vectors to target submucosal glands in a xenograft model system. Findings from these studies demonstrated successful gene targeting to progenitor cells of submucosal gland buds which was stable throughout subsequent gland development. In summary, these studies have provided evidence for the existence of phenotypically distinct submucosal gland progenitor cells which represent appropriate targets for gene therapy of submucosal glands in CF.

Amino Acid Sequence↗

PubMed1998

A biomechanical analysis of unilateral hooked-sulcated external fixator on osteomined tibia.

Four tibiae removed from 30-40 years males, who died of accidents in less than 12 hours, were osteomized at medium part. Then these tibiae were fixed by an unilateral hooked-sulcated external fixator (UHSEF), and the bone-fixator system was used as a model of external fixation of tibial fracture. The axial compression, distraction, torsion, antero-posterior and lateral bending rigidity and the strain of the pins were determined in this system. Based on the results, we found that compared with the configuration of four paralleled pins, the rigidity of the fan-like configuration didn't decrease significantly if the angle between lateral and medium pins was less than 45 degrees. But the reverse was true when the frame separation increased from 5 to 8 cm. What' more, the pin strain decreased if the rigidity of the system was improved. These data provided a theoretical basis of biomechanics for the improvement of UHSEF.

Adult↗