Biomedical subjects

Lan Chen

Publications and source records attributed to Lan Chen.

16 recordsLinked to original sources

PubMed2003

Novel SIN-1 reactive intermediates modulate chloride secretion across murine airway cells.

We examined the effects of reactive oxygen-nitrogen intermediates on chloride (Cl-) currents across murine tracheal epithelial (MTE) cells isolated from CD-1 mice. MTE cells were cultured on permeable supports until they formed water-tight monolayers with transepithelial resistances (Rt)>500 Omega/cm2 and then were mounted in Ussing chambers. Baseline short-circuit current (ISC) values, prior to and following the addition of 10 microM amiloride (an inhibitor of sodium-transport pathways) into the apical side, were 65 +/- 1.9 microA/cm2 and 7.6 +/- 0.51 microA/cm2, respectively (X +/- 1 SE, n=32). The addition of 3-morpholinosydnominine (SIN-1, 1 mM), which generates both superoxide and nitric oxide anions, to amiloride-treated monolayers resulted in a transient increase of ISC to a peak value of 35 +/- 1.3 microA/cm2 (X +/- SE, n=14) within the next 30-60 min. After this, the ISC decreased gradually and returned to its pre-SIN-1 value. These changes were blocked by glibenclamide (200 microM), an inhibitor of cystic fibrosis transmembrane regulator, or reduced by glutathione (GSH, 5 mM), a scavenger of peroxynitrite. Forskolin (10 microM) augmented the SIN-1 effect when added at the peak of the SIN-1 response but not when ISC had returned to its baseline value. Perfusion of MTE cells with SIN-1 also increased whole cell Cl- currents 4-fold and the open probability of CFTR-type single-channel currents from 0.041 to 0.92 in a transient fashion. Decomposed SIN-1, but not pure SIN-1c (the stable decomposition product of SIN-1), also increased ISC with an EC50 of 5 microM. Electrospray mass spectroscopy revealed several unique and uncharacterized compounds formed during the decomposition of SIN-1 as well as the reaction of SIN-1c with peroxynitrite. Formation of these compounds was inhibited by GSH. We conclude that compounds formed by the reaction of peroxynitrite with by-products of SIN-1, rather than reactive oxygen-nitrogen species per se, were responsible for the modulation of Cl- secretion across primary cultures of MTE cells.

Animals↗

PubMed2003

Ramoplanin inhibits bacterial transglycosylases by binding as a dimer to lipid II.

Ramoplanin is a lipglycodepsipeptide antibiotic that inhibits peptidoglycan biosynthesis. Its mechanism of action has been the subject of debate. It was originally proposed to inhibit the MurG step of peptidoglycan synthesis by binding Lipid I. In this paper, we report that ramoplanin inhibits bacterial transglycosylases by binding to Lipid II, the substrate for these enzymes. The inhibition curves reveal that the inhibitory species has a stoichiometry of 2:1 ramoplanin:Lipid II. A Job titration confirms that ramoplanin binds as a dimer to Lipid II. The apparent dissociation constant is in the nanomolar range, which is unusually low given the nature of the interacting species. We show that Lipid II binding is coupled to the formation of a higher order species, which may explain the tight binding. We also present a testable model for the binding-competent dimeric conformation of ramoplanin.

Anti-Bacterial Agents↗

PubMed2003

Vancomycin analogues active against vanA-resistant strains inhibit bacterial transglycosylase without binding substrate.

Bacterial transglycosylases are enzymes that couple the disaccharide subunits of peptidoglycan to form long carbohydrate chains. These enzymes are the target of the pentasaccharide antibiotic moenomycin as well as the proposed target of certain glycopeptides that overcome vancomycin resistance. Because bacterial transglycosylases are difficult enzymes to study, it has not previously been possible to evaluate how moenomycin inhibits them or to determine whether glycopeptide analogues directly target them. We have identified transglycosylase assay conditions that enable kinetic analysis of inhibitors and have examined the inhibition of Escherichia coli penicillin-binding protein 1b (PBP1b) by moenomycin as well as by various glycopeptides. We report that chlorobiphenyl vancomycin analogues that are incapable of binding substrates nevertheless inhibit E. coli PBP1b, which shows that these compounds interact directly with the enzyme. These findings support the hypothesis that chlorobiphenyl vancomycin derivatives overcome vanA resistance by targeting bacterial transglycosylases. We have also found that moenomycin is not competitive with respect to the lipid II substrate of PBP1b, as has long been believed. With the development of suitable methods to evaluate bacterial transglycosylases, it is now possible to probe the mechanism of action of some potentially very important antibiotics.

Bacterial Proteins↗

PubMed2003

Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases.

MurG is an essential glycosyltransferase that forms the glycosidic linkage between N-acetyl muramyl pentapeptide and N-acetyl glucosamine in the biosynthesis of the bacterial cell wall. This enzyme is a member of a major superfamily of NDP-glycosyltransferases for which no x-ray structures containing intact substrates have been reported. Here we present the 2.5-A crystal structure of Escherichia coli MurG in complex with its donor substrate, UDP-GlcNAc. Combined with genomic analysis of other superfamily members and site-specific mutagenesis of E. coli MurG, this structure sheds light on the molecular basis for both donor and acceptor selectivity for the superfamily. This structural analysis suggests that it will be possible to evolve new glycosyltransferases from prototypical superfamily members by varying two key loops while maintaining the overall architecture of the family and preserving key residues.

Amino Acid Sequence↗

PubMed2003

Functional ablation of the mouse Ldb1 gene results in severe patterning defects during gastrulation.

The LIM domain-binding protein 1 (Ldb1) is found in multi-protein complexes containing various combinations of LIM-homeodomain, LIM-only, bHLH, GATA and Otx transcription factors. These proteins exert key functions during embryogenesis. Here we show that targeted deletion of the Ldb1 gene in mice results in a pleiotropic phenotype. There is no heart anlage and head structures are truncated anterior to the hindbrain. In about 40% of the mutants, posterior axis duplication is observed. There are also severe defects in mesoderm-derived extraembryonic structures, including the allantois, blood islands of the yolk sack, primordial germ cells and the amnion. Abnormal organizer gene expression during gastrulation may account for the observed axis defects in Ldb1 mutant embryos. The expression of several Wnt inhibitors is curtailed in the mutant, suggesting that Wnt pathways may be involved in axial patterning regulated by Ldb1.

Animals↗

PubMed2002

Rethinking ramoplanin: the role of substrate binding in inhibition of peptidoglycan biosynthesis.

Ramoplanin is a cyclicdepsipeptide antibiotic that inhibits peptidoglycan biosynthesis. It was proposed in 1990 to block the MurG step of peptidoglycan synthesis by binding to the substrate of MurG, Lipid I. The proposed mechanism of MurG inhibition has become widely accepted even though it was never directly tested. In this paper, we disprove the accepted mechanism for how ramoplanin functions, and we present an alternative mechanism. This work has implications for the design of ramoplanin derivatives and may influence how other proposed substrate binding antibiotics are studied.

Anti-Bacterial Agents↗

PubMed2002

Ssdp proteins interact with the LIM-domain-binding protein Ldb1 to regulate development.

The LIM-domain-binding protein Ldb1 is a key factor in the assembly of transcriptional complexes involving LIM-homeodomain proteins and other transcription factors that regulate animal development. We identified Ssdp proteins (previously described as sequence-specific, single-stranded-DNA-binding proteins) as components of Ldb1-associated nuclear complexes in HeLa cells. Ssdp proteins are associated with Ldb1 in a variety of additional mammalian cell types. This association is specific, does not depend on the presence of nucleic acids, and is functionally significant. Genes encoding Ssdp proteins are well conserved in evolution from Drosophila to humans. Whereas the vertebrate Ssdp gene family has several closely related members, the Drosophila Ssdp gene is unique. In Xenopus, Ssdp encoded by Drosophila Ssdp or mouse Ssdp1 mRNA enhances axis induction by Ldb1 in conjunction with the LIM-homeobox gene Xlim1. Furthermore, we were able to demonstrate an interaction between Ssdp and Chip (the fly homolog of Ldb1) in Drosophila wing development. These findings indicate functional conservation of Ssdp as a cofactor of Ldb1 during invertebrate and vertebrate development.

3T3 Cells↗

PubMed2002

Reactive oxygen nitrogen species decrease cystic fibrosis transmembrane conductance regulator expression and cAMP-mediated Cl- secretion in airway epithelia.

We investigated putative mechanisms by which nitric oxide modulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and function in epithelial cells. Immunoprecipitation followed by Western blotting, as well as immunocytochemical and cell surface biotinylation measurements, showed that incubation of both stably transduced (HeLa) and endogenous CFTR expressing (16HBE14o-, Calu-3, and mouse tracheal epithelial) cells with 100 microm diethylenetriamine NONOate (DETA NONOate) for 24-96 h decreased both intracellular and apical CFTR levels. Calu-3 and mouse tracheal epithelial cells, incubated with DETA NONOate but not with 100 microm 8-bromo-cGMP for 96 h, exhibited reduced cAMP-activated short circuit currents when mounted in Ussing chambers. Exposure of Calu-3 cells to nitric oxide donors resulted in the nitration of a number of proteins including CFTR. Nitration was augmented by proteasome inhibition, suggesting a role for the proteasome in the degradation of nitrated proteins. Our studies demonstrate that levels of nitric oxide that are likely to be encountered in the vicinity of airway cells during inflammation may nitrate CFTR resulting in enhanced degradation and decreased function. Decreased levels and function of normal CFTR may account for some of the cystic fibrosis-like symptoms that occur in chronic inflammatory lung diseases associated with increased NO production.

Animals↗

PubMed2002

Intrinsic lipid preferences and kinetic mechanism of Escherichia coli MurG.

MurG, the last enzyme involved in the intracellular phase of peptidoglycan synthesis, is a membrane-associated glycosyltransferase that couples N-acetyl glucosamine to the C4 hydroxyl of a lipid-linked N-acetyl muramic acid derivative (lipid I) to form the beta-linked disaccharide (lipid II) that is the minimal subunit of peptidoglycan. Lipid I is anchored to the bacterial membrane by a 55 carbon undecaprenyl chain. Because this long lipid chain impedes kinetic analysis of MurG, we have been investigating alternative substrates containing shortened lipid chains. We now describe the intrinsic lipid preferences of MurG and show that the optimal substrate for MurG in the absence of membranes is not the natural substrate. Thus, while the undecaprenyl carrier lipid may be critical for certain steps in the biosynthetic pathway to peptidoglycan, it is not required-in fact, is not preferred-by MurG. Using synthetic substrate analogues and products containing different length lipid chains, as well as a synthetic dead-end acceptor analogue, we have also shown that MurG follows a compulsory ordered Bi Bi mechanism in which the donor sugar binds first. This information should facilitate obtaining crystals of MurG with substrates bound, an important goal because MurG belongs to a major superfamily of NDP-glycosyltransferases for which no structures containing intact substrates have yet been solved.

Bacterial Outer Membrane Proteins↗

PubMed2002

A remote upstream element regulates tissue-specific expression of the rat aggrecan gene.

The regulation of chondrogenesis and of the genes expressed as markers of chondrocyte differentiation is poorly understood. The hyaluronan-binding proteoglycan aggrecan is an essential and specific component of cartilage, but the aggrecan proximal promoter is expressed in an unregulated fashion in vitro. DNA comprising the rat aggrecan gene (83 kb including the 30-kb first intron) was surveyed for active elements, which would impart selective expression to the aggrecan promoter in transfection assays in vitro. A 4.7-kb DNA fragment (P3) with cell-specific enhancer activity was discovered approximately 12 kb upstream of the transcription start site; this active DNA fragment is position- and orientation-independent, and strongly stimulates aggrecan promoter expression in chondrocytes, while weakly suppressing transcription in fibroblasts. Most of this activity has been localized to P3-7, a 2.3-kb internal fragment of P3. Another enhancer element (A23), which is not tissue-specific, was discovered about 70 kb downstream of the transcription start site. Several lines of transgenic mice were created using combinations of these DNA elements to drive the lacZ reporter gene. Neither a short (900 bp) nor a long (3.7 kb) promoter alone showed detectable expression in 14.5-day embryos, whereas placing the P3 tissue-specific enhancer together with P0 gave strong expression restricted to embryonic cartilage of transgenic mice. The A23 downstream enhancer in conjunction with P0 did not confer expression. This is the first report of a gene control region which confers authentic tissue-specific regulation of aggrecan in vitro or in vivo and should greatly facilitate understanding the coordinate regulation of chondrocytic genes.

3T3 Cells↗

PubMed2002

Spatial and temporal 'knock down' of gene expression by electroporation of double-stranded RNA and morpholinos into early postimplantation mouse embryos.

Here we report the use of double-stranded RNA (dsRNA) and morpholino technologies to specifically 'knock down' gene expression in early postimplantation mouse embryos. Sequence specific interference mediated by either dsRNA or by morpholino has been a useful tool for studying gene function in several organisms. However, specifically for the dsRNA, doubts have been raised about whether it could successfully be applied on vertebrate embryos. We demonstrate that electroporation of dsRNA directed against Otx2 or Foxa2 into postimplantation mouse embryos results in specific knock down of the expression of the respective endogenous genes in a region- and germ-layer specific manner. We also show that electroporation of morpholino directed against Foxa2 into the node of mouse embryos leads to a specific down regulation of Foxa2 expression in the floor plate. Our results demonstrate for the first time that dsRNA and morpholino technologies can be successfully applied in early postimplantation mouse embryos to specifically knock down gene expression.

Animals↗

PubMed2002

Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks.

The anterior visceral endoderm plays a pivotal role in establishing anterior-posterior polarity of the mouse embryo, but the molecular nature of the signals required remains to be determined. Here, we demonstrate that Cerberus-like(-/-);Lefty1(-/-) compound mutants can develop a primitive streak ectopically in the embryo. This defect is not rescued in chimeras containing wild-type embryonic, and Cerberus-like(-/-);Lefty1(-/-) extraembryonic, cells but is rescued in Cerberus-like(-/-); Lefty1(-/-) embryos after removal of one copy of the Nodal gene. Our findings provide support for a model whereby Cerberus-like and Lefty1 in the anterior visceral endoderm restrict primitive streak formation to the posterior end of mouse embryos by antagonizing Nodal signaling. Both antagonists are also required for proper patterning of the primitive streak.

Animals↗

PubMed2002

cAMP regulation of Cl(-) and HCO(-)(3) secretion across rat fetal distal lung epithelial cells.

We isolated and cultured fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses and assayed for anion secretion in Ussing chambers. With symmetrical Ringer solutions, basal short-circuit currents (I(sc)) and transepithelial resistances were 7.9 +/- 0.5 microA/cm(2) and 1,018 +/- 73 Omega.cm(2), respectively (means +/- SE; n = 12). Apical amiloride (10 microM) inhibited basal I(sc) by approximately 50%. Subsequent addition of forskolin (10 microM) increased I(sc) from 3.9 +/- 0.63 microA/cm(2) to 7.51 +/- 0.2 microA/cm(2) (n = 12). Basolateral bumetanide (100 microM) decreased forskolin-stimulated I(sc) from 7.51 +/- 0.2 microA/cm(2) to 5.62 +/- 0.53, whereas basolateral 4,4'-dinitrostilbene-2,2'-disulfonate (5 mM), an inhibitor of HCO secretion, blocked the remaining I(sc). Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl(-)- or HCO(-)(3)-free solutions; however, no response was seen using HCO(-)(3)- and Cl(-)-free solutions. The forskolin-stimulated I(sc) was inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced I(sc) across monolayers having nystatin-permeablized basolateral membranes. Immunolocalization studies were consistent with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in FDLE cells. In aggregate, these findings indicate the presence of cAMP-activated Cl(-) and HCO(-)(3) secretion across rat FDLE cells mediated via CFTR.

Amiloride↗

PubMed2002

[Primary adenocarcinoma of vagina--attaching 24 cases clinical analysis].

BACKGROUND & OBJECTIVES: The incidence of primary vaginal adenocarcinoma was lower and its treatment was difficult and its prognosis was worse. This study was designed to explore the clinical characters, treatment, prognosis of primary vaginal adenocarcinoma in this paper as the incidence of primary vaginal adenocarcinoma was lower, its treatment was difficult and its prognosis was worse. METHODS: From 1964 to 1999, twenty-four patients were reviewed retrospectively in our hospital. RESULTS: The main clinical symptoms were vaginal bleeding and discharge. The median age was 49 years old. Five patients were classified as stage I, 10 patients were stage II, 5 patients were stage III, and 4 patients were stage IV. The lesion was located in the anterior wall in 8(33.3%) patients, the posterior wall in 5(20.8%) patients, the left wall in 1 patient and the multiple walls in 10(41.7%) patients. Twenty-four patients were generally received radiotherapy, including external-beam and intracavitary irradiation. Two patients were concurrently received chemotherapy and irradiation, Eight patients were received chemotherapy as the tumor was not control and recurrent or metastatic. The survival rate of 3 years was 47.3% and that of 5 years was 34.2%. The survival mean time of patients with stage I and stage II was 60.3 months obviously much longer than 14.8 months of patients with stage III and stage IV, and they were significantly statistical difference (P < 0.01). But the survival mean time of patients more than 40 years and less than or equal to 40 years was not significantly statistical difference. Eight patients had local recurrence, which is the main reason of failure treatment. CONCLUSIONS: Primary vaginal adenocarcinomas should mainly be adopted combined treatment depending on radiotherapy, but the prognosis was worse, the stage was the important factors affecting on the prognosis.

Adenocarcinoma↗

PubMed2002

[A colorimetric method of HEC toxin hemolysis test for diagnosis of paroxysmal nocturnal hemoglobinuria].

The purpose of the study is to establish a colorimetric method of HEC toxin hemolysis test for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). RBCs from normal persons and patients with PNH and non-PNH anemia were treated with HEC toxin secreted by Aeromonas hydrophila J-1 strain and the absorbance at 630 nm was measured to quantitate the extent of hemolysis. The results demonstrated that the RBCs from PNH patients showed resistance to the toxin hemolysis, which was in accord with the percentages of CD59(-) cells, while the RBCs from normal persons and non-PNH anemic patients were nearly totally lysed. It is concluded that the method can be considered as a simple, specific and reliable method for the diagnosis of PNH.

Bacterial Toxins↗

PubMed1999

Expression Purification and Activity ofhuman Myristoyl-CoA:N-myristoyltransferase.

A gene encodinghuman myristoyl-CoA: protein N-myristoyltransferase (hNMT) from a brain cDNA libraryhas been obtained by PCR amplification and DNA sequencing. Then the mature-type and His(6)-fusion-type expression plasmids (pMF-hNMT(3) and pMFHT-hNMT(2)) containing the hNMT gene under control of T7 promoter have been constructed and transformed into E. coli BL21(DE3). SDS-PAGE analysis showed that the recombinant hNMT products expressed at 37 degrees were almost unsoluble but most of the His(6)-hNMT product expressed at lower temperature (22 degrees ) was soluble and its yield was about 7% of the total soluble cellular proteins. By immobilized metal (Ni(2+)) chelation affinity chromatography up to 80% His(6)-hNMT was purified by one step from bacterial lysate. The labelling experiment in vitro domonstrated that the expressed and purified His(6)-hNMT had an obvious catalytic activity to transferring myristoyl group.

Journal Article↗