Biomedical subjects

J Qin

Publications and source records attributed to J Qin.

At least 73 records · Page 4Linked to original sources

PubMed2000

Molecular views of redox regulation: three-dimensional structures of redox regulatory proteins and protein complexes.

The last decade has witnessed the explosion of research on redox-controlled cellular and biochemical processes. Whereas the vast majority of these studies have centered on clinical, genetic, and biochemical aspects of redox signaling and regulation inside and outside the cell, a significant number of nuclear magnetic resonance (NMR) and crystallographic studies have been undertaken to obtain an atomic-level understanding of the mechanisms of the redox regulation. This review highlights the recent progress of three-dimensional structure determination of key proteins and protein complexes involved in redox regulation. An increased list of such class of protein structures and their complexes with ligands will provide invaluable insight into the molecular basis of redox-regulatory processes and may be useful for the future development of therapeutic agents for redox-related diseases.

Amino Acid Sequence↗

PubMed2000

Relation among serum and tissue concentrations of lutein and zeaxanthin and macular pigment density.

BACKGROUND: Lutein and zeaxanthin are the only carotenoids in the macular region of the retina (referred to as macular pigment [MP]). Foods that are rich in lutein and zeaxanthin can increase MP density. Response to dietary lutein and zeaxanthin in other tissues has not been studied. OBJECTIVE: The objective of this study was to examine tissue responses to dietary lutein and zeaxanthin and relations among tissues in lutein and zeaxanthin concentrations. DESIGN: Seven subjects consumed spinach and corn, which contain lutein and zeaxanthin, with their daily diets for 15 wk. At 0, 4, 8, and 15 wk and 2 mo after the study, serum, buccal mucosa cells, and adipose tissue were analyzed for carotenoids, and MP density was measured. RESULTS: Serum and buccal cell concentrations of lutein increased significantly from baseline during dietary modification. Serum zeaxanthin concentrations were greater than at baseline only at 4 wk, whereas buccal cell and adipose tissue concentrations of zeaxanthin did not change. Adipose tissue lutein concentrations peaked at 8 wk. Changes in adipose tissue lutein concentration were inversely related to the changes in MP density, suggesting an interaction between adipose tissue and retina in lutein metabolism. To investigate the possibility of tissue interactions, we examined cross-sectional relations among serum, tissue, and dietary lutein concentrations, anthropometric measures, and MP density in healthy adults. Significant negative correlations were found between adipose tissue lutein concentrations and MP for women, but a significant positive relation was found for men. CONCLUSION: Sex differences in lutein metabolism may be an important factor in tissue interactions and in determining MP density.

Adipose Tissue↗

PubMed2000

[The expression of P16 and Rb proteins in 106 cases of lung cancer].

OBJECTIVE: To observe the relationship between the expression of P16 and Rb proteins and the proliferation of lung cancer cells. METHODS: The expressions of P16 and Rb proteins were measured in 66 cases of NSCLC and 40 cases of SCLC by means of immunohistochemistry technique. RESULTS: The inverse correlation of Rb and P16 proteins expression was confirmed in 66 cases of NSCLC (chi 2 = 10.52, P < 0.01). No relationship between the expression of P16 and differentiation, stage, lymph node metastasis or pathological type was found (P > 0.05). There was no correlation between the expression of Rb and differentiation, stage, lymph node metastasis or pathological pattern (P > 0.05). The frequency of the loss of Rb protein expression in SCLC (78%) was significantly higher than in NSCLC (21%, chi 2 = 32.3, P < 0.01). CONCLUSIONS: Loss of Rb protein expression in SCLC may be an important marker of the diagnosis of SCLC. The expression of P16 adversely correlates with that of Rb. This study provides strong confirmation about that there might be a negative feedback between P16 and Rb in phase G1.

Aged↗

PubMed2000

[Expression of recombinant human stem cell factor in insect cells].

The cDNA of human stem cell factor(hSCF) containing signal sequence was cloned into the transfer vector pVL941 of AcNPV to construct a recombinant transfer vector pVL941-SCF. Sf9 cells were cotransfected with wild type viral DNA and pVL914-SCF to produce the recombinant virus AcNPV-SCF by homologous recombination in cell. Southern-hybridization analysis suggested that the recombinant viral DNA contained hSCF cDNA fragment. The Sf9 cells infected with the recombinant baculovirus AcNPV-SCF expressed biologically active rhSCF which was secreted into the cell culture. The synergistic activities of SCF in conjunction with human interleukin-3(hIL-3) was measured by MTT colorimetric method and TF-1 cell line. The expression level of Sf9 cells reached its highest at about 1970 units/ml in the 3rd day after the infection with AcNPV-SCF. Three SCF bands with molecular masses of 18 x 10(3), 20 x 10(3) and 22 x 10(3) were detected by immunoblotting.

Animals↗

PubMed1999

Recruitment of the protein tyrosine phosphatase CSW by DOS is an essential step during signaling by the sevenless receptor tyrosine kinase.

The pleckstrin homology (PH) domain-containing protein Daughter of Sevenless (DOS) is an essential component of the Sevenless receptor tyrosine kinase (SEV) signaling cascade, which specifies R7 photoreceptor development in the Drosophila eye. Previous results have suggested that DOS becomes tyrosine phosphorylated during SEV signaling and collaborates with the protein tyrosine phosphatase CSW. We have investigated this possibility by identifying tyrosine residues 801 and 854 of DOS as the phosphorylated binding sites for the CSW SH2 domains. We show that these sites become phosphorylated in response to SEV activation and that phosphorylation of both sites is required to allow CSW to bind DOS. Mutant DOS proteins in which either Y801 or Y854 of DOS has been changed to phenylalanine are unable to function during signaling by SEV and other receptor tyrosine kinases. In contrast, we find that a mutant DOS protein in which all tyrosine phosphorylation sites except Y801 and Y854 have been removed is able effectively to provide DOS function during SEV signaling and to rescue the lethality associated with dos loss-of-function mutations. These results indicate that a primary role for DOS during signaling by SEV and other receptor tyrosine kinases is to become phosphorylated at Y801 and Y854 and then recruit CSW.

Amino Acid Sequence↗

PubMed1999

A role for a melanosome transport signal in accessing the MHC class II presentation pathway and in eliciting CD4+ T cell responses.

Melanosomal membrane proteins are frequently recognized by the immune system of patients with melanoma and vitiligo. Melanosomal glycoproteins are transported to melanosomes by a dileucine-based melanosomal transport signal (MTS). To investigate whether this sorting signal could be involved in presentation of melanosome membrane proteins to the immune system, we devised a fusion construct containing the MTS from the mouse brown locus product gp75/tyrosinase-related protein-1 and full-length OVA as a reporter Ag. The fusion protein was expressed as an intracellular membrane protein, sorted to the endocytic pathway, processed, and presented by class II MHC molecules. DNA immunization with this construct elicited CD4+ T cell proliferative responses in vivo. Ag presentation and T cell responses in vitro and in vivo required a functional MTS. Mutations of either the upstream leucine in MTS or elimination of the entire MTS negated in vitro Ag presentation and in vivo T cell responses. In a mouse melanoma model, DNA immunization with MTS constructs protected mice from tumor challenge in a CD4+ T cell-dependent manner, but complete deletion of MTS decreased tumor rejection. Therefore, MTS can target epitopes to the endocytic pathway leading to presentation by class II MHC molecules to helper T cells.

Amino Acid Sequence↗

PubMed1999

Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks.

The Brca1 (breast cancer gene 1) tumor suppressor protein is phosphorylated in response to DNA damage. Results from this study indicate that the checkpoint protein kinase ATM (mutated in ataxia telangiectasia) was required for phosphorylation of Brca1 in response to ionizing radiation. ATM resides in a complex with Brca1 and phosphorylated Brca1 in vivo and in vitro in a region that contains clusters of serine-glutamine residues. Phosphorylation of this domain appears to be functionally important because a mutated Brca1 protein lacking two phosphorylation sites failed to rescue the radiation hypersensitivity of a Brca1-deficient cell line. Thus, phosphorylation of Brca1 by the checkpoint kinase ATM may be critical for proper responses to DNA double-strand breaks and may provide a molecular explanation for the role of ATM in breast cancer.

Amino Acid Sequence↗

PubMed1999

Purification and characterization of the serum amyloid A3 enhancer factor.

Serum amyloid A (SAA) is a major acute-phase protein synthesized and secreted mainly by the liver. In response to acute inflammation, its expression may be induced up to 1000-fold, primarily as a result of a 200-fold increase in the rate of SAA gene transcription. We showed previously that cytokine-induced transcription of the SAA3 gene promoter requires a transcriptional enhancer that contains three functional elements: two CCAAT/enhancer-binding protein (C/EBP)-binding sites and a third site that interacts with a constitutively expressed transcription factor, SAA3 enhancer factor (SEF). Each of these binding sites as well as cooperation among their binding factors is necessary for maximum transcription activation by inflammatory cytokines. Deletion or site-specific mutations in the SEF-binding site drastically reduced SAA3 promoter activity, strongly suggesting that SEF is important in SAA3 promoter function. To further elucidate its role in the regulation of the SAA3 gene, we purified SEF from HeLa nuclear extracts to near homogeneity by using conventional liquid chromatography and DNA affinity chromatography. Ultraviolet cross-linking and Southwestern experiments indicated that SEF consisted of a single polypeptide with an apparent molecular mass of 65 kDa. Protein sequencing and antibody supershift experiments identified SEF as transcription factor LBP-1c/CP2/LSF. Cotransfection of SEF expression vector with SAA3-luciferase reporter resulted in approximately a 5-fold increase in luciferase activity. Interestingly, interleukin-1 treatment of SEF-transfected cells caused dramatic synergistic activation (31-fold) of the SAA3 promoter. In addition to its role in regulating SAA3 gene expression, we provide evidence that SEF could also bind in a sequence-specific manner to the promoters of the alpha(2)-macroglobulin and Aalpha-fibrinogen genes and to an intronic enhancer of the human Wilm's tumor 1 gene, suggesting a functional role in the regulation of these genes.

Binding Sites↗

PubMed1999

Oligomerization of a MutS mismatch repair protein from Thermus aquaticus.

The MutS DNA mismatch protein recognizes heteroduplex DNAs containing mispaired or unpaired bases. We have examined the oligomerization of a MutS protein from Thermus aquaticus that binds to heteroduplex DNAs at elevated temperatures. Analytical gel filtration, cross-linking of MutS protein with disuccinimidyl suberate, light scattering, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry establish that the Taq protein is largely a dimer in free solution. Analytical equilibrium sedimentation showed that the oligomerization of Taq MutS involves a dimer-tetramer equilibrium in which dimer predominates at concentrations below 10 microM. The DeltaG(0)(2-4) for the dimer to tetramer transition is approximately -6.9 +/- 0.1 kcal/mol of tetramer. Analytical gel filtration of native complexes and gel mobility shift assays of an maltose-binding protein-MutS fusion protein bound to a short, 37-base pair heteroduplex DNA reveal that the protein binds to DNA as a dimer with no change in oligomerization upon DNA binding.

Adenosine Triphosphatases↗

PubMed1999

A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase.

A new method was developed for tracking the stereochemical path of enzymatic cleavage of DNA. DNA with a phosphorothioate of known chirality at the scissile bond is cleaved by the enzyme in H218O. The cleavage produces a DNA molecule with the 5'-[16O,18O, S]-thiophosphoryl group, whose chirality depends on whether the cleavage reaction proceeds by a single-step hydrolysis mechanism or by a two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorothioate dictates whether the 18O is retained or lost in the ligation product, which can be determined by mass spectrometry. This method has advantages over previous methods in that it is not restricted to particular DNA sequences, requires substantially less material, and avoids purification of the products at intermediate stages in the procedure. The method was validated by confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposase. In all three cases, DNA cleavage proceeded with inversion of configuration, indicating direct hydrolysis of the phosphodiester bond by water as opposed to a reaction involving a covalent enzyme-DNA intermediate.

5' Untranslated Regions↗

PubMed1999

An on-line preconcentrator and the evaluation of electrospray interfaces for the capillary electrophoresis/mass spectrometry of peptides.

A unique preconcentrator was constructed for on-line preconcentration using capillary electrophoresis generating concentration detection limits for peptides in the mid-picomolar range. Several common sheathless electrospray configurations were constructed in conjunction with the preconcentrator and were compared. It was found that a palladium wire interface was the most reliable interface and the problems encountered using this interface are discussed. A tryptic digest of the myosin I heavy chain kinase that contains two phosphopeptides was separated by capillary electrophoresis using this interface and the on-line preconcentrator.

Amino Acid Sequence↗

PubMed1999

High expression of doublecortin and KIAA0369 protein in fetal brain suggests their specific role in neuronal migration.

The X-linked subcortical laminar heterotopia and lissencephaly syndrome is a disorder of neuronal migration caused by a mutation in XLIS, a recently cloned gene on chromosome Xq22.3-q23. The predicted protein product for XLIS, doublecortin (DC), shows high homology to a putative calcium calmodulin-dependent kinase, KIAA0369 protein (KI). Here we identified DC and KI in the brains of human and rat fetuses by immunochemical and immunohistochemical means. In this study, Western blotting demonstrated that both DC and KI are specific to the nervous system and are abundant during the fetal period, around 20 gestational weeks in humans and embryonic days 17 to 20 in rats. Immunostaining of the developing neocortex disclosed localization of DC and KI immunoreactivities in neuronal cell bodies and processes in the zones of ongoing neuronal migration. Although KI showed a somewhat wider distribution than DC, the temporal and spatial patterns of their expression were similar. These results suggest that DC and KI participate in a common signaling pathway regulating neuronal migration.

Animals↗

PubMed1999

A novel human SRB/MED-containing cofactor complex, SMCC, involved in transcription regulation.

A novel human complex that can either repress activator-dependent transcription mediated by PC4, or, at limiting TFIIH, act synergistically with PC4 to enhance activator-dependent transcription has been purified. This complex contains homologs of a subset of yeast mediator/holoenzyme components (including SRB7, SRB10, SRB11, MED6, and RGR1), homologs of other yeast transcriptional regulatory factors (SOH1 and NUT2), and, significantly, some components (TRAP220, TRAP170/hRGR1, and TRAP100) of a human thyroid hormone receptor-associated coactivator complex. The complex shows direct activator interactions but, unlike yeast mediator, can act independently of the RNA polymerase II CTD. These findings demonstrate both positive and negative functional capabilities for the human complex, emphasize novel (CTD-independent) regulatory mechanisms, and link the complex to other human coactivator complexes.

Cloning, Molecular↗

PubMed1999

Identity between TRAP and SMCC complexes indicates novel pathways for the function of nuclear receptors and diverse mammalian activators.

The human thyroid hormone receptor-associated protein (TRAP) complex, an earlier described coactivator for nuclear receptors, and an SRB- and MED-containing cofactor complex (SMCC) that mediates activation by Gal4-p53 are shown to be virtually the same with respect to specific polypeptide subunits, coactivator functions, and mechanisms of action (activator interactions). In parallel with ligand-dependent interactions of nuclear receptors with the TRAP220 subunit, p53 and VP16 activation domains interact directly with a newly cloned TRAP80 subunit. These results indicate novel pathways for the function of nuclear receptors and other activators (p53 and VP16) through a common coactivator complex that is likely to target RNA polymerase II. Identification of the TRAP230 subunit as a previously predicted gene product also suggests a coactivator-related transcription defect in certain disease states.

Amino Acid Sequence↗

PubMed1999

Modulation of GT-1 DNA-binding activity by calcium-dependent phosphorylation.

The analysis of pea rbcS-3A promoter sequence showed that BoxII was necessary for the control of rbcS-3A gene expression by light. GT-1, a DNA-binding protein that interacts with BoxII in vitro, is a good candidate for being a light-modulated molecular switch controlling gene expression. However, the relationship between GT-1 activity and light-responsive gene activation still remains hypothetical. Because no marked de novo synthesis was detected after light treatment, light may induce post-translational modifications of GT-1 such as phosphorylation or dephosphorylation. Here, we show that recombinant GT-1 (hGT-1) of Arabidopsis can be phosphorylated by various mammalian kinase activities in vitro. Whereas phosphorylation by casein kinase II had no apparent effect on hGT-1 DNA binding, phosphorylation by calcium/calmodulin kinase II (CaMKII) increased the binding activity 10-20-fold. Mass spectrometry analyses of the phosphorylated hGT-1 showed that amongst the 6 potential phosphorylatable residues (T86, T133, S175, T179, S198 and T278), only T133 and S198 are heavily modified. Analyses of mutants altered at T86, T133, S175, T179, S198 and T278 demonstrated that phosphorylation of T133 can account for most of the stimulation of DNA-binding activity by CaMKII, indicating that this residue plays an important role in hGT-1/BoxII interaction. We further showed that nuclear GT-1 DNA-binding activity to BoxII was reduced by treatment with calf intestine phosphatase in extracts prepared from light-grown plants but not from etiolated plants. Taken together, our results suggest that GT-1 may act as a molecular switch modulated by calcium-dependent phosphorylation and dephosphorylation in response to light signals.

Amino Acid Sequence↗

PubMed1999

Green and yellow vegetables can maintain body stores of vitamin A in Chinese children.

BACKGROUND: Vitamin A activity of plant provitamin A carotenoids is uncertain. OBJECTIVE: The objective was to determine whether plant carotenoids can sustain or improve vitamin A nutrition during the fall season in kindergarten children in the Shandong province of China. DESIGN: The serum vitamin A concentration of 39% of the children was <1.05 micromol/L and of 61% of the children was > or = 1.05 micromol/L. For 5 d/wk for 10 wk, 22 children were provided approximately 238 g green-yellow vegetables/d and 34 g light-colored vegetables/d. Nineteen children maintained their customary dietary intake, which included 56 g green-yellow vegetables/d and 224 g light-colored vegetables/d. Octadeuterated and tetradeuterated vitamin A were given before and after the interventions, respectively, and their enrichments in the plasma were determined by gas chromatography-mass spectrometry. Serum retinol and carotenoid concentrations were measured by HPLC. RESULTS: Carotenoid nutrition improved after consumption of green-yellow vegetables. Serum concentrations of retinol were sustained in the group fed green-yellow vegetables but decreased in the group fed light-colored vegetables (P < 0.01). The isotope-dilution tests confirmed that total-body vitamin A stores were sustained in the group fed green-yellow vegetables, but decreased 27 micromol (7700 microg retinol) per child, on average, in the group fed light-colored vegetables (P < 0.06). CONCLUSION: Green-yellow vegetables can provide adequate vitamin A nutrition in the diet of kindergarten children and protect them from becoming vitamin A deficient during seasons when the provitamin A food source is limited.

Analysis of Variance↗

PubMed1999

Technical solution for an interactive functional MR imaging examination: application to a physiologic interview and the study of cerebral physiology.

Studies with functional magnetic resonance (MR) imaging produce large unprocessed raw data sets in minutes. The analysis usually requires transferring of the data to an off-line workstation, and this process frequently occurs after the subject has left the MR unit. The authors describe a hardware configuration and processing software that captures whole-brain raw data files as they are being produced from the MR unit. It then performs the reconstruction, registration, and statistical analysis, and displays the results in seconds after completion of the MR image acquisition.

Brain↗

PubMed1999

Enhancing effects of silkworm expressed recombinant human macrophage colony-stimulating factor on hematopoietic recovery of irradiation-injured mice.

OBJECTIVE: To study the hematopoietic enhancing effects of recombinant human macrophage colony-stimulating factor (rhM-CSF) expressed in silkworm. METHOD: Balb/c mice were irradiated with sublethal dose of 60 Co gamma-rays and then administered intraperitoneally with silkworm expressed rhM-CSF (1000 U per individual for 7 days) in treatment group or with normal saline (for 7 days as well) in control group. The hematopoietic recovery of irradiation mice was observed by comparing peripheral white blood cell (WBC) counts, differential counts of WBC and bone marrow hematopoietic progenitor cell colony forming assay in soft agar at different time after irradiation. RESULTS: The total WBC counts (x 10(9)/L) of treatment group at day 15 and 20 after irradiation(3.42 +/- 1.20, 5.56 +/- 2.50, respectively) were significantly higher than those of control group (2.03 +/- 0.90, 3.72 +/- 2.30; both P < 0.05). On days 10, 15, 20, 25 and 30 after irradiation, the monocyte counts (x 10(9)/L) of treatment group (0.08 +/- 0.06, 0.16 +/- 0.10, 0.48 +/- 0.35, 0.47 +/- 0.21 and 0.33 +/- 0.17, respectively) were all significantly higher than those of control group (0.025 +/- 0.016, 0.05 +/- 0.04, 0.23 +/- 0.16, 0.33 +/- 0.19 and 0.17 +/- 0.13; all P < 0.05). On days 15, 20 and 25, the granulocyte count (X 10(9)/L) of treatment group (1.03 +/- 0.61, 2.18 +/- 1.19 and 3.28 +/- 1.09) were also higher than those of control group (0.62 +/- 0.37, 1.40 +/- 0.99 and 2.20 +/- 0.74; all P < 0.05). On day 9 after irradiation, the bone marrow CFU-GM yield of control group (19 +/- 11/10(6) cells) was significantly lower than that of treatment group (78 +/- 30/10(6) cells, P < 0.05). CONCLUSION: rhM-CSF expressed in silkworm could accelerate hematopoietic recovery in irradiated mice.

Animals↗