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Biomedical subjects

L Sun

Publications and source records attributed to L Sun.

At least 271 records · Page 15Linked to original sources
PubMed1998

Identification of a long variant of mRNA encoding the NR3 subunit of the NMDA receptor: its regional distribution and developmental expression in the rat brain.

A longer variant of rat mRNA encoding the NR3 subunit of the NMDA receptor has been identified. It contains a 60-bp insertion at the nucleotide position 3007 in the intracellular domain of the C-terminal of the previously cloned variant. Therefore, the NR3 mRNA exists in at least two variants--with the insert (NR3-long; NR3-l) and without the insert (NR3-short; NR3-s). The NR3-l variant is expressed throughout the adult rat brain. Moreover, this variant predominates in the occipital and entorhinal cortices, thalamus and cerebellum. Analysis of NR3-l development indicates that it is regulated in a region-specific manner.

Animals↗
PubMed1998

Crystal structure of an RNA octamer duplex r(CCCIUGGG)2 incorporating tandem I.U wobbles.

The crystal structure of the RNA octamer duplex r(CCCIUGGG)2has been elucidated at 2.5 A resolution. The crystals belong to the space group P21and have unit cell constants a = 33.44 A, b = 43.41 A, c = 49.39 A and beta = 104.7 degrees with three independent duplexes (duplexes 1-3) in the asymmetric unit. The structure was solved by the molecular replacement method and refined to an Rwork/Rfree of 0.185/0.243 using 3765 reflections between 8.0 and 2.5 A. This is the first report of an RNA crystal structure incorporating I.U wobbles and three molecules in the asymmetric unit. Duplex 1 displays a kink of 24 degrees between the mismatch sites, while duplexes 2 and 3 have two kinks each of 19 degrees and 27 degrees, and 24 degrees and 29 degrees, respectively, on either side of the tandem mismatches. At the I.U/U.I mismatch steps, duplex 1 has a twist angle of 33.9 degrees, close to the average for all base pair steps, but duplexes 2 and 3 are underwound, with twist angles of 24.4 degrees and 26.5 degrees, respectively. The tandem I.U wobbles show intrastrand purine-pyrimidine stacking but exhibit interstrand purine-purine stacking with the flanking C.G pairs. The three independent duplexes are stacked non-coaxially in a head-to-tail fashion to form infinite pseudo-continuous helical columns which form intercolumn hydrogen bonding interactions through the 2'-hydroxyl groups where the minor grooves come together.

Base Pairing↗
PubMed1998

Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR.

It is well known that the functional activity of the diphtheria toxin repressor DtxR is controlled by iron, which serves as an essential cofactor necessary for activation of target DNA binding by this regulatory element. In this communication, we describe the isolation and characterization of a unique series of DtxR mutants that are constitutively active and repress the expression of beta-galactosidase from a diphtheria tox promoter/operator-lacZ transcriptional fusion, even in the absence of iron. These self-activating mutants of DtxR (SAD) were isolated through the use of a positive selection system for the cloning of functional dtxR alleles and target DNA operator sites. Of the four independently isolated SAD mutants that were characterized, two (SAD2 and SAD11) were found to carry a single missense mutation (E175K) in their respective C-terminal SH3-like domains. In contrast, the mutant allele encoding SAD3 was found to carry a total of six missense mutations distributed throughout the N- and C-terminal domains of the repressor. Partial diploid analysis of strains carrying both native dtxR and alleles encoding either SAD2 or SAD3 demonstrate that these iron-independent mutants possess a positive dominant phenotype in the regulation of beta-galactosidase expression from a diphtheria tox promoter/operator-lacZ transcriptional fusion.

Bacterial Proteins↗
PubMed1998

Continuous, flow-through immunomagnetic cell sorting in a quadrupole field.

A flow-through quadrupole magnetic cell separator has been designed, built, and evaluated by using a cell model system of human peripheral T lymphocytes (CD4+, CD8+, and CD45+ cells). The immunomagnetic labeling was accomplished by using a sandwich of mouse anti-human monoclonal antibody conjugated to fluorescein isothiocyanate and rat anti-mouse polyclonal antibody conjugated to a colloidal magnetic nanoparticle. The feed and sorted fractions were analyzed by FACScan flow cytometry. The magnetically labeled cells were separated from nonlabeled ones in a flow-through cylindrical column within a quadrupole field, which exerted a radial, outward force on the magnetic cells. The flow rate of the cell samples was 0.1-0.75 ml/min, and the flow rate of sheath fluid was 1.5-33.3 times that of the sample flow rate. The maximum shear stress exerted on the cell was less than 1 dyne/cm2, which was well below the level that would threaten cell integrity and membrane disruption. The maximum magnetic field was 0.765 T at the channel wall, and the gradient was 0.174 T/mm. The highest purity of selected cells was 99.6% (CD8 cells, initial purity of 26%), and the highest recovery of selected cells was 79% (CD4 cells, initial purity of 20%). The maximum throughput of the quadrupole magnetic cell separator was 7,040 cells/s (CD45 cells, initial purity of 5%). Theoretical calculations showed that the throughput can be increased to 10(6) cells/s by a scale-up of the current prototype.

Animals↗
PubMed1998

[Study on induced mutagenesis interaction of the high temperature and/or cigarette smoke].

The study aim is to research the genotoxicity and interaction of the High temperature and/or cigarette smoke. The micronucleus of human lymphocytes and bone marrow in mice were observed following heat stress and/or watersoluble cigarette smoke substances. The results indicated that the high temperature (40 degrees C, 41 degrees C and 42 degrees C for 5 min, respectively) significantly induced micronuclei in human lymphocytes in vitro (P < 0.01). A significant synergistic effect of the high temperature and cigarette was found. When high temperature combining with high concentration cigarette smoke (1.0-2.0 cigarette/20 g/d, for 10 d) was used, a significant induction and interaction (P < 0.01) on micronucleus of the bone marrow were also observed. The results indicated that high temperature substanance aberrations would be enhanced when the high temperature combines with cigarette smoke.

Animals↗
PubMed1998

Reduced p27Kip1 expression in hepatocellular carcinomas.

This study was designed to determine whether changes in p27Kip1 expression are involved in human hepatocarcinogenesis. We examined the p27Kip1 mRNA expression in hepatocellular carcinomas and surrounding non-cancerous liver tissue samples from 21 patients using a reverse-transcriptase polymerase chain reaction assay. The mean p27Kip1 mRNA expression levels (ratio of p27Kip1/beta-actin mRNA) were significantly lower in hepatocellular carcinomas than in non-cancerous liver tissues (0.49 +/- 0.24 versus 0.57 +/- 0.22, P < 0.05). p27Kip1 mRNA expression was reduced in 11 (52%) of the 21 hepatocellular carcinomas when compared with the surrounding non-cancerous liver tissues. These findings suggest that reduced p27Kip1 expression may be at least partly responsible for human hepatocarcinogenesis.

Adult↗
PubMed1998

Lymphocyte fractionation using immunomagnetic colloid and a dipole magnet flow cell sorter.

The relationship between cell function and surface marker expression is a subject of active investigation in biology and medicine. These investigations require separating cells of a homogeneous subset into multiple fractions of varying marker expression. We have developed a novel cell sorter, the dipole magnet flow sorter (DMFS), which separates selected T lymphocyte subpopulations, targeted by immunomagnetic colloid, into multiple fractions according to cell surface marker expression, as determined by flow cytometry. A narrow stream of cells is introduced into a sheath of carrier fluid in a rectangular channel while subjected to a perpendicular magnetic force. The special design of the pole pieces ensures a constant magnetic force acting on the magnetically labeled cells in the separation area. Cells are spread across the flow in relation to their magnetophoretic mobility. Separation is achieved by control of the positions of the effluent stream boundaries, which separate fluid volumes with cells of different magnetophoretic mobility. CD4 and CD8 T lymphocytes labeled with primary antibody-fluorescein isothiocyanate (FITC) conjugate and anti-FITC-magnetic colloid are the chosen cell systems. Flow cytometry analysis shows that, for CD4 cells, a three-fold increase in total marker number per cell is observed when comparing the highest to the lowest fluorescence fractions. Similarly, a four-fold increase in total marker number is observed for CD8 cells. We also observed the separation of two dissimilar cell types that differed in expression of the CD4 marker, monocytes and T helper lymphocytes. We believe that this type of separation is applicable to any cells in suspension for which a suitable antibody exists and, due to the comparatively gentle nature of the process, is particularly suitable for the sorting of fragile cells.

Biomedical Engineering↗
PubMed1998

Mode of action of interleukin-6 on mature osteoclasts. Novel interactions with extracellular Ca2+ sensing in the regulation of osteoclastic bone resorption.

We describe a physiologically significant mechanism through which interleukin-6 (IL-6) and a rising ambient Ca2+ interact to regulate osteoclastic bone resorption. VOXEL-based confocal microscopy of nonpermeabilized osteoclasts incubated with anti- IL-6 receptor antibodies revealed intense, strictly peripheral plasma membrane fluorescence. IL-6 receptor expression in single osteoclasts was confirmed by in situ reverse transcriptase PCR histochemistry. IL-6 (5 ng/l to 10 microg/l), but not IL-11 (10 and 100 microg/l), reversed the inhibition of osteoclastic bone resorption induced by high extracellular Ca2+ (15 mM). The IL-6 effect was abrogated by excess soluble IL-6 receptor (500 microg/l). Additionally, IL-6 (5 pg/l to 10 microg/l) inhibited cytosolic Ca2+ signals triggered by high Ca2+ or Ni2+. In separate experiments, osteoclasts incubated in 10 mM Ca2+ or on bone released more IL-6 than those in 1.25 mM Ca2+. Furthermore, IL-6 mRNA histostaining was more intense in osteoclasts in 10 or 20 mM Ca2+ than cells in 1.25 mM Ca2+. Similarly, IL-6 receptor mRNA histostaining was increased in osteoclasts incubated in 5 or 10 mM Ca2+. Thus, while high Ca2+ enhances IL-6 secretion, the released IL-6 attenuates Ca2+ sensing and reverses inhibition of resorption by Ca2+. Such an autocrine-paracrine loop may sustain osteoclastic activity in the face of an inhibitory Ca2+ level generated locally during resorption.

Animals↗
PubMed1998

Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV), a betaherpesvirus, has developed several ways to evade the immune system, notably downregulation of cell surface expression of major histocompatibility complex class I heavy chains. Here we report that HCMV has devised another means to compromise immune surveillance mechanisms. Extracellular accumulation of both constitutively produced monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor-superinduced RANTES (regulated on activation, normal T cell expressed and secreted) was downregulated in HCMV-infected fibroblasts in the absence of transcriptional repression or the expression of polyadenylated RNA for the cellular chemokine receptors CCR-1, CCR-3, and CCR-5. Competitive binding experiments demonstrated that HCMV-infected cells bind RANTES, MCP-1, macrophage inflammatory protein (MIP)-1beta, and MCP-3, but not MCP-2, to the same receptor as does MIP-1alpha, which is not expressed in uninfected cells. HCMV encodes three proteins with homology to CC chemokine receptors: US27, US28, and UL33. Cells infected with HCMV mutants deleted of US28, or both US27 and US28 genes, failed to downregulate extracellular accumulation of either RANTES or MCP-1. In contrast, cells infected with a mutant deleted of US27 continues to bind and downregulate those chemokines. Depletion of chemokines from the culture medium was at least partially due to continuous internalization of extracellular chemokine, since exogenously added, biotinylated RANTES accumulated in HCMV-infected cells. Thus, HCMV can modify the chemokine environment of infected cells through intense sequestering of CC chemokines, mediated principally by expression of the US28-encoded chemokine receptor.

Binding Sites↗
PubMed1998

Theoretical analysis of cell separation based on cell surface marker density.

A theoretical analysis was performed to determine the number of fractions a multidisperse, immunomagnetically labeled cell population can be separated into based on the surface marker (antigen) density. A number of assumptions were made in this analysis: that there is a proportionality between the number of surface markers on the cell surface and the number of immunomagnetic labels bound; that this surface marker density is independent of the cell diameter; and that there is only the presence of magnetic and drag forces acting on the cell. Due to the normal distribution of cell diameters, a "randomizing" effect enters into the analysis, and an analogy between the "theoretical plate" analysis of distillation, adsorption, and chromatography can be made. Using the experimentally determined, normal distribution of cell diameters for human lymphocytes and a breast cancer cell line, and fluorescent activated cell screening data of specific surface marker distributions, examples of theoretical plate calculations were made and discussed.

Antigens, Surface↗
PubMed1998

Synthesis and biological evaluations of 3-substituted indolin-2-ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity toward particular receptor tyrosine kinases.

3-Substituted indolin-2-ones have been designed and synthesized as a novel class of tyrosine kinase inhibitors which exhibit selectivity toward different receptor tyrosine kinases (RTKs). These compounds have been evaluated for their relative inhibitory properties against a panel of RTKs in intact cells. By modifying the 3-substituted indolin-2-ones, we have identified compounds which showed selective inhibition of the ligand-dependent autophosphorylation of various RTKs at submicromolar levels in cells. Structure-activity analysis for these compounds and their relative potency and selectivity to inhibit particular RTKs has determined that (1) 3-[(five-membered heteroaryl ring)methylidenyl]indolin-2-ones are highly specific against the VEGF (Flk-1) RTK activity, (2) 3-(substituted benzylidenyl)indolin-2-ones containing bulky group(s) in the phenyl ring at the C-3 position of indolin-2-ones showed high selectivity toward the EGF and Her-2 RTKs, and (3) the compound containing an extended side chain at the C-3 position of the indolin-2-one (16) exhibited high potency and selectivity when tested against the PDGF and VEGF (Flk-1) RTKs. Recent published crystallographic data for two of these 3-substituted indolin-2-ones provides a rationale to suggest that these compounds may bind in the ATP binding pocket of RTKs. The structure-activity analysis supports the use of subsets of these compounds as specific chemical leads for the development of RTK-specific drugs with broad application for the treatment of human diseases.

Cells, Cultured↗
PubMed1998

Epidermal growth factor activation of NF-kappaB is mediated through IkappaBalpha degradation and intracellular free calcium.

The transcription factor NF-kappa-B is normally sequestered in the cytoplasm by its inhibitory subunit IkappaB. Most extracellular signals activate NF-kappa-B through a mechanism involving the phosphorylation and proteasome-dependent degradation of IkappaB. EGF activates NF-kappaB in A-431 carcinoma cells, which overexpress EGF receptors and in mouse embryo fibroblasts, which have a normal complement of receptors. Supershift experiments indicate that the NF-kappa-B complexes induced by EGF are composed of p50/p50 homodimers and p65/p50 heterodimers, but not c-rel. EGF stimulation enhances the degradation of IkappaBalpha, but not IkappaBbeta nor an N-terminal deletion mutant of IkappaBalpha. Treatment of cells with a proteasome inhibitor, such as ALLN or MG132, blocks EGF-mediated NF-kappaB activation, indicating that EGF-induced NF-kappa-B activation requires proteasome-dependent IkappaB degradation. Also, Bapta A/M (a cell-permeable chelator of intracellular calcium) blocks EGF-induced NF-kappa-B activation and IkappaBalpha degradation, suggesting a requirement of intracellular free Ca2+ for this growth factor response. Protein kinase C inhibition, in contrast, did not influence EGF activation of NF-kappaB.

Animals↗
PubMed1998

A novel 52 kDa protein induces apoptosis and concurrently activates c-Jun N-terminal kinase 1 (JNK1) in mouse C3H10T1/2 fibroblasts.

A 52 kDa protein (p52) was purified from chicken embryos and its corresponding cDNA was cloned. The p52 cDNA is 1768 bp long and has an open reading frame of 465 amino acids. The sequence of the p52 cDNA shows significant homology with mouse and human cDNAs from the EST database, so do the deduced amino acid sequences, indicating the existence of human and mouse homologues of p52. Northern blot hybridization showed that the p52 mRNA was expressed in a wide range of embryonic and adult tissues. There was more p52 mRNA in embryonic heart and liver than in the brain or muscle. The adult testis had the highest level of p52 mRNA, whereas adult liver had the lowest. Expression of p52 in mouse C3H10T1/2 fibroblasts caused apoptotic cell death, upregulation of transcription factor c-Jun and activation of c-Jun N-terminal kinase 1 (JNK1). In addition, expression of Bcl-2, but not of the dominant negative mutant JNK1, can block the p52-mediated apoptosis. These results indicate that p52 may represent a new cell-death protein inducing apoptosis and activating JNK1 through different pathways.

Amino Acid Sequence↗
PubMed1998

Multiple domains are involved in the targeting of the mouse DNA methyltransferase to the DNA replication foci.

It has been shown that, during the S-phase of the cell cycle, the mouse DNA methyltransferase (DNA MTase) is targeted to sites of DNA replication by an amino acid sequence (aa 207-455) lying in the N-terminal domain of the enzyme [Leonhardt, H., Page, A. W., Weier, H. U. and Bestor, T. H. (1992) Cell , 71, 865-873]. In this paper it is shown, by using enhanced green fluorescent protein (EGFP) fusions, that other peptide sequences of DNA MTase are also involved in this targeting. The work focuses on a sequence, downstream of the reported targeting sequence (TS), which is homologous to the Polybromo-1 protein. This motif (designated as PBHD) is separated from the reported targeting sequence by a zinc-binding motif [Bestor , T. H. (1992) EMBO J , 11, 2611-2617]. Primed in situ extension using centromeric-specific primers was used to show that both the host DNA MTase and EGFP fusion proteins containing the targeting sequences were localized to centromeric, but not telomeric, regions during late S-phase and mitosis. Also found was that, in approximately 10% of the S-phase cells, the EGFP fusions did not co-localize with the centromeric regions. Mutants containing either, or both, of these targeting sequences could act as dominant negative mutants against the host DNA MTase. EGFP fusion proteins, containing the reported TS (aa 207-455), were targeted to centromeric regions throughout the mitotic stage which lead to the discovery of a similar behavior of the endogenous DNA MTase although the host MTase showed much less intense staining than in S-phase cells. The biological role of the centromeric localization of DNA MTase during mitosis is currently unknown.

Animals↗
PubMed1998

Prediction of molecular recognition-enhanced phenobarbital extraction based on solvatochromic analysis.

The extraction efficiency for organic molecules using synthetic receptors is highly dependent on solvent properties. Solvent influences the partitioning of both the desired compound and the interfering species. Solvent also influences the solubility of the receptor and its affinity for the substrate. Therefore the free energy involved in extraction can be optimized by using a carefully selected solvent. In this paper we demonstrate the use of a solvatochromic model to predict the influence of solvent dipolarity, H-bond acidity and H-bond basicity on selectivity and yield of phenobarbital extraction. We also used this method to estimate the purity and yield of phenobarbital extraction in 12 poly(vinyl chloride) plasticizers and solvents. This approach can be generalized for assisting the selection of optimal solvent and provide insight into the rational design of solvent and receptor for industrial extractions.

Drug Contamination↗
PubMed1998

Jak-STAT pathway is involved in the induction of TNF-beta gene during stimulation by IL-2.

IL-2 is the major regulatory cytokine of the immune system. It plays a key role in T cell survival, growth and activation. IL-2 may induce the expression of multiple genes including some cytokine genes. The induction of these genes is triggered by different signal pathways, one of them being the Jak-STAT signal pathway. The genes regulated by this pathway remain to be determined. By studying IL-2-inducible genes, we have confirmed that the TNF-beta gene is one of the immediate early genes activated by IL-2. By analysis of the DNA sequences around 180-300 bases upstream of the transcription initiation point of the mouse TNF-beta gene, we demonstrate that there is a STAT5 binding site which is essential to the inducibility of the TNF-beta gene. Furthermore, in BA/F3 cells co-transfected with the STAT5A gene and IL-2R beta gene, the activation of the TNF-beta gene promoter by IL-2 was greatly promoted, whereas the TNF-beta gene promoter became IL-2-non-inducible if the STAT5A gene was substituted with a dominant negative STAT5A, i.e. a C-terminally truncated mutant. Taken together, our results show that the Jak-STAT signal pathway is involved in induction of the TNF-beta gene in cells stimulated by IL-2.

Animals↗