Biomedical subjects

J Song

Publications and source records attributed to J Song.

At least 37 records · Page 2Linked to original sources

PubMed2001

Two consecutive zinc fingers in Sp1 and in MAZ are essential for interactions with cis-elements.

The zinc finger proteins Sp1 and Myc-associated zinc finger protein (MAZ) are transcription factors that control the expression of various genes. Regulation of transcription by these factors is based on interactions between GC-rich DNA-binding sites (GGGCGG for Sp1 and GGGAGGG for MAZ) and the carboxyl-terminal zinc finger motifs of the two proteins. Sp1 and MAZ have three and six zinc fingers, respectively, and the details of their interactions with cis-elements remain to be clarified. We demonstrate here that Sp1 and MAZ interact with the same GC-rich DNA-binding sites, apparently sharing DNA-binding sites with each other. We found that the DNA binding activities of Sp1 and MAZ depended mainly on consecutive zinc fingers, namely the second and third zinc fingers in Sp1 and the third and fourth zinc fingers in MAZ. Furthermore, the interactions of the zinc finger proteins with the same cis-elements appear to play a critical role in the regulation of gene expression. It seems plausible that two consecutive zinc finger motifs in a zinc finger protein might be essential for interaction of the protein with DNA.

3T3 Cells↗

PubMed2001

High-yield expression and functional analysis of Escherichia coli glycerol-3-phosphate transporter.

The glycerol-3-phosphate (G3P) transporter, GlpT, from Escherichia coli mediates G3P and inorganic phosphate exchange across the bacterial inner membrane. It possesses 12 transmembrane alpha-helices and is a member of the Major Facilitator Superfamily. Here we report overexpression, purification, and characterization of GlpT. Extensive optimization applied to the DNA construct and cell culture has led to a protocol yielding approximately 1.8 mg of the transporter protein per liter of E. coli culture. After purification, this protein binds substrates in detergent solution, as measured by tryptophan fluorescence quenching, and its dissociation constants for G3P, glycerol-2-phosphate, and inorganic phosphate at neutral pH are 3.64, 0.34, and 9.18 microM, respectively. It also shows transport activity upon reconstitution into proteoliposomes. The phosphate efflux rate of the transporter in the presence of G3P is measured to be 29 micromol min(-1) mg(-1) at pH 7.0 and 37 degrees C, corresponding to 24 mol of phosphate s(-1) (mol of protein)(-1). In addition, the glycerol-3-phosphate transporter is monomeric and stable over a wide pH range and in the presence of a variety of detergents. This preparation of GlpT provides ideal material for biochemical, biophysical, and structural studies of the glycerol-3-phosphate transporter.

Biological Transport↗

PubMed2001

A new-generation stable inducible packaging cell line for lentiviral vectors.

We have successfully generated and characterized a stable packaging cell line for HIV-1-based vectors. To allow safe production of vector, a minimal packaging construct carrying only the coding sequences of the HIV-1 gag-pol, tat, and rev genes was stably introduced into 293G cells under the control of a Tet(o) minimal promoter. 293G cells express the chimeric Tet(R)/VP16 trans-activator and contain a tetracycline-regulated vesicular stomatitis virus protein G (VSV-G) envelope gene. When the cells were grown in the presence of tetracycline the expression of both HIV-1-derived and VSV-derived packaging functions was suppressed. On induction, approximately 50 ng/ml/24 hr of Gag p24 equivalent of vector was obtained. After introduction of the transfer vector by serial infection, vector could be collected for several days with a transduction efficiency similar or superior to that of vector produced by transient transfection both for dividing and growth-arrested cells. The vector could be effectively concentrated to titers reaching 10(9) transducing units/ml and allowed for efficient delivery and stable expression of a GFP transgene in the mouse brain. The packaging cell line and all vector producer clones described here were shown to be free from replication-competent recombinants, and from recombinants between packaging and vector constructs that transfer the viral gag-pol genes. The packaging cell line and the assays developed will advance lentiviral vectors toward the stringent requirements of clinical applications.

Animals↗

PubMed2001

Monomeric state and ligand binding of recombinant GABA transporter from Escherichia coli.

The gamma-aminobutyric acid (GABA) transporter from Escherichia coli was homologously overexpressed and purified to homogeneity with a yield of 1.0 mg per liter culture. The purification procedure consists of a cobalt affinity column, proteolytic cleavage of His- and myc-tags, and size-exclusion chromatography. The purified transporter exists as a monomer in FOS-Choline 12 detergent, with a Stokes radius of 45 A for the protein-detergent complex. In detergent solution the protein binds substrates, as indicated by tryptophan fluorescence quenching. Its dissociation constants (K(d)) for GABA, muscimol and nipecotic acid are 13.8, 13.3 and 27.9 microM, respectively. This protein preparation provides ideal starting materials for future biochemical, biophysical and structural studies of the GABA transporter.

Bacterial Proteins↗

PubMed2001

Modulating artificial membrane morphology: pH-induced chromatic transition and nanostructural transformation of a bolaamphiphilic conjugated polymer from blue helical ribbons to red nanofibers.

Design and characterization of helical ribbon assemblies of a bolaamphiphilic conjugated polymer and their color-coded transformation into nanofibers are described. An L-glutamic acid modified bolaamphiphilic diacetylene lipid was synthesized and self-assembled into right-handed helical ribbons with micron scale length and nano scale thickness under mild conditions. The ribbon structures were further stabilized by polymerizing well-aligned diacetylene units to form bisfunctional polydiacetylenes (PDAs). Transitions from flat sheets to helical ribbons and tubes were observed by transmission electron microscopy. The helical ribbons appear to originate from the rupture of flat sheets along domain edges and the peeling off between stacked lipid layers. These results point to the applicability of chiral packing theory in bolaamphiphilic supramolecular assemblies. Contact mode atomic force microscopy observations revealed that high order existed in the surface packing arrangement. Hexagonal and pseudorectangular packings were observed in flat and twisted regions of the ribbons, respectively, suggesting a correlation between microscopic morphologies and nanoscopic packing arrangements. The tricarboxylate functionalities of the bolaamphiphilic lipid provide a handle for the manipulation of the bisfunctional PDAs' morphology. Increasing solution pH caused the fraying of helical ribbons into nanofibers accompanied by a sharp blue-to-red chromatic transition. A dramatic change in circular dichroism spectra was observed during this process, suggesting the loss of chirality in packing. A model is proposed to account for the pH-induced morphological change and chromatic transition. The color-coded transition between two distinct microstructures would be useful in the design of sensors and other "smart" nanomaterials requiring defined molecular templates.

Acetylene↗

PubMed2001

Solution structure of the toluene 4-monooxygenase effector protein (T4moD).

Toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina catalyzes the NADH- and O(2)-dependent hydroxylation of toluene to form p-cresol. The complex consists of an NADH oxidoreductase (T4moF), a Rieske ferredoxin (T4moC), a diiron hydroxylase [T4moH, with (alphabetagamma)(2) quaternary structure], and a catalytic effector protein (T4moD). The solution structure of the 102-amino acid T4moD effector protein has been determined from 2D and 3D (1)H, (13)C, and (15)N NMR spectroscopic data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space (DYANA software) with input from 1467 experimental constraints, comprising 1259 distance constraints obtained from NOEs, 128 dihedral angle constraints from J-couplings, and 80 hydrogen bond constraints. Of 60 conformers that met the acceptance criteria, the 20 that best satisfied the input constraints were selected to represent the solution structure. With exclusion of the ill-defined N- and C-terminal segments (Ser1-Asn11 and Asp99-Met102), the atomic root-mean-square deviation for the 20 conformers with respect to the mean coordinates was 0.71 A for the backbone and 1.24 A for all non-hydrogen atoms. The secondary structure of T4moD consists of three alpha-helices and seven beta-strands arranged in an N-terminal betaalphabetabeta and a C-terminal betaalphaalphabetabetabeta domain topology. Although the published NMR structures of the methane monooxygenase effector proteins from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath) have a similar secondary structure topology, their three-dimensional structures differ from that of T4moD. The major differences in the structures of the three effector proteins are in the relative orientations of the two beta-sheets and the interactions between the alpha-helices in the two domains. The structure of T4moD is closer to that of the methane monooxygenase effector protein from M. capsulatus (Bath) than that from M. trichosporium OB3b. The specificity of T4moD as an effector protein was investigated by replacing it in reconstituted T4MO complexes with effector proteins from monooxygenases from other bacterial species: Pseudomonas pickettii PKO1 (TbuV, toluene 3-monooxygenase); Pseudomonas species JS150 (TbmC, toluene 2-monooxygenase); and Burkeholderia cepacia G4 (S1, toluene 2-monooxygenase). The results showed that the closely related TbuV effector protein (55% sequence identity) provided partial activation of the complex, whereas the more distantly related TbmC (34% sequence identity) and S1 (29% sequence identity) did not. The (1)H NMR chemical shifts of the side-chain amide protons of Asn34, a conserved, structurally relevant amino acid, were found to be similar in spectra of effector proteins T4moD and TbuV but not in the spectrum of TbmC. This suggests that the region around Asn34 may be involved in structural aspects contributing to functional specificity.

Amino Acid Sequence↗

PubMed2001

Independent repression of a GC-rich housekeeping gene by Sp1 and MAZ involves the same cis-elements.

The transcription factors Sp1 and MAZ (Myc-associated zinc finger protein) contain several zinc finger motifs, and each functions as both a positive and a negative regulator of gene expression. In this study, we characterized the extremely GC-rich promoter of the human gene for MAZ, which is known as a housekeeping gene. Unique symmetrical motifs in the promoter region (nucleotides -383 to -334) were essential for the expression of the gene for MAZ, whereas an upstream silencer element (nucleotides -784 to -612) was found to act in a position-dependent but orientation-independent manner. Sp1 and MAZ bound to the same cis-elements in the GC-rich promoter, apparently sharing DNA-binding sites. The relative extent of binding of Sp1 and MAZ to these cis-elements corresponded to the extent of negative regulation of the expression of the gene for MAZ in various lines of cells. Furthermore, novel repressive domains in both Sp1 (amino acids 622-788) and MAZ (amino acids 127-292) were identified. Suppression by Sp1 and suppression by MAZ were independent phenomena; histone deacetylases were involved in the autorepression by MAZ itself, whereas DNA methyltransferase 1 was associated with suppression by Sp1. Our results indicate that both deacetylation and methylation might be involved in the regulation of expression of a single gene via the actions of different zinc finger proteins that bind to the same cis-elements.

Animals↗

PubMed2001

Reversible inhibition of Hsp70 chaperone function by Scythe and Reaper.

Protein folding mediated by the Hsp70 family of molecular chaperones requires both ATP and the co-chaperone Hdj-1. BAG-1 was recently identified as a bcl-2-interacting, anti-apoptotic protein that binds to the ATPase domain of Hsp70 and prevents the release of the substrate. While this suggested that cells had the potential to modulate Hsp70-mediated protein folding, physiological regulators of BAG-1 have yet to be identified. We report here that the apoptotic regulator Scythe, originally isolated through binding to the potent apoptotic inducer Reaper, shares limited sequence identity with BAG-1 and inhibits Hsp70- mediated protein refolding. Scythe-mediated inhibition of Hsp70 is reversed by Reaper, providing evidence for the regulated reversible inhibition of chaperone activity. As Scythe functions downstream of Reaper in apoptotic induction, these findings suggest that Scythe/Reaper may signal apoptosis, in part through regulating the folding and activity of apoptotic signaling molecules.

Adenosine Triphosphatases↗

PubMed2001

Frequent germline deletion polymorphism of chromosomal region 8p12-p21 identified as a recurrent homozygous deletion in human tumors.

A number of carcinomas show high frequency of loss of heterozygosity (LOH) at chromosome 8p, suggesting that putative tumor suppressor genes are present in this region. While searching for homozygous deletions in a panel of pancreatic and biliary tumors, we discovered a homozygous deletion at the microsatellite AFMa224wh5 in chromosome region 8p12-p21. We applied a six-step algorithm comprising germline analysis, breakpoint sequencing, population screening, online gene mapping, allelic discrimination of tumor-associated LOH, and family history analysis. The results indicated that the deletion was likely due to a normal 102-bp deletion polymorphism present in nearly 10% of the study population, not likely to involve a recessive cancer-associated gene. Researchers need to be aware that germline insertion/deletion polymorphisms can affect the results of positional cloning efforts in human neoplasms. This problem would be accentuated in studies of cell lines where a paired sample of constitutional DNA is often unavailable.

Chromosomes, Human, Pair 8↗

PubMed2001

Characterization and promoter analysis of the mouse gene for transcription factor Sp4.

Transcription factor Sp4 is a member of the Sp1 family. It functions differently from other members of this family, such as Sp1 and Sp3, and the gene for Sp4 is transcribed in a tissue-specific manner. Recent studies in mice suggest that Sp4 might play an important role in growth, viability, and male fertility. We report here the isolation and characterization of the gene for Sp4 from a mouse genomic library. The mouse gene for Sp4 was about 80 kb in length and it consisted of six exons and five introns. The promoter was found in a CpG island and had a high G+C content. The proximal promoter contained multiple putative binding sites for the transcription factors Sp1 and MAZ but lacked a consensus TATA box. Multiple sites for the initiation of transcription were mapped in a GC-rich region from 286 bp to 211 bp upstream of the ATG triplet at the site of initiation of translation, and all of the sites were either C or G. Transfection experiments and deletion analysis allowed us to localize the promoter to a region that was no more than 93 bp upstream from the first site of initiation of transcription. We also found that ectopic expression of Sp1 and of MAZ, but not of Sp3, suppressed expression of the Sp4 promoter in a dose-dependent manner.

Amino Acid Sequence↗

PubMed2001

Identification of mouse Jun dimerization protein 2 as a novel repressor of ATF-2.

A mouse cDNA that encodes a DNA-binding protein was identified by yeast two-hybrid screening, using activating transcription factor-2 (ATF-2) as the bait. The protein contained a bZIP (basic amino acid-leucine zipper region) domain and its amino acid sequence was almost identical to that of rat Jun dimerization protein 2 (JDP2). Mouse JDP2 interacted with ATF-2 both in vitro and in vivo via its bZIP domain. It was encoded by a single gene and various transcripts were expressed in all tested tissues of adult mice, as well as in embryos, albeit at different levels in various tissues. Furthermore, mouse JDP2 bound to the cAMP-response element (CRE) as a homodimer or as a heterodimer with ATF-2, and repressed CRE-dependent transcription that was mediated by ATF-2. JDP2 was identified as a novel repressor protein that affects ATF-2-mediated transcription.

3T3 Cells↗

PubMed2001

Arthritis prevalence and activity limitations in older adults.

OBJECTIVE: To evaluate the prevalence of arthritis and activity limitations among older Americans by assessing their demographic, ethnic, and economic characteristics. METHODS: Data from the Asset and Health Dynamic Survey Among the Oldest Old (AHEAD), a national probability sample of community-dwelling adults born before 1924, were analyzed cross-sectionally. Arthritis that resulted in a physician's visit or a joint replacement not associated with a hip fracture was ascertained by self-report. RESULTS: The prevalence of arthritis in older adults ranged from 25% in non-Hispanic whites to 40% in non-Hispanic blacks to 44% in Hispanics. A higher prevalence of arthritis was associated with less education as well as lower income and less wealth. The prevalence of limitations in activities of daily living (ADL) among non-Hispanic white, non-Hispanic black, and Hispanic adults who reported arthritis only was 29%, 30%, and 37%, respectively, and increased to 48%, 57%, and 56%, respectively, among those reporting arthritis plus other chronic conditions, after adjustment for age and sex. CONCLUSION: Non-Hispanic black and Hispanic older adults reported having arthritis at a substantially higher frequency than did non-Hispanic whites. In addition, Hispanics reported higher rates of ADL limitations than did non-Hispanic whites with comparable disease burden. Further study is needed to confirm and elucidate the reasons for these racial and economic disparities in older populations.

Activities of Daily Living↗

PubMed2001

Oligoclonal B lymphocyte expansion in the synovium of a patient with Behçet's disease.

OBJECTIVE: Plasma cell infiltration is observed in recurrent arthritis associated with Behçet's disease (BD). The immune mechanism underlying B lymphocyte proliferation in the synovium is unclear. One hypothesis involves nonspecific polyclonal activation and another involves antigen-driven activation. The present study was undertaken to test both hypotheses and identify immunoglobulin genes that are clonally expanded in the synovium. METHODS: Peripheral blood lymphocytes (PBL) and synovial cells from a patient with BD and PBL from a healthy control subject were obtained. Complementarity-determining region 3 (CDR3) fingerprinting analysis and nucleotide sequence analysis of Ig transcripts derived from clonally expanded B lymphocytes were performed in parallel. RESULTS: Of 44 mu heavy chain clones of the VH4 family identified in the synovial tissue from the BD patient, 8 clones showed identical nucleotide sequences, and therefore, 18.2% were clonally expanded. For y heavy chain, 4 of 50 clones of the VH3 family showed nearly identical sequences; therefore, 4-8% were clonally expanded. The kappa light chain did not show a dominant band, but a clone with a 12-amino acid CDR3 showed 3% clonal expansion. Somatic mutations were frequently observed, with a high ratio of replacement to silent mutations in the CDRs compared with the framework regions. Three Ig genes expressed in the clonally expanded B lymphocytes were derived from germline gene segments reported to be involved in the production of autoantibodies. CONCLUSION: These results support the hypothesis that antigen-driven clonal B lymphocyte proliferation occurs in the synovium in BD. Immunoglobulin transcripts clonally expanded in the synovium were identified.

Adult↗

PubMed2001

Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins.

Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. "Contractile" state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (alpha-SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, beta-NM actin was localised to the cell periphery and basal cortex. The dense body protein alpha-actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In "synthetic" state SMC (passages 2-3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (beta-non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic changes in their distribution. The distinct compartmentalisation of structural proteins observed in "contractile" state SMC was no longer obvious, with proteins more evenly distributed throughout the cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation.

Actinin↗

PubMed2001

Reorganization of structural proteins in vascular smooth muscle cells grown in collagen gel and basement membrane matrices (Matrigel): a comparison with their in situ counterparts.

When smooth muscle cells are enzyme-dispersed from tissues they lose their original filament architecture and extracellular matrix surrounds. They then reorganize their structural proteins to accommodate a 2-D growth environment when seeded onto culture dishes. The aim of the present study was to determine the expression and reorganization of the structural proteins in rabbit aortic smooth muscle cells seeded into 3-D collagen gel and Matrigel (a basement membrane matrix). It was shown that smooth muscle cells seeded in both gels gradually reorganize their structural proteins into an architecture similar to that of their in vivo counterparts. At the same time, a gradual decrease in levels of smooth muscle-specific contractile proteins (mainly smooth muscle myosin heavy chain-2) and an increase in beta-nonmuscle actin occur, independent of both cell growth and extracellular matrix components. Thus, smooth muscle cells in 3-D extracellular matrix culture and in vivo have a similar filament architecture in which the contractile proteins such as actin, myosin, and alpha-actinin are organized into longitudinally arranged "myofibrils" and the vimentin-containing intermediate filaments form a meshed cytoskeletal network. However, the myofibrils reorganized in vitro contain less smooth muscle-specific and more nonmuscle contractile proteins.

Actinin↗

PubMed2001

Cytoskeletal reorganization during the formation of oligodendrocyte processes and branches.

During oligodendrocyte development, signals relevant to process formation must be transduced into appropriate changes in cytoskeletal organization. We have explored how microtubules and microfilaments interact during the outgrowth and branching of oligodendrocyte processes in culture. We observed that microfilaments are enriched in the peripheral region beneath the plasma membrane and constitute the major cytoskeletal element at the leading edge of the process, which is also enriched in the B-isoform of the non-muscle myosin II heavy chain. Microtubules form a dense bundle within the process and splay before extending into the leading edge and branches, following tracks laid by microfilaments. Pharmacologic disruption of microfilaments and microtubules compromised normal process outgrowth and branching. However, microtubules rapidly reinvaded most processes after removal of both antimicrotubule and antimicrofilament drugs, but the reinvasion was severely compromised if the antimicrofilament drug was retained. These results are consistent with the hypothesis that microfilaments guide the local reorganization of microtubules for the elongation of oligodendrocyte processes and the formation of new branches.

Actin Cytoskeleton↗

PubMed2001

Genetic variation of the methylenetetrahydrofolate reductase and cystathionine beta-synthase genes in Korean patients with coronary artery disease and a new polymorphism in intron 7.

We investigated polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and cystathionine beta-synthase (CBS) genes in Korean patients with coronary artery disease (CAD) and identified a new polymorphism (c-->t) in intron 7 of the CBS gene using the single-strand conformation polymorphism method. No significant differences were found in allele frequencies for either gene between the CAD and normal groups although the frequency of the so-called thermolabile MTHFR polymorphism, a proposed risk factor for CAD, was higher in Koreans than in other populations studied. The relatively low incidence of CAD in Korea suggests that this MTHFR polymorphism is not causative in this population.

Coronary Disease↗

PubMed2001

Ligustrazini inhibits endotoxin induced PAI-1 expression in human umbilical vein endothelial cells.

Plasminogen activator inhibitor 1 (PAI-1) is one of important coagulant factors. Endotoxin lipopolysaccharide (LPS) induces thrombosis by stimulating PAI-1 secretion of vascular cells (EC). Using sandwich enzyme-linked immunosorbent assay (ELISA) and Northern blot, was investigated the effects of Chinese medicine ligustrazini on PAI-1 expression in EC and LPS-stimulated EC. The results showed that ligustrazini inhibited both basal and LPS-induced PAI-1 mRNA expression in EC. The effect of ligustrazini on LPS-induced PAI-1 secretion worked in a dose-dependent manner. This study provided theoretic and experimental evidence for use of ligustrazini against septic shock and cardiovascular diseases.

Anti-Inflammatory Agents, Non-Steroidal↗