Biomedical subjects

L Chen

Publications and source records attributed to L Chen.

At least 163 records · Page 9Linked to original sources

PubMed2001

Roles of aggrecan domains in biosynthesis, modification by glycosaminoglycans and product secretion.

Aggrecan is a member of the chondroitin sulphate (CS) proteoglycan family, which also includes versican/PG-M, neurocan and brevican. Members of this family exhibit structural similarity: a G1 domain at the N-terminus and a G3 domain at the C-terminus, with a central sequence for modification by CS chains. A unique feature of aggrecan is the insertion of three additional domains, an inter-globular domain (IGD), a G2 domain and a keratan sulphate (KS) domain (sequence modified by KS chains), between the G1 domain and the CS domain (sequence modified by CS chains). The G1 and G3 domains have been implicated in product secretion, but G2, although structurally similar to the tandem repeats of G1, performs an unknown function. To define the functions of each aggrecan domain in product processing, we cloned and expressed these domains in various combinations in COS-7 cells. The results indicated that the G3 domain enhanced product secretion, alone or in combination with the KS or CS domain, and promoted glycosaminoglycan (GAG) chain attachment. Constructs containing the G1 domain were not secreted. Addition of a CS domain sequence to G1 reduced this inhibition, but GAG chain attachment was still decreased. The potential GAG chain attachment site in the IGD was occupied by GAGs, and IGD product was secreted efficiently. The KS domain was modified by GAG chains and secreted. Finally, the G2 domain was expressed but not secreted, and inhibited secretion of the IGD when expressed as an IGD-G2 combination.

Aggrecans↗

PubMed2001

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine.

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca(2+) release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200-500 microM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, P(o) < 0.005) and brief (<250 micros) gating events and insensitivity to Ca(2+). Addition of ryanodine restores Ca(2+)-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

Allosteric Regulation↗

PubMed2001

Molecular cloning of pituitary tumor transforming gene 1 from ovarian tumors and its expression in tumors.

Pituitary tumor transforming gene 1 (PTTG1) recently cloned from human testis is a potent oncogene and is highly expressed in all the tumors analyzed to date. However, primary structure of PTTG1 and the cell types that express PTTG1 in tumors remained undescribed. We have used the reverse transcriptase-polymerase chain reaction technique to clone PTTG1 from ovarian tumors. Nucleotide sequencing of the PTTG1 cDNAs from various ovarian tumors showed identity with that of the human testis PTTG1. To determine the cell types that express PTTG1 in normal and tumor tissues, we performed in situ hybridization using digoxigenin-labeled cRNA as a probe. Our studies revealed a high level of expression of PTTG1 mRNA in both seminomatous and non-seminomatous testicular tumors; epithelial, sex-cord and stromal cell, and germ cell tumors of the ovary; and invasive ductal, ductal in situ and infiltrating ductal carcinoma of the breast. In normal tissues, expression of PTTG1 mRNA was very low or undetectable except in testis, where PTTG1 mRNA was found to be localized to spermatocytes and spermatids. Tumors that expressed high levels of PTTG1 mRNA also exhibited high levels of expression of basic fibroblast growth factor (bFGF), suggesting a correlation between PTTG1 and bFGF expression, and further suggesting that the PTTG1 protein may be involved in tumor angiogenesis and mitogenesis.

Amino Acid Sequence↗

PubMed2001

Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase.

A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

Amino Acid Sequence↗

PubMed2001

Versican modulates embryonic chondrocyte morphology via the epidermal growth factor-like motifs in G3.

This investigation was designed to characterize the effect of the extracellular matrix molecule versican on chondrocyte morphology, using the well-studied chondrocyte cell culture system. When cultured chondrocytes reverted or "dedifferentiated" to a fibroblast-like morphology, we found that versican expression was significantly enhanced. Transfection of chondrocytes, isolated from embryonic chicken sterna, with a chicken miniversican construct accelerated the reversion process, while expression of an antisense construct inhibited it. A mutant miniversican lacking two epidermal growth factor-like motifs (versicanDeltaEGF) promoted differentiation, as shown by morphological changes and changes in the expression of other extracellular matrix molecules. A truncated versican mutant, the G3DeltaEGF, a G3 domain lacking its two epidermal growth factor-like motifs, also enhanced differentiation. This effect is related to G3DeltaEGF-induced change in cytoskeleton, since transfected cells exhibited misassembly of actin filaments. This article thus provides the first evidence that versican modulates chondrocyte morphology via changes in cytoskeletal structure, and may imply that extracellular matrix molecules play an important role in cell differentiation.

Actins↗

PubMed2001

Identification of the motif in versican G3 domain that plays a dominant-negative effect on astrocytoma cell proliferation through inhibiting versican secretion and binding.

This study was designed to investigate the mechanisms by which mutant versican constructs play a dominant-negative effect on astrocytoma cell proliferation. Although a mini-versican or a versican G3 construct promoted growth of U87 astrocytoma cells, a mini-versican lacking epidermal growth factor (EGF) motifs (versicanDeltaEGF) and a G3 mutant (G3DeltaEGF) exerted a dominant-negative effect on cell proliferation. G3DeltaEGF-transfected cells formed smaller colonies, arrested cell cycle at G(1) phase, inhibited expression of cell cycle proteins cdk4 and cyclin D1, and contained multiple nucleoli. In cell surface binding assays, G3 products expressed in COS-7 cells and bacteria bound to U87 cell surface. G3DeltaEGF products exhibited decreased binding activity, but higher levels of G3DeltaEGF products were able to inhibit the binding of G3 to the cell surface. G3DeltaEGF expression inhibited secretion of endogenous versican in astrocytoma cells and also inhibited the secretion of mini-versican in COS-7 cells co-transfected with the mini-versican and G3DeltaEGF constructs. The effect seems to depend on the expression efficiency of G3DeltaEGF, and it occurred via the carbohydrate recognition domain.

Amino Acid Motifs↗

PubMed2001

Syntheses, structures, and luminescence of novel lanthanide complexes of tripyridylamine, N,N,N',N'-tetra(2-pyridyl)-1,4-phenylenediamine and N,N,N',N'-tetra(2-pyridyl)biphenyl-4,4'-diamine.

Two novel blue luminescent bridging ligands N,N,N',N'-tetra(2-pyridyl)-1,4-phenylenediamine (tppd) and N,N,N',N'-tetra(2-pyridyl)-1,1-biphenyl-4,4'-diamine (tpbpd) have been synthesized. Several novel lanthanide complexes containing 2,2',2"-tripyridylamine (2,2',2"-tpa), 2,2',3"-tpa, tppd, or tpbpd ligands have been synthesized and characterized structurally, which include Pr(hfa)3(2,2',2"-tpa), I, Ln(tmhd)3(2,2',3"-tpa), 2 (Ln = Dy, 2a; Eu, 2b; Tb, 2c; Sm, 2d), [Eu(tmhd)3][Pr(hfa)3](2,2',3"-tpa), 3, [Pr(hfa)3]2(tppd), 4, and [Ln(hfa)3]2(tpbpd), 5, where Ln = Pr (5a), Eu (5b), tmhd = 2,2,6,6-tetramethyl-3,5-heptanedionato, and hfa = hexafluoroacetylacetonate. The Dy(III), Eu(III), and Tb(III) complexes display a bright photoluminescence, which can be achieved by either a direct excitation process or an indirect excitation process. Compounds 2a-2d can be sublimed readily.

Journal Article↗

PubMed2001

Herbicide resistance from a divided EPSPS protein: the split Synechocystis DnaE intein as an in vivo affinity domain.

We report that the N- and C-terminal splicing domains of the intein found in the dnaE gene of Synechocystis sp. PCC6803 (Ssp DnaE intein) are capable of association in vivo and in vitro, even with key splicing residues changed to alanine (Cys(1), Asn(159), and Cys(+1) to Ala). These studies utilized the herbicide resistant form of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Salmonella typhimurium and an Escherichia coli strain with the EPSPS gene deleted from its genome (E. coli strain ER2799). EPSPS was mapped to identify potential split sites using a facile Tn7 linker scanning procedure. Forty positions were found to tolerate a five amino acid insertion while 21 sites did not, as assayed by the rescue of growth of E. coli strain ER2799. Further characterization of these sites by inserting a full length Ssp DnaE intein identified residue 235 of EPSPS as the optimal position. The EPSPS gene was then divided into amino acids 1-235 and 236-427 which were fused to residues 1-123 and 124-159 of a splicing defective Ssp DnaE intein, respectively. Expression of the EPSPS-intein fusions from separate DNA molecules conferred resistance to the herbicide glyphosate, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. As a control the split EPSPS without the intein-affinity domain did not allow cell growth. The use of an intein as an in vivo affinity domain was termed intein-mediated protein complementation (IPC). Intein fragment assembly was verified in vitro by immobilizing the C-terminal splicing domain of the Ssp DnaE intein on a resin and demonstrating that the N-terminal 235 amino acids of EPSPS only bound to the resin when fused to the N-terminal splicing domain of the Ssp DnaE intein. As chloroplast DNA is not transmitted by pollen in plants such as corn and soybean, transgene spread via pollen may be controlled in the future by expressing inactive gene fragments from separate DNA locations, such as the nuclear and chloroplast genome, and using the split intein to generate protein activity.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2001

Crystal structure and catalytic mechanism of the MJ0109 gene product: a bifunctional enzyme with inositol monophosphatase and fructose 1,6-bisphosphatase activities.

Inositol monophosphatase (EC 3.1.3.25) in hyperthermophilic archaea is thought to play a role in the biosynthesis of di-myo-inositol-1,1'-phosphate (DIP), an osmolyte unique to hyperthermophiles. The Methanococcus jannaschii MJ109 gene product, the sequence of which is substantially homologous to that of human inositol monophosphatase, exhibits inositol monophosphatase activity but with substrate specificity that is broader than those of bacterial and eukaryotic inositol monophosphatases (it can also act as a fructose bisphosphatase). To understand its substrate specificity as well as the poor inhibition by Li(+) (a potent inhibitor of the mammalian enzyme), we have crystallized the enzyme and determined its three-dimensional structure. The overall fold, as expected, is similar to that of the mammalian enzyme, but the details suggest a closer relationship to fructose 1,6-bisphosphatases. Three complexes of the MJ0109 protein with substrate and/or product and inhibitory as well as activating metal ions suggest that the phosphatase mechanism is a three-metal ion assisted catalysis which is in variance with that proposed previously for the human inositol monophosphatase.

Amino Acid Sequence↗

PubMed2001

Survival analysis of clinical, pathologic, and genetic features in neuroblastoma presenting as locoregional disease.

BACKGROUND: Locoregional neuroblastoma is a clinical subgroup characterized by the absence of distant metastasis (International Neuroblastoma Staging System Stages 1, 2, and 3). Although these patients generally have an excellent survival with minimal therapy, some do experience recurrence with lethal consequences. METHODS: To identify risk factors for disease progression, the authors performed a retrospective analysis of clinical (age and stage) and tumor biologic markers (histology, MYCN, DNA index, and allelic analysis of chromosomes 1p, 11q12-qter, and 14q12-q32) in 44 patients (10 Stage 1, 18 Stage 2, and 16 Stage 3). Allelic analysis was performed using polymorphic polymerase chain reaction markers in a semiautomated, fluorescent detection system. RESULTS: Sixteen patients (38%) were younger than 365 days at diagnosis. Seventeen of 39 tumors (43%) had unfavorable histology, 6 (13%) were MYCN amplified, 14 (31%) were diploid, 17 (38%) had 1p36 loss of heterozygosity (LOH), 11 (25%) had 1p22 LOH, 10 (22%) had 11q LOH, and 13 (29%) had 14q LOH. Seventeen patients (38%) progressed, including 6 who progressed to Stage 4 disease (13%). Sixteen patients with progressive disease received cytotoxic therapy. Thirty-seven patients are alive (84%) with a median follow-up of 51 months. By permutation log rank test, both MYCN amplification and diploidy were associated with overall survival (OS), but only diploidy was associated with progression free survival (PFS) and progression to Stage 4 disease. LOH of 1p36, 1p22, 11q, or 14q did not show correlation with either OS or PFS. CONCLUSIONS: Locoregional neuroblastoma tumors with diploid DNA index, regardless of other biologic features, have increased risk of local recurrence and Stage 4 progression.

Child↗

PubMed2001

High-speed counter-current chromatography separation and purification of resveratrol and piceid from Polygonum cuspidatum.

High-speed counter-current chromatography (HSCCC) was applied to the separation and purification of resveratrol and piceid from the dried roots (20.0 g) of Polygonum cuspidatium. The EtOAc extracts were separated with chloroform-methanol-water (4:3:2, v/v). Resveratrol was identified in fraction 5. The water extracts were separated first with EtOAc-EtOH-water (10:1:10, v/v) and then with the same solvent system at the modified volume ratio of 70:1:70. Yields of resveratrol and piceid obtained were 2.18% and 1.07%. Chemical structures of the purified resveratrol and piceid were confirmed by electrospray ionization MS and 1H nuclear magnetic resonance spectroscopy.

Chromatography, High Pressure Liquid↗

PubMed2001

Stabilization of the MDM2 oncoprotein by mutant p53.

MDM2 is a short-lived protein that regulates p53 degradation. We report here that transient coexpression of MDM2 and several p53 hotspot mutants resulted in stabilization and increased expression of MDM2. Ectopic expression of the mutant p53(175H) allele by recombinant adenovirus infection or stable transfection also stabilized endogenous MDM2 in p53-null cells. A panel of human tumor cell lines expressing different endogenous mutant p53 alleles also contained stabilized nuclear MDM2 at elevated levels when compared with p53-null cells. MDM2 was present in complexes with mutant p53 in tumor cells, and stabilization of MDM2 required direct binding to mutant p53. These results reveal a novel property of mutant p53 and a unique feature of tumors with p53 missense mutations. Accumulation of stable MDM2 may contribute to tumorigenesis through its p53-independent transforming functions.

Humans↗

PubMed2001

Neutrophil depletion exacerbates experimental Chagas' disease in BALB/c, but protects C57BL/6 mice through modulating the Th1/Th2 dichotomy in different directions.

To elucidate the roles of neutrophils in experimental Chagas' disease, we depleted the peripheral neutrophils in BALB/c and C57BL/6 mice with a monoclonal antibody 1 day before Trypanosoma cruzi infection. Neutrophil depletion in BALB/ c mice resulted in exacerbation of the disease and decreased expression of mRNA for Th1 cytokines, including IL-2 and IFN-gamma, IL-12p40 and TNF-alpha in their spleens after the infection, while a Th2 cytokine, IL-10, increased especially 1 day after infection. Neutrophils from infected BALB / c mice expressed mRNA for IL-12p40, IFN-gamma, TNF-alpha and Th1 chemoattractive chemokines, monokine induced by IFN-gamma (MIG) and macrophage inflammatory protein-1alpha (MIP-1alpha ). In contrast, in C57BL/6 mice neutrophil depletion induced resistance to the disease and enhanced the expression of the above Th1 cytokines, although IL-10 mRNA in neutrophil-depleted C57BL/6 mice was also higher than in control mice. Neutrophils from C57BL/6 mice did not express IL-12p40, IFN-gamma and MIG but expressed TNF-alpha, MIP-1alpha and IL-10. Therefore, neutrophils may play opposite roles in these two strains of mice with respect to protection versus exacerbation of T. cruzi infection, possibly through modulating the Th1/Th2 dichotomy in different directions.

Animals↗

PubMed2001

MRI study of immediate cell viability in focused ultrasound lesions in the rabbit brain.

The purpose of this study was to evaluate cell viability in MR imaged focused ultrasound (FUS) lesions using cell-viability staining with triphenyl tetrazolium chloride (TTC) and both light and electron microscopy. Ten paired ultrasonic lesions were created in 5 rabbit brains in vivo with an ultrasound beam of 1.5 MHz electrical power input to the transducer of 50 W and exposure duration of 15 seconds. T2-weighted fast spin-echo (FSE) MRI was performed to detect the FUS lesions in the brain 4 hours after treatment, after which the animals were immediately euthanized. Lesion sizes were measured on TTC-stained specimens, histological sections stained with hematoxylin and eosin (H&E), and T2-weighted MR images. The differences between the lesion diameters measured with the three methods were within the range of 0.1--0.7 mm. The lesion sizes measured from MRI correlated well with those seen from H&E sections. The measurements from MRI slightly overestimated lesion sizes on TTC-stained wet tissues by approximately one MRI pixel (0.31 mm). Electron microscopy demonstrated nuclear and cytoplasmic ultrastructural damage within the grey-white, non-TTC-stained lesion zone, whereas the TTC-stained normal tissue showed preservation of neuronal ultrastructure. Therefore, MR-imaged lesions represent a cell-death zone in rabbit brain 4 hours after FUS ablation, with slight overestimation by approximately one MRI pixel. J. Magn. Reson. Imaging 2001;13:23-30.

Animals↗

PubMed2001

Regulation of gene expression by small molecules in rice.

A system for the regulation of gene expression by small molecules in transgenic rice was developed. This gene switch system consists of two components: (1) a hybrid chemically activated transcription factor, and (2) a synthetic target promoter. The two elements were transformed into rice suspension cells and transgenic plants were regenerated. A luciferase reporter under control of the gene switch system displayed as high as 10,000-fold inducibility following exposure to the small molecule ligand. The dose-response and induction time-course were determined. Regulated luciferase activity in activated plants decreased one day following removal of ligand and could be reactivated multiple times without apparent cosuppression. Analysis of luciferase activity following ligand application to media surrounding the roots suggests that ligand can be absorbed and transported systemically. In contrast, reporter activation was limited to a small area when ligand was applied directly to the leaf surface. The described gene switch system represents an important tool for situations requiring conditional gene expression in a monocot species.

Biological Transport↗

PubMed2001

Characterization of a spontaneously arising murine squamous cell carcinoma (SCC VII) as a prerequisite for head and neck cancer immunotherapy.

BACKGROUND: To develop novel therapeutic approaches for patients with head and neck malignancies, poorly immunogenic murine models of squamous cell carcinoma (SCC) need to be defined. METHODS: The phenotype, growth characteristics, and responsiveness to tumor-specific T-cell transfer of a spontaneously arising murine SCC (SCC VII) were characterized. RESULTS: SCC VII expresses major histocompatibility complex (MHC) class I molecules yet is resistant to tumor-specific T-cell killing and relatively insensitive to killing mediated by lymphokine-activated killer (LAK) cells. Intradermal tumors are reproducibly established after vaccination of 5 x 10(4) cells, and systemic micrometastases are apparent after intravenous administration of 2.5 x 10(4) cells. Immunotherapy of 3-day lung metastases using tumor-specific T cells and systemic interleukin-2 (IL-2) was ineffective in reducing the number of metastases in vivo. CONCLUSIONS: SCC VII is a poorly immunogenic murine squamous cell cancer, which represents an ideal model for preclinical testing of immunotherapeutic approaches for patients with SCC of the upper aerodigestive tract.

Animals↗

PubMed2001

Sequence-structure homology recognition by iterative alignment refinement and comparative modeling.

Our approach to fold recognition for the fourth critical assessment of techniques for protein structure prediction (CASP4) experiment involved the use of the FUGUE sequence-structure homology recognition program (http://www-cryst.bioc.cam.ac.uk/fugue), followed by model building. We treat models as hypotheses and examine these to determine whether they explain the available data. Our method depends heavily on environment-specific substitution tables derived from our database of structural alignments of homologous proteins (HOMSTRAD, http://www-cryst.bioc.cam.ac.uk/homstrad/). FUGUE uses these tables to incorporate structural information into profiles created from HOMSTRAD alignments that are matched against a profile created for the target from multiple sequence alignment. In addition, environment-specific substitution tables are used throughout the modeling procedure and as part of the model evaluation. Annotation of sequence alignments with JOY, to reflect local structural features, proved valuable, both for modifying hypotheses, and for rejecting predictions when the expected pattern of conservation is not observed. Our stringency in rejecting incorrect predictions led us to submit a relatively small number of models, including only a low number of false positives, resulting in a high average score.

Amino Acid Sequence↗

PubMed2001

TNF-alpha receptor 1 (p55) on islets is necessary for the expression of LIGHT on diabetogenic T cells.

Insulin-dependent diabetes mellitus results from T-cell-mediated destruction of pancreatic islet beta cells. Both CD4 and CD8 T cells have been shown to be independently capable of beta cell destruction. However, the mechanism of beta cell destruction has remained elusive. It has previously been shown that the absence of TNF-alpha receptor 1 (p55) on the islets protected islets from CD4 T-cell-mediated destruction as long as the T cells did not have access to wild-type islets in vivo. Wild-type and TNF-alpha receptor 1 (p55) deficient islets induce similar levels of proliferation of BDC2.5 T cells. In this study, we demonstrate that islet TNF-alpha receptor 1 (p55) influences the expression of LIGHT (TNFSF-14), a TNF family member with both cytolytic and costimulatory properties, on BDC2.5 T cells and the expression of its receptor HVEM (TNFRSF-14) by islets, indicating a role for LIGHT-HVEM interactions in autoimmune diabetes.

Animals↗