Biomedical subjects
Publications and source records attributed to Ming Li.
Extending the single optimized spaced seed of PatternHunter to multiple ones, PatternHunter II simultaneously remedies the lack of sensitivity of Blastn and the lack of speed of Smith-Waterman, for homology search. At Blastn speed, PatternHunter II approaches Smith-Waterman sensitivity, bringing homology search technology back to a full circle.
OBJECTIVE: To establish the HLF-02 and provide a new material to the study of geronics, oncology, virology, pharmacology, and biogenetics. METHODS: The lung tissue was digested with 0.15% trypsin. More than 90% of the dissociated cell exhibited initial viability (trypan blue dye exclusion and the ability of adhesion). The surviving cells were analyzed by morphology, growth curves, karyotype analysis, electron microscopic observation and heterotransplantation. RESULTS: A newly established cell strain, which was designated HLF-02 had been maintained in continuous culture for over 50 generations for 10 months. Morphological observation, electron microscopic observation, karyotype analysis and heterotransplantation showed that it was corresponding to diploid cells. The doubling time of the cells was 75.5 hours. Chromosome analyses showed the human cell strain to be typical diploid appearance, and the tumorigenecity assays of nude mice were negative. CONCLUSION: The HLF-02 is a human dipolid cell strain and can serve the further studies of cell gerontics, oncology, virology, pharmacology, and biogenetics.
Human apolipoprotein A- I, the major protein component of high density lipoproteins and the main activator of the enzyme lecithin: cholesterol acyltransferase, defines the structure and stability and functions of HDL. It is clearly demonstrated that high concentration of the apoA- I not only inhibits the initiation and progression of atherosclerosis, but also makes the preexisting atherosclerotic lesions regress. This review gives an overview of the apoA- I structure, production, relation between apoA- I and HDL, and several mechanisms of the apoA-I anti-atherosclerosis. These mechanisms include directing excess celluar cholesterol from the peripheral tissues to the liver in reverse cholesterol transport, inhibiting oxidative modification of LDL, and modulating inflammatory responses to favour vasoprotection.
GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of approximately 0.5 microm. Fusions between GAT1 and green fluorescent protein (GFP) were tested in heterologous expression systems, and a construct was selected that shows function, expression level, and trafficking similar to that of wild-type (WT) GAT1. A strain of knock-in mice was constructed that expresses this mGAT1-GFP fusion in place of the WT GAT1 gene. The pattern of fluorescence in brain slices agreed with previous immunocytochemical observations. [3H]GABA uptake, synaptic electrophysiology, and subcellular localization of the mGAT1-GFP construct were also compared with WT mice. Quantitative fluorescence microscopy was used to measure the density of mGAT1-GFP at presynaptic structures in CNS preparations from the knock-in mice. Fluorescence measurements were calibrated with transparent beads and gels that have known GFP densities. Surface biotinylation defined the fraction of transporters on the surface versus those in the nearby cytoplasm. The data show that the presynaptic boutons of GABAergic interneurons in cerebellum and hippocampus have a membrane density of 800-1300 GAT1 molecules per square micrometer, and the axons that connect boutons have a linear density of 640 GAT1 molecules per micrometer. A cerebellar basket cell bouton, a pinceau surrounding a Purkinje cell axon, and a cortical chandelier cell cartridge carry 9000, 7.8 million, and 430,000 GAT1 molecules, respectively; 61-63% of these molecules are on the surface membrane. In cultures from hippocampus, the set of fluorescent cells equals the set of GABAergic interneurons. Knock-in mice carrying GFP fusions of membrane proteins provide quantitative data required for understanding the details of synaptic transmission in living neurons.
Synthesis of a dibenzothiazole thioether ligand that coordinates Ag(I) yielding an asymmetric dinuclear complex with three unique intermolecular pi-stacking interactions, which influence the first coordination spheres of the silver centers, is reported.
The paper presents a novel on-line transient moving chemical reaction boundary method (tMCRBM) for simply but efficiently stacking ionizable analytes in high-salt matrix in capillary zone electrophoresis (CZE). The powerful function and stability of the tMCRBM are elucidated with the ionizable test analytes of L-phenylalanine (Phe) and L-tryptophan (Trp) in the matrix with 85.6-165.6 mM sodium ion and further compared with the normal CZE of Phe and Trp samples dissolved in running buffer. The results verify that (1) the on-line tMCRBM mode can evidently increase separation efficiency, peak height, and resolution, (2) with the mode, the analytes in a 28-cm high-salt matrix plug can be stacked successfully and further separated well, (3) the values of relative standard deviation of peak height, peak area, and migrating time range from 3.9% to 6.1%; the results indicate the high stability of the technique of tMCRBM-CZE. The techniques implies obvious potential significance for those ionizable analytes, e.g., protein, peptide, and weak alkaline or acidic compound, in such matrixes as serum, urine, seawater, and wastewater, with high salt, which has a deleterious effect on isotachophoresis (ITP) and especially on electrostacking and field-amplified sample injection (FASI). The mechanism of stacking of zwitterionic analytes in a high-salt matrix by the tMCRBM relies on non-steady-state isoelectric focusing (IEF) but not on transient ITP, electrostacking, and FASI.
The oil bodies of rapeseeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with abundant structural alkaline proteins termed oleosins and some other minor proteins. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain flanked by relatively polar C- and N-terminal domains. Although the hydrophilic N-terminal domain had been studied, the structural feature of the central hydrophobic domain remains unclear due to its high hydrophobicity. In the present study, we reported the generation, purification, and characterization of a 9-kDa central hydrophobic domain from rapeseed oleosin (19 kDa). The 9-kDa central hydrophobic domain was produced by selectively degrading the N and C termini with enzymes and then purifying the digest by SDS-PAGE and electroelution. We have also reconstituted the central domain into liposomes and synthetic oil bodies to determine the secondary structure of the domain using CD and Fourier transform infrared (FTIR) spectroscopy. The spectra obtained from CD and FTIR were analyzed with reference to structural information of the N-terminal domain and the full-length rapeseed oleosin. Both CD and FTIR analysis revealed that 50-63% of the domain was composed of beta-sheet structure. Detailed analysis of the FTIR spectra indicated that 80% of the beta-sheet structure, present in the central domain, was arranged in parallel to the intermolecular beta-sheet structure. Therefore, interactions between adjacent oleosin proteins would give rise to a stable beta-sheet structure that would extend around the surface of the seed oil bodies stabilizing them in emulsion systems. The strategies used in our present study are significant in that it could be generally used to study difficult proteins with different independent structural domains, especially with long hydrophobic domains.
Two new pentadentate, pendent arm macrocyclic ligands of the type 1-alkyl-4,7-bis(4-tert-butyl-2-mercaptobenzyl)-1,4,7-triazacyclononane where alkyl represents an isopropyl, (L(Pr))(2-), or an ethyl group, (L(Et))(2-), have been synthesized. It is shown that they bind strongly to ferric ions generating six-coordinate species of the type [Fe(L(alk))X]. The ground state of these complexes is governed by the nature of the sixth ligand, X: [Fe(III)(L(Et))Cl] (2) possesses an S = 5/2 ground state as do [Fe(III)(L(Et))(OCH(3))] (3) and [Fe(III)(L(Pr))(OCH(3))] (4). In contrast, the cyano complexes [Fe(III)(L(Et))(CN)] (5) and [Fe(III)(L(Pr))(CN)] (6) are low spin ferric species (S = 1/2). The octahedral [FeNO](7) nitrosyl complex [Fe(L(Pr))(NO)] (7) displays spin equilibrium behavior S = 1/2<==>S = (3)/(2) in the solid state. Complexes [Zn(L(Pr))] (1), 4.CH(3)OH, 5.0.5toluene.CH(2)Cl(2), and 7.2.5CH(2)Cl(2) have been structurally characterized by low-temperature (100 K) X-ray crystallography. All iron complexes have been carefully studied by zero- and applied-field Mössbauer spectroscopy. In addition, Sellmann's complexes [Fe(pyS(4))(NO)](0/1+) and [Fe(pyS(4))X] (X = PR(3), CO, SR(2)) have been studied by EPR and Mössbauer spectroscopies and DFT calculations (pyS(4) = 2,6-bis(2-mercaptophenylthiomethyl)pyridine(2-)). It is concluded that the electronic structure of 7 with an S = 1/2 ground state is low spin ferrous (S(Fe) = 0) with a coordinated neutral NO radical (Fe(II)-NO) whereas the S = 3/2 state corresponds to a high spin ferric (S(Fe) = 5/2) antiferromagnetically coupled to an NO(-) anion (S = 1). The S = 1/2<==>S = 3/2 equilibrium is then that of valence tautomers rather than that of a simple high spin<==>low spin crossover.
We calculate the x-ray scattering from coupled capillary fluctuations of thin liquid films, taking into account an asymmetric interfacial interaction potential. Harmonic expansion of the potential around its minimum produces the well-known Kiessig fringes in both specular reflectivity and longitudinal diffuse scattering. The addition of a cubic term to the expansion, representing the asymmetry, leads to q(z)-dependent changes of the modulation period of the Kiessig fringes. The cubic term produces a relative phase shift between the interference fringes of the specular reflectivity and the off-specular longitudinal diffuse scattering. It is suggested that these effects may be used to estimate, via x-ray scattering, the interfacial potential of thin liquid films.
Protein refolding at high concentrations always leads to aggregation, which limits commercial application. An ion-exchange chromatography process with gradient changes in urea concentration and pH was developed to refold denatured lysozyme at high concentration. After adsorption of the denatured protein onto an ion-exchange medium, elution was carried out in combination with a gentle decrease in urea concentration and elevation of pH. Protein would gradually refold along the column with high activity yield. Denatured and reduced lysozyme at 40 mg/ml was loaded into a column filled with SP Sepharose Fast Flow, resulting in 95% activity recovery and 98% mass yield within a short period of time.
Sox9 is a transcription factor related to campomelic dysplasia and sex reversal in human patients. Earlier studies in zebrafish led to the identification of two homologues of mammalian sox9, sox9a and sox9b. The present study represents the first evaluation of expression patterns of zebrafish sox9b during early embryogenesis. Our analyses reveal that sox9b transcripts are present throughout the life-cycle of the zebrafish, but exhibit tissue-specific distribution during embryogenesis. Zygotic expression of sox9b occurs in the anterolateral margins and the midline of the prospective dorsal neuroectoderm during late gastrulation. During early segmentation, the transcript is expressed in pairs of longitudinal bands in the prospective midbrain, hindbrain, and trunk, which identify the cranial and trunk neural crest progenitors. Neural crest cells cease expression of sox9b during migration, but some of their derivatives resume sox9b expression. Sox9b can serve as a marker for neural crest precursors.
Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.
The hyperpolarization-activated transient current of mammalian 5-hydroxytryptamine transporters (SERT) expressed in Xenopus oocytes was studied. Human (h) and rat (r) SERT transient currents are blocked by HEPES with changes in the waveform kinetics, and the blockade of hSERT has use-dependent properties. HEPES also changes the time course of the prepriming step, especially for hSERT. Transient currents at hSERT and rSERT are also blocked by spermine and spermidine in the mM range, and by fluoxetine, cocaine, QX-314, and QX-222 in the microM range. These pharmacological and kinetic properties of transient current blockade emphasize the similarities between the transient current and phenomena at ion channels.
The transgalactosylation rate catalyzed by the lipid-coated beta-d-galactosidase in supercritical fluoroform (scCHF3) can be reversibly controlled by changing temperature or pressure (reflecting polarity changes) without damaging enzymes.
This study was conducted to get an insight into monomethoxypolyethylene glycol (MPEG) modified recombinant human TNF-alpha (rhTNF-alpha) derived from gene cloning and expression. The purification and modification processes were integrated together to make the scale-up production of PEG-modified rhTNF-alpha practical. Capillary electrophoresis was demonstrated to be a highly efficient tool in the biochemical characterization of the PEGylated products compared to slab electrophoresis. The cytotoxicity of MPEG-modified rhTNF-alpha was studied in vitro towards L929 cell line and found to decrease gradually along with the increase in the reaction ratio between the activated MPEG and the native rhTNF-alpha. The MPEG-modified rhTNF-alpha was more resistant to proteinase degradation in vitro than the native. When MPEG chains were released partially from the MPEG-modified rhTNF-alpha by alkaline pretreatment, the cytotoxicity of the MPEG-modified rhTNF-alpha was enhanced, which was in contrast to the native. These results would be helpful to explain the disagreement between the high bioavailability of MPEG-modified TNF-alpha in vivo and its decreased cytotoxicity in vitro.
Since retinoic acid receptor (RAR)-beta mRNA is frequently lost during esophageal carcinogenesis and esophageal cancer cells that do not express RAR-beta are resistant to retinoic acid (RA), we stably transfected RAR-beta expression vector into an esophageal cancer cell line TE-8 and an antisense RAR-beta into TE-3 cells. Transfection of RAR-beta decreased cell growth and colony formation and induced apoptosis in TE-8 cells. Antisense RAR-beta-transfected TE-3 cells had a shorter doubling time and became resistant to RA. Induction of RAR-beta decreased COX-2 expression in RAR-beta transfected TE-8 cells, whereas antisense RAR-beta transfected TE-3 cells increased COX-2 expression. The inhibitory effect of RAR-beta on COX-2 expression was further enhanced in the presence of RA, which was blocked by an RAR antagonist. The synthetic retinoid N-(4-hydroxyphenyl)retinamide, which does not bind effectively to RAR-beta, had no effect on COX-2 suppression. Furthermore, RA blocked bile acid-induced COX-2 expression and prostaglandin E(2) production only in the RAR-beta positive cells. Our data demonstrated that anticancer effect of RAR-beta may be related to its ability to suppress COX-2 expression and support that the loss of RAR-beta expression may contribute to esophageal carcinogenesis.
BACKGROUND: Progressive osseous heteroplasia (POH), an autosomal dominant disorder, is characterized by extensive dermal ossification during childhood, followed by disabling and widespread heterotopic ossification of skeletal muscle and deep connective tissue. Occasional reports of mild heterotopic ossification in Albright's hereditary osteodystrophy (AHO) and a recent report of two patients with AHO who had atypically extensive heterotopic ossification suggested a common genetic basis for the two disorders. AHO is caused by heterozygous inactivating mutations in the GNAS1 gene that result in decreased expression or function of the alpha subunit of the stimulatory G protein (Gsalpha) of adenylyl cyclase. METHODS: We tested the hypothesis that GNAS1 mutations cause POH, using the polymerase chain reaction to amplify GNAS1 exons and exon-intron boundaries in 18 patients with sporadic or familial POH. RESULTS: Heterozygous inactivating GNAS1 mutations were identified in 13 of the 18 probands with POH. The defective allele in POH is inherited exclusively from fathers, a result consistent with a model of imprinting for GNAS1. Direct evidence that the same mutation can cause either POH or AHO was observed within a single family, in which the phenotype correlated with the parental origin of the mutant allele. CONCLUSIONS: Paternally inherited inactivating GNAS1 mutations cause POH. This finding extends the range of phenotypes derived from haplo insufficiency of GNAS1, provides evidence that imprinting is a regulatory mechanism for GNAS1 expression, and suggests that Gsalpha is a critical negative regulator of osteogenic commitment in nonosseous connective tissues.
Adhesive interactions between integrin receptors and the extracellular matrix (ECM) are intimately involved in regulating development of a variety of tissues within the organism. In the present study, we have investigated the relationships between beta(1) integrin receptors and focal adhesion associated proteins during eye development. We used specific antibodies to examine the distribution of beta(1) integrin ECM receptors and the cytoplasmic focal adhesion associated proteins, talin, vinculin, and paxillin in the developing Xenopus retina. Immunoblot analysis confirmed antibody specificity and indicated that beta(1) integrins, talin, vinculin, and paxillin were expressed in developing retina and in the retinal-derived Xenopus XR1 glial cell line. Triple-labeling immunocytochemistry revealed that talin, vinculin, paxillin, and phosphotyrosine proteins colocalized with beta(1) integrins at focal adhesions located at the termini of F-actin filaments in XR1 cells. In the retina, these focal adhesion proteins exhibited developmentally regulated expression patterns during eye morphogenesis. In the embryonic retina, immunoreactivities for focal adhesion proteins were expressed in neuroepithelial cells, and immunoreactivity was especially strong at the interface between the optic vesicle and overlying ectoderm. At later stages, these proteins were expressed throughout all retinal layers with higher levels of expression observed in the plexiform layers, optic fiber layer, and in the region of the inner and outer limiting membrane. Strong immunoreactivities for beta(1) integrin, paxillin, and phosphotyrosine were expressed in the radially oriented Müller glial cells at later stages of development. These results suggest that focal adhesion-associated proteins are involved in integrin-mediated adhesion and signaling and are likely to be essential in regulating retinal morphogenesis.