Biomedical subjects

Jian Yao

Publications and source records attributed to Jian Yao.

At least 19 recordsLinked to original sources

PubMed2006

The role of cytosolic alpha-glucan phosphorylase in maltose metabolism and the comparison of amylomaltase in Arabidopsis and Escherichia coli.

Transitory starch of leaves is broken down hydrolytically, making maltose the predominant form of carbon exported from chloroplasts at night. Maltose metabolism in the cytoplasm of Escherichia coli requires amylomaltase (MalQ) and maltodextrin phosphorylase (MalP). Possible orthologs of MalQ and MalP in the cytosol of Arabidopsis (Arabidopsis thaliana) were proposed as disproportionating enzyme (DPE2, At2g40840) and alpha-glucan phosphorylase (AtPHS2, At3g46970). In this article, we measured the activities of recombinant DPE2 and AtPHS2 proteins with various substrates; we show that maltose and a highly branched, soluble heteroglycan (SHG) are excellent substrates for DPE2 and propose that a SHG is the in vivo substrate for DPE2 and AtPHS2. In E. coli, MalQ and MalP preferentially use smaller maltodextrins (G(3)-G(7)) and we suggest that MalQ and DPE2 have similar, but nonidentical, roles in maltose metabolism. To study this, we complemented a MalQ(-) E. coli strain with DPE2 and found that the rescue was not complete. To investigate the role of AtPHS2 in maltose metabolism, we characterized a T-DNA insertion line of the AtPHS2 gene. The nighttime maltose level increased 4 times in the Atphs2-1 mutant. The comparison of maltose metabolism in Arabidopsis with that in E. coli and the comparison of the maltose level in plants lacking DPE2 or AtPHS2 indicate that an alternative route to metabolize the glucan residues in SHG exists. Other plant species also contain SHG, DPE2, and alpha-glucan phosphorylase, so this pathway for maltose metabolism may be widespread among plants.

Arabidopsis↗

PubMed2006

Secreted protein-based reporter systems for monitoring inflammatory events: critical interference by endoplasmic reticulum stress.

A number of recent reports have used secreted protein-based reporter assays for monitoring intercellular and intracellular events involved in inflammation. However, we found that these assay systems are critically affected by endoplasmic reticulum (ER) stress. When reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of NF-kappaB were exposed to IL-1beta or TNF-alpha, induction of SEAP activity was markedly reduced under ER stress conditions. Downregulation of SEAP activity was observed regardless of cell types and type of regulatory elements; e.g., when reporter hepatocytes that express SEAP under the control of the dioxin responsive elements were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin or benzo[a]pyrene, ER stress similarly suppressed the increase in SEAP activity despite its transcriptional upregulation. Activity of constitutively expressed SEAP in various cells was also reduced by ER stress in a magnitude-dependent manner, and it was associated with disturbed subcellular transport of SEAP to the Golgi. Furthermore, interference by ER stress was similarly observed in other reporter assay using secreted luciferase. These results evidenced critical interference by ER stress in secreted protein-based reporter systems. The suppression of reporter responses by ER stress should be considered carefully for experimental design and interpretation of data when secreted protein-based reporter systems are used for investigation.

Alkaline Phosphatase↗

PubMed2006

Real-time detection and continuous monitoring of ER stress in vitro and in vivo by ES-TRAP: evidence for systemic, transient ER stress during endotoxemia.

Activity of secreted alkaline phosphatase (SEAP) produced by transfected cells is rapidly down-regulated by endoplasmic reticulum (ER) stress independent of transcriptional regulation. This phenomenon was observed in a wide range of cell types triggered by various ER stress inducers. The magnitude of the decrease in SEAP was proportional to the extent of ER stress and inversely correlated with the induction of endogenous ER stress markers grp78 and grp94. In contrast to SEAP, activity of secreted luciferase was less susceptible to ER stress. The decrease in SEAP activity by ER stress was caused by abnormal post-translational modification, accelerated degradation and reduced secretion of SEAP protein. In transgenic mice constitutively producing SEAP, systemic induction of ER stress led to reduction in serum SEAP. In these mice, administration with lipopolysaccharide caused rapid, transient decrease in serum SEAP activity, and it was correlated with up-regulation of grp78 in several organs including the spleen, lung, kidney, liver and heart. These results elucidated for the first time a possible involvement of transient, systemic ER stress in endotoxemia and provided evidence for usefulness of ER stress responsive alkaline phosphatase for real-time monitoring of ER stress in vitro and in vivo.

Alkaline Phosphatase↗

PubMed2006

High levels of dioxin-like potential in cigarette smoke evidenced by in vitro and in vivo biosensing.

Cigarette smoke contains low levels of agonists for the aryl hydrocarbon receptor (AhR; also called the dioxin receptor). However, little is understood about the whole potential of cigarette smoke for activating AhR. In this report, we evaluated the total "dioxin-like" activity of cigarette smoke using in vitro and in vivo reporter systems. Cigarette smoke extract (CSE) was prepared from seven cigarette brands (1-20 mg tar content) and subjected to in vitro bioassay based on the xenobiotic-responsive element (XRE) as the sensor and secreted alkaline phosphatase (SEAP) as the reporter. Exposure of reporter cells to CSE triggered activation of XRE in a dose-dependent manner, which was suppressed by functional inhibition of AhR. Direct, brief exposure of the cells to cigarette smoke similarly induced activation of XRE. Using 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) as the standard, the XRE-activating potential (XAP) of individual smoke was evaluated quantitatively. Positive correlation was observed between the tar content and XAP values. The XAP values estimated were extremely high with a range from 18.5 to 51.2 ng 2,3,7,8-TCDD equivalent per cigarette. To further estimate XAP of cigarette smoke in vivo, we generated transgenic reporter mice that secrete SEAP under the control of XRE. After exposure of the mice to smoke, serum levels of SEAP were significantly elevated within 12 hours, peaked at 24 hours, and declined thereafter. These results evidenced for the first time that cigarette smoke has unexpectedly high dioxin-like potential that triggers the AhR-XRE pathway in vitro and in vivo.

Alkaline Phosphatase↗

PubMed2006

Novel potential of tunicamycin as an activator of the aryl hydrocarbon receptor -- dioxin responsive element signaling pathway.

Tunicamycin is a well-known inhibitor of protein glycosylation and used as an inducer of endoplasmic reticulum (ER) stress. We found that tunicamycin induced expression of cytochrome P450 1A1 in a dose-dependent manner. Like dioxin, the transcriptional induction was associated with dose-dependent activation of the dioxin responsive element (DRE). This effect was independent of inhibition of protein glycosylation or induction of ER stress. Pharmacological and genetic inhibition of the aryl hydrocarbon receptor (AhR) significantly attenuated activation of DRE by tunicamycin. These results elucidated the novel potential of tunicamycin as an activator of the AhR -- DRE signaling pathway.

Animals↗

PubMed2006

Profiling of functional phosphodiesterase in mesangial cells using a CRE-SEAP-based reporting system.

1. Phosphodiesterases (PDEs) are critically implicated in the regulation of mesangial cell function, but profile of functional PDEs in mesangial cells is still unclear. In this study, we investigated roles of individual PDEs in the regulation of mesangial cell behavior by the cAMP pathway. 2. Reporter mesangial cells that express secreted alkaline phosphatase (SEAP) under the control of the cAMP response element (CRE) were exposed to selective PDE inhibitors in the presence or absence of cAMP, and activity of CRE, expression of CRE-regulated protein, mitogenesis and cell survival were examined. 3. Exposure of reporter cells to cAMP-elevating agents resulted in time- and concentration-dependent activation of CRE. Treatment of the cells with any PDE inhibitors alone did not induce CRE activation. Under stimulation with 8-bromo-cAMP or 8-bromo-cGMP, however, inhibitors of PDE2, PDE3, PDE4 and PDE5 enhanced activation of CRE. Inhibition of PDE1 or PDE6 did not affect the CRE activation. 4. Among different combinations tested, only inhibitors of PDE3 and PDE4 cooperatively increased the level of intracellular cAMP, activity of protein kinase A, activation of CRE, and CRE-regulated protein, connexin43. 5. Concomitant inhibition of PDE3 and PDE4 attenuated mitogen-induced activation of extracellular signal-regulated kinases and cell proliferation. Under serum deprivation, combinational inhibition of PDE3 and PDE4 exclusively caused activation of caspase-3 and apoptosis. 6. The present data elucidated that PDE3 and PDE4 play critical roles in the regulation of mesangial cell function. PDE3 and PDE4 were identified as the novel, antiapoptotic machinery that supports survival of mesangial cells.

1-Methyl-3-isobutylxanthine↗

PubMed2006

Spontaneous activation of the NF-kappaB signaling pathway in isolated normal glomeruli.

In this report, we describe that NF-kappaB is spontaneously activated in isolated, normal glomeruli. Ex vivo incubation of isolated rat glomeruli triggered expression of a NF-kappaB-dependent gene, monocyte chemoattractant protein-1 (MCP-1), in parallel with downregulation of IkappaBalpha and IkappaBbeta proteins and activation of the p65 NF-kappaB subunit. The induction of MCP-1 was also observed in mesangial cells coincubated with isolated glomeruli or exposed to media conditioned by isolated glomeruli (GCM), which was abrogated by inhibition of NF-kappaB. The activation of NF-kappaB by glomerulus-derived factors was confirmed using reporter mesangial cells that produce secreted alkaline phosphatase (SEAP) under the control of the kappaB enhancer element. When the reporter cells were adoptively transferred into normal glomeruli, expression of SEAP mRNA and activity of SEAP were also upregulated in the explanted glomeruli. The molecular weight of factors responsible for activation of NF-kappaB was >50 kDa, and TNF-alpha was identified as one of glomerulus-derived activators. To examine upstream events involved, we focused on MAP kinases that are spontaneously activated in explanted glomeruli. Selective suppression of ERK or p38 MAP kinase significantly attenuated activation of NF-kappaB in mesangial cells triggered by coculture with isolated glomeruli. Interestingly, the suppressive effects by MAP kinase inhibitors were not observed in mesangial cells treated with GCM. These data suggested that NF-kappaB was spontaneously activated in explanted glomeruli via autocrine/paracrine factors including TNF-alpha and that the production of NF-kappaB activators by glomeruli was, at least in part, through MAP kinase pathways.

Animals↗

PubMed2006

Priming of glomerular mesangial cells by activated macrophages causes blunted responses to proinflammatory stimuli.

Macrophage-mesangial cell interaction plays a crucial role in the pathogenesis of glomerulonephritis. Activated macrophages trigger mesangial cells to express an array of inflammation-associated genes via activation of NF-kappaB and AP-1. However, this inflammatory response is often transient and subsides spontaneously. We found that mesangial cells activated by bystander macrophages showed blunted responses of NF-kappaB to subsequent macrophage exposure. It was associated with sustained levels of IkappaBbeta, but not IkappaBalpha. The tolerance observed was reversible and reproduced by conditioned media from activated macrophages (macrophage-conditioned medium (MphiCM)). In vivo priming of mesangial cells by activated glomerular macrophages also caused the tolerance of mesangial cells. The macrophage-derived tolerance inducers were heat-labile, and multiple molecules were involved. Among inflammatory cytokines produced by macrophages, TNF-alpha and IL-1beta were able to induce mesangial cell tolerance dose-dependently. The mesangial cell tolerance was also observed in activation of the MAPK-AP-1 pathway; i.e., phosphorylation of ERK, JNK, and p38 MAPK by macrophages was blunted when the cells were pre-exposed to MphiCM. Induction of c-fos and c-jun was also abrogated in mesangial cells pre-exposed to MphiCM, and the suppression was attenuated by blockade of MAPK activation during the first exposure to MphiCM. These data elucidated that mesangial cells, once exposed to macrophages, become insensitive to subsequent activation by macrophages and proinflammatory stimuli. This self defense of glomerular cells may play a role in the resolution of macrophage-mediated, acute glomerulonephritis.

Animals↗

PubMed2006

Gap junction-mediated intercellular communication between dendritic cells (DCs) is required for effective activation of DCs.

Gap junctions, formed by members of the connexin (Cx) family, are intercellular channels allowing direct exchange of signaling molecules. Gap junction-mediated intercellular communication (GJIC) is a widespread mechanism for homeostasis in organs. GJIC in the immune system is not yet fully understood. Although dendritic cells (DC) reportedly form cell-to-cell contact between DCs in nonlymphoid and lymphoid organs, GJIC between DCs remains unknown. In this study we examined whether DCs form GJIC. XS52 and bone marrow-derived DCs (BMDCs) were tested for GJIC by counting intercellular transfer of Lucifer Yellow microinjected into a cell. Either DC became effectively dye-coupled when activated with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma. LPS- plus IFN-gamma-induced dye-coupling was mediated by DC-derived TNF-alpha. In addition, CpG plus IFN-gamma induced dye-coupling in BMDCs, which was also mediated by DC-derived TNF-alpha. LPS- plus IFN-gamma-induced activation of DCs (assessed by CD40 expression) was observed when there was cell-to-cell contact and was significantly blocked by heptanol, a gap junction blocker. These results indicate that cell-to-cell contact and GJIC are required for effective DC activation. In addition, heptanol significantly inhibited the LPS- plus IFN-gamma-induced up-regulation of the other costimulatory (i.e., CD80 and CD86) and MHC class II molecules expressed by BMDCs, and it significantly reduced their allostimulatory capacity. Among Cx members, Cx43 was up-regulated in dye-coupled BMDCs, and Cx mimetic peptide, a blocker of Cx-mediated GJIC, significantly inhibited the dye-coupling and activation, suggesting the involvement of Cx43. Thus, our study provides the first evidence for GJIC between DCs, which is required for effective DC activation.

Animals↗

PubMed2006

Study of in vivo differentiation of rat bone marrow stromal cells into schwann cell-like cells.

In order to raise an abundant and accessible reservoir for Schwann cells (SCs), which are used as seed cells for constructing tissue-engineered nerve grafts, we investigated the feasibilty of in vivo differentiation of bone marrow stromal cells (MSCs) into SC-like cells. In this study, MSCs were harvested from adult rats' bone marrow, culture-expanded, and characterized. Subcultured MSCs were then labeled with Hoechst 33342, followed by transplantation into the nerve regeneration chamber, which was made of a silicone tube bridging the sciatic nerve defect of the rats. Four weeks after surgery, some of the differentiated MSCs turned into SC-like cells immunopositive to S-100 protein, accompanied by myelination of the regenerated nerve fibers. Walking-track analyses provided evidence that transplantation of MSCs contributed to reconstruction of the sciatic nerve and reinnervation of target tissues. The experimental results suggest that MSCs are capable of differentiating into SC-like cells in vivo, making them a promising candidate for cell transplantation in peripheral nerve repair.

Animals↗

PubMed2006

Effect of methamidophos and urea application on microbial communities in soils as determined by microbial biomass and community level physiological profiles.

In this study, we evaluated the effect of the application by two agrochemicals, methamidophos (O,S-dimethyl phosphoroamidothioate) and urea, on microbial diversity in soil, using the combined approaches of soil microbial biomass analysis and community level physiological profiles (CLPPs). The results showed that both a low and a high level of methamidophos application (CS2 and CS3) and urea application (CS4) significantly decreased microbial biomass C (Cmic) by 41-83% compared with the control (CS1). The soil organic C (Corg) values of CS3 and CS4 were significantly higher and lower by 24% and 14%, respectively, than that of CS1. Similarly to Cmic, the values of Cmic/Corg of the three applied soils which decreased were lower by 31-84% than that of CS1. In contrast, the respiration activity of the three applied soils were significantly higher than the control. Agrochemical application also significantly increased the soil total of N and P (Ntol and Ptol) and decreased the Corg/Ntol and Corg/Ptol values. The CLPPs results showed that the AWCD (average well color development) of the three applied soils were significantly higher than that of CS1 during the incubation period. Substrate richness, Shannon and Simpson indices of microbial communities under chemical stresses, increased significantly. In addition, the CFU (colony-forming unit) numbers of methamidophos metabolized bacteria in CS2 and CS3 also increased significantly by 86.1% and 188.9% compared with that of CS1. The combined results suggest that agrochemicals reduce microbial biomass and enhance functional diversities of soil microbial communities; meanwhile, some species of bacteria may be enriched in soils under methamidophos stress.

Biomass↗

PubMed2006

Identification of open reading frames unique to a select agent: Ralstonia solanacearum race 3 biovar 2.

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Arginine↗

PubMed2006

Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum.

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.

Chemotaxis↗

PubMed2005

ER stress disrupts Ca2+-signaling complexes and Ca2+ regulation in secretory and muscle cells from PERK-knockout mice.

Disruption of protein synthesis and folding results in ER stress, which is associated with the pathophysiology of diverse diseases affecting secretory and muscle cells. Cells are protected against ER stress by activation of the unfolded protein response (UPR) that is regulated by the protein kinase PERK, which phosphorylates the translation initiation factor 2 eIF2alpha to attenuate protein synthesis. PERK-/- cells are unable to modulate ER protein load and experience high levels of ER stress. In addition to its role in protein synthesis, the ER also orchestrates many signaling events essential for cell survival, prominent among which is Ca2+ signaling. It is not known, however, whether there is a relationship between ER stress and the function of the Ca2+-signaling pathway in muscle and non-muscle cells. To directly address this question we characterized Ca2+ signaling in the secretory pancreatic and parotid acinar cells and in urinary bladder smooth muscle (UBSM) cells obtained from PERK-/- and wild-type mice. Deletion of PERK that results in high levels of ER stress, and distention and fragmentation of the ER slowed the rate of agonist-mediated Ca2+ release from the ER and reduced Ca2+-induced Ca2+ release, although IP3 production, localization of the IP3 receptors, IP3-mediated Ca2+ release, Ca(v)1.2 current and RyRs activity remained unaltered. On the other hand, ER stress disrupted the integrity of the Ca2+-signaling complexes in both secretory and UBSM cells, as revealed by markedly reduced co-immunoprecipitation of plasma membrane- and ER-resident Ca2+-signaling proteins. These findings establish a relationship between the unfolding protein response, ER stress and Ca2+ signaling and highlight the importance of communication within the terminal ER-plasma membrane microdomain for propagation of the Ca2+ signal from the plasma membrane into the cell.

Animals↗

PubMed2005

Synergistic effects of PDGF-BB and cAMP-elevating agents on expression of connexin43 in mesangial cells.

The gap junction plays an important role in the regulation of cell growth, migration, and differentiation. Platelet-derived growth factor (PDGF) is reported to be a potent inhibitor of gap junctional intercellular communication (GJIC). Short-term exposure of cells to PDGF causes rapid and transient disruption of GJIC without altering connexin43 (Cx43) protein level. In this study, we investigated long-term effects of PDGF-BB on Cx43 expression in mesangial cells (MCs). Exposure of MCs to PDGF-BB affected neither the Cx43 protein level nor GJIC. However, in the presence of cAMP-elevating agents, PDGF-BB dramatically increased the expression of Cx43, which was accompanied by obviously augmented membrane distribution of Cx43 and functional GJIC. The increased expression of Cx43 was closely correlated with reduction in alpha-actin, a dedifferentiation marker of MCs. The effect of PDGF on Cx43 was largely prevented by inhibitors of phosphatidylinositol 3'-kinase or mitogen-activated protein kinase, but not by inhibition of protein kinase C. Exposure of MCs to PDGF-BB caused elevation in intracellular cAMP, and it was abolished by indomethacin, a cyclooxygenase inhibitor. However, indomethacin did not affect the synergistic effect. In addition, PDGF-BB also did not affect the degradation of Cx43. With the use of MCs transfected with a Cx43 promoter-luciferase vector, cooperative activation of Cx43 promoter by PDGF and cAMP was found. Together, our data reveal, for the first time, unexpected synergy between PDGF-BB and cAMP-elevating agents in the induction of Cx43 and MC differentiation. Regulation of GJIC could be an important mechanism via which PDGF modulates MC phenotypes.

Animals↗

PubMed2005

Bioassay-based screening of microorganisms that degrade dioxin using substrate-immobilized microtubes.

In the current study, we attempted to develop a method for bioassay-based screening of microorganisms that degrade dioxin. However, a crucial problem encountered was that the standard dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) added to bacterial medium immediately disappeared from the liquid phase due to its adsorption onto polypropylene (PP) tubes. Among other aromatic hydrocarbons, adsorption onto PP tubes was also observed in beta-naphthoflavone but not in benzo[a]pyrene. Adsorption of TCDD was observed not only onto PP tubes but also onto polystyrene, glass, and PP tubes with low affinity for DNA or protein. Silanization was not effective at preventing adsorption of TCDD. TCDD immobilized onto PP tubes was recovered by organic solvents, including ethanol, methanol, and dimethyl sulfoxide (DMSO). The elution efficiency of the immobilized TCDD by DMSO was approximately 85%. Based on these findings, screening of bacteria that degrade dioxin was attempted as follows. First, TCDD was immobilized onto PP tubes. Second, bacterial suspension was added to the tubes and incubated for biodegradation of TCDD. Third, remaining, immobilized TCDD was eluted by DMSO and subjected to a reporter bioassay to evaluate the level of TCDD. Using this method, we demonstrated successful screening of bacteria that have the potential for degradation of dioxin.

Bacteria↗

PubMed2005

Influence of cAMP on reporter bioassays for dioxin and dioxin-like compounds.

In reporter assays for detection of dioxins, the dioxin-responsive element (DRE) is generally used as a sensor sequence. In several systems, the CYP1A1 promoter containing DREs (DRE(cyp)) is inserted into a part of the long terminal repeat of mouse mammary tumor virus (LTR(MMTV)) to improve sensitivity of assays. We found that DRE(cyp)-LTR(MMTV) responds not only to dioxins and dioxin-like compounds but also to forskolin, a cAMP-elevating agent. This effect was dose-dependent and reproduced by other cAMP-elevating agents including 8-bromo-cAMP and 3-isobutyl-methylxanthine. The cAMP response element (CRE) and CRE-like sequences were absent in DRE(cyp)-LTR(MMTV) and not involved in this process. In contrast to the effect of dioxin, the activation of DRE(cyp)-LTR(MMTV) by cAMP was independent of the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor for DRE. Furthermore, neither DRE(cyp), LTR(MMTV) nor the consensus sequence of DRE alone was activated in response to cAMP. These data elucidated for the first time that the combination of DRE(cyp) with LTR(MMTV) causes a peculiar response to cAMP and suggested that use of AhR antagonists is essential to exclude false-positive responses of DRE(cyp)-LTR(MMTV)-based bioassays for detection and quantification of dioxins and dioxin-like compounds.

Animals↗

PubMed2005

Dog sciatic nerve regeneration across a 30-mm defect bridged by a chitosan/PGA artificial nerve graft.

We have developed a dual-component artificial nerve graft comprising an outer microporous conduit of chitosan and internal oriented filaments of polyglycolic acid (PGA). The novel graft was used for bridging sciatic nerve across a 30-mm defect in six Beagle dogs, which were used as a chitosan/PGA graft group. The other Beagle dogs were divided into an autograft group (n = 6) as the positive control and a non-grafted group (n = 5) as the negative control. All animals of three groups were monitored for changes in their appearance and locomotion activities after surgery. Their posture and gait were recorded regularly with the aid of photographs and videotapes for each dog. Six months post-operatively, a combination of electrophysiological examination, FluoroGold retrograde tracing, histological assessment including light microscopy and transmission electron microscopy, immunohistochemistry as well as morphometric analyses to both regenerated nerves and target muscles was utilized to investigate the nerve repair effects of our artificial nerve graft. The results demonstrated that, in the chitosan/PGA graft group, the dog sciatic nerve trunk had been reconstructed with restoration of nerve continuity and functional recovery, and its target skeletal muscle had been re-innervated, improving locomotion activities of the operated limb. This study proves the feasibility of the chitosan/PGA artificial nerve graft for peripheral nerve regeneration by bridging a longer defect in a large animal model.

Action Potentials↗