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Biomedical subjects

X Ji

Publications and source records attributed to X Ji.

At least 19 recordsLinked to original sources
PubMed2005

Phenylephrine acts via IP3-dependent intracellular NO release to stimulate L-type Ca2+ current in cat atrial myocytes.

This study determined the effects of alpha1-adrenergic receptor (alpha1-AR) stimulation by phenylephrine (PE) on L-type Ca2+ current (I(Ca,L)) in cat atrial myocytes. PE (10 microm) reversibly increased I(Ca,L) (51.3%; n = 40) and shifted peak I(Ca,L) activation voltage by -10 mV. PE-induced stimulation of I(Ca,L) was blocked by each of 1 microm prazocin, 10 microm L-NIO, 10 microm W-7, 10 microm ODQ, 2 microm H-89 or 10 microm LY294002, and was unaffected by 10 microm chelerythrine or incubating cells in pertussis toxin (PTX). PE-induced stimulation of I(Ca,L) also was inhibited by each of 10 microm ryanodine or 5 microm thapsigargin, by blocking IP3 receptors with 2 microm 2-APB or 10 microm xestospongin C or by intracellular dialysis of heparin. In field-stimulated cells, PE increased intracellular NO (NOi) production. PE-induced NOi release was inhibited by each of 1 microm prazocin, 10 microm L-NIO, 10 microm W-7, 10 microm LY294002, 2 microm H-89, 10 microm ryanodine, 5 microm thapsigargin, 2 microm 2-APB or 10 microm xestospongin C, and unchanged by PTX. PE (10 microm) increased phosphorylation of Akt, which was inhibited by LY294002. Confocal microscopy showed that PE stimulated NOi release from subsarcolemmal sites and this was prevented by 2 mm methyl-beta-cyclodextrin, an agent that disrupts caveolae formation. PE also increased local, subsarcolemmal SR Ca2+ release via IP3-dependent signalling. Electron micrographs of atrial myocytes show peripheral SR cisternae in close proximity to clusters of caveolae. We conclude that in cat atrial myocytes PE acts via alpha1-ARs coupled to PTX-insensitive G-protein to release NOi, which in turn stimulates I(Ca,L). PE-induced NOi release requires stimulation of both PI-3K/Akt and IP3-dependent Ca2+ signalling. NO stimulates I(Ca,L) via cGMP-mediated cAMP-dependent PKA signalling. IP3-dependent Ca2+ signalling may enhance local SR Ca2+ release required to activate Ca2+-dependent eNOS/NOi production from subsarcolemmal caveolae sites.

Adrenergic alpha-Agonists↗
PubMed2004

Presence of plastid and absence of mitochondrial DNA in male reproductive cells as evidence for cytoplasmic inheritance in Turnera ulmifolia and Zantedeschia aethiopica.

Epifluorescence microscopy of mature pollen grains of Turnera ulmifolia and Zantedeschia aethiopica stained with 4',6-diamidino-2-phenylindole demonstrated the presence of fluorescent cytoplasmic DNA aggregates in the male reproductive cells of both species. Double staining of the cells with 4',6-diamidino-2-phenylindole and 3,3'-dihexyloxacarbocyanine iodide in Technovit resin sections showed that the mitochondria of these cells did not correspond to the fluorescent cytoplasmic DNA aggregates. Electron microscopy studies revealed both plastids and mitochondria in the cells of these species. In addition, immunoelectron microscopy using an anti-DNA monoclonal antibody showed clear labeling of plastids but not mitochondria. These data provide cytological evidence for biparental plastid inheritance and maternal mitochondrial inheritance in these species.

Coloring Agents↗
PubMed2004

Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme.

Anti-MUC1 single-chain Fv (scFv) fragments generated from the humanised antibody huHMFG1 had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue V(H)-71, previously described as a key residue for maintaining the CDR-H2 main-chain conformation and thus important for antigen binding, markedly stabilised the scFv while having only a minor effect on the binding affinity of the molecule. Because of its improved stability, the engineered fragment exhibited immunoreactivity with tumour cells even after 7 days of incubation in human serum at 37 degrees C. It also showed, in contrast to the wild-type scFv, a concentration-dependent binding to the target antigen when displayed on phage. When fusing the scFv to the recombinant ribonuclease rapLRI, only the fusion protein generated with the stable mutant scFv was able to kill MUC1(+) tumour cells with an IC(50) of 80 nM. We expect this novel immunoenzyme to become a promising tool for the treatment of MUC1(+) malignancies.

Amino Acid Sequence↗
PubMed2003

The Bcl-2 family pro-apoptotic molecule, BNIP3 regulates activation-induced cell death of effector cytotoxic T lymphocytes.

BNIP3 is a recently described pro-apoptotic member of the Bcl-2 family and in BNIP3 cDNA-transfected cell lines, cell death occurs via a caspase-independent pathway with opening of the mitochondrial permeability transition (PT) pore and rapid loss of mitochondrial transmembrane potential (Delta psi m). However, its expression or function in physiologic cell types is not known. Our results using the T-cell receptor transgenic mice P14, specific for lymphocyte choreomeningitis virus (LCMV) glycoprotein, show that in contrast to the other Bcl-2 family pro-apoptotic molecules, BNIP3 is transcriptionally highly up-regulated in effector cytotoxic T lymphocytes (CTL). Because CTL have a propensity to undergo activation-induced cell death (AICD) upon restimulation, we tested for other features associated with BNIP3-induced cell death. AICD of CTL was caspase-independent as determined by measuring caspase activation during target cell killing as well as by lack of inhibition with caspase inhibitors. Moreover, similar to BNIP3-induced cell death, CTL apoptosis was associated with increased production of reactive oxygen species and decreased Delta psi m. Finally, retroviral transduction of BNIP3 antisense RNA diminished AICD in effector CTL. These results suggest that BNIP3 may play an important role in T-cell homeostasis by regulating effector CTL numbers.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Molecular motions and conformational changes of HPPK.

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) belongs to a class of catalytic enzymes involved in phosphoryl transfer and is a new target for the development of novel antimicrobial agents. In the present study, the fundamental consideration is to view the overall structure of HPPK as a network of interacting residues and to extract the most cooperative collective motions that define its global dynamics. A coarse-grained model, harmonically constrained according to HPPK's crystal structure is used. Four crystal structures of HPPK (one apo and three holo forms with different nucleotide and pterin analogs) are studied with the goal of providing insights about the function-dynamic correlation and ligand induced conformational changes. The dynamic differences are examined between HPPK's apo- and holo-forms, because they are involved in the catalytic reaction steps. Our results indicate that the palm-like structure of HPPK is nearly rigid, whereas the two flexible loops: L2 (residues 43-53) and L3 (residues 82-92) exhibit the most concerted motions for ligand recognition and presumably, catalysis. These two flexible loops are involved in the recognition of HPPKs nucleotide and pterin ligands, whereas the rigid palm region is associated with binding of these cognate ligands. Six domains of collective motions are identified, comprised of structurally close but not necessarily sequential residues. Two of these domains correspond to the flexible loops (L2 and L3), whereas the remaining domains correspond to the rigid part of the molecule.

Amino Acid Sequence↗
PubMed2002

Therapeutic advantage from combining 5-fluorouracil with the hypoxia-selective cytotoxin NLCQ-1 in vivo; comparison with tirapazamine.

PURPOSE: The antitumor effect and bone marrow toxicity of 5-fluorouracil (5FU) in combination with the hypoxia-selective cytotoxins NLCQ-1 or tirapazamine (TPZ) were investigated in vivo. METHODS: Using appropriate intraperitoneal administration schedules for optimal synergistic interactions, the antitumor effect and the bone marrow toxicity of combinations of NLCQ-1 or TPZ and 5FU were determined in EMT6/BALB/c and SCCVII/C3H models in terms of dose modification factors (DMF) using the in vivo-in vitro clonogenic assay as endpoint. Bone marrow toxicity studies were performed in parallel using a modified CFU-GM assay. The antitumor efficacies of each combination treatment under optimal administration conditions were evaluated in the SCCVII/C3H model using also the tumor regrowth assay as endpoint. RESULTS: A schedule-dependent and tumor-specific synergistic interaction was observed for NLCQ-1 plus 5FU and DMFs of 2.0-2.3 and 1.0 were obtained for the antitumor effect and bone marrow toxicity, respectively, in both tumor models. The antitumor effect of 5FU was slightly potentiated (DMF 1.2) by TPZ in the EMT6/BALB/c model but not in the SCCVII/C3H model when the in vivo-in vitro assay was used as the endpoint. Significant additional tumor regrowth delays (about 11 and 6 days for NLCQ-1 and TPZ, respectively) were observed, compared to the effect of 5FU alone, when an equitoxic dose of NLCQ-1 (10 mg/kg) or TPZ (23 mg/kg) was administered 1 h before 5FU (50 mg/kg) twice a day at 4-h intervals on days 0 and 9. CONCLUSIONS: These results corroborate the therapeutic advantage of combining hypoxia-selective cytotoxins such as NLCQ-1 and TPZ with chemotherapy.

Animals↗
PubMed2002

The humoral immune responses elicited in mice by inoculations with a recombinant protein or DNA based on the circumsporozoite-protein gene of Plasmodium falciparum.

The humoral responses elicited in mice by inoculation, in various doses and by several routes, with plasmid DNA containing the gene coding for the circumsporozoite protein (CSP) of Plasmodium falciparum FCC1/HN were compared with those evoked by inoculation with a recombinant expressed protein based on the CSP. With the DNA vaccine, intramuscular inoculations appeared the most effective, followed by intravenous and then subcutaneous injections, the responses in each case being dose-dependent. In both standard ELISA and dot-ELISA, sera from the mice immunized with the DNA were found to have much lower titres of antimalarial antibodies than the corresponding sera from mice immunized with the recombinant protein. Although both 'vaccines' elicited humoral immune responses in BALB/c mice, that based on plasmid DNA took much longer than the recombinant protein to induce high-titre antibody responses.

Animals↗
PubMed2001

Crystallization and preliminary X-ray diffraction analysis of shikimate kinase from Mycobacterium tuberculosis in complex with MgADP.

Shikimate kinase (SK) from Mycobacterium tuberculosis (Mt) was overexpressed in Escherichia coli, purified and cocrystallized with MgADP in hanging drops using the vapor-diffusion procedure with PEG 4000 and 2-propanol as precipitants at pH 7.5. The crystal of MtSK-MgADP, which diffracted to 2.2 A resolution, belonged to space group P3(2)21 or P3(1)21, with unit-cell parameters a = b = 64.01, c = 92.41 A. There was one MtSK molecule in the asymmetric unit. Molecular-replacement trials with the crystal structure of SK from Erwinia chrysanthemi (PDB code 1shk) and adenylate kinase (PDB code 1ake) as search models were not successful. Heavy-atom derivative screening is in progress.

Adenosine Diphosphate↗
PubMed2001

Constructing parton convolution in effective field theory.

Parton convolution models have been used extensively in describing the sea quarks in the nucleon and explaining quark distributions in nuclei (the EMC effect). From the effective field theory point of view, we construct the parton convolution formalism which has been the underlying conception of all convolution models. We explain the significance of scheme and scale dependence of the auxiliary quantities such as the pion distributions in a nucleon. As an application, we calculate the complete leading nonanalytic chiral contribution to the isovector component of the nucleon sea.

Journal Article↗
PubMed2001

Synthesis and purine receptor affinity of 6-oxopurine nucleosides and nucleotides containing (N)-methanocarba-pseudoribose rings.

6-Oxopurine derivatives containing a northern (N) methanocarba modification (i.e., fused cyclopropane and cyclopentane rings in place of the ribose) were synthesized and the adenosine receptor affinity measured. Guanine or hypoxanthine was coupled at the 7-position, or 1,3-dibutylxanthine was coupled at the 9-position. The pseudoribose ring was also substituted at the 5'-position with an N-methyluronamide or with phosphate groups.

Calcium↗
PubMed2001

Unusual conformational changes in 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as revealed by X-ray crystallography and NMR.

The crystal structure of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) in complex with MgADP has been determined at 1.5-A resolution with a crystallographic R factor of 0.191. The solution structure of HPPK in complex with Mg(2+) and beta,gamma-methyleneadenosine 5'-triphosphate (MgAMPPCP) has been determined using a simulated annealing protocol with 3,523 experimental NMR restraints. The root mean square deviation of the ensemble of 20 refined conformers that represent the solution structure from the mean coordinate set derived from them is 0.74 +/- 0.26 A for all backbone atoms and 0.49 +/- 0.22 A when residues Pro(14), Pro(44)-Gln(50), and Arg(84)-Pro(91) are excluded. Binding of MgADP causes significant changes in the conformation and dynamical property of three loops of HPPK that are involved in catalysis. A dramatic, unusual conformational change is that loop 3 moves away from the active center significantly with some residues moving by >17 A. The binding of MgADP also stabilizes loop 1 and loop 3 but makes loop 2 more mobile. Very similar conformational and dynamical changes are observed in the NMR solution structure of HPPK.MgAMPPCP. The conformational and dynamical changes may play important roles in both substrate binding and product release in the catalytic cycle.

Adenosine Diphosphate↗
PubMed2001

[Histopathological changes of prostate cancer after castration therapy and correlative factors].

OBJECTIVE: To study the histopathological changes of prostate cancer after castration therapy among Chinese and correlative factors. METHODS: Needle biopsy specimens and prostatectomy specimens were obtained from 17 cases of prostate cancer and examined by light microscopy and image analysis system. RESULTS: Glandular atrophy, cytoplasmic vacuolization, nuclear pyknosis, and relative increase of stroma could be seen after orchectomy. There was a close correlation between atrophy of neoplastic tissue in prostate gland and patients' age (P < 0.05). The atrophy of neoplastic tissue in prostate gland was not correlated with the serum PSA, tumor staging and grading before castration therapy (P > 0.05). CONCLUSION: Castration is effective for treatment of prostate cancer, especially among the elderly patients. It is important for pathologists to recognize the histopathological changes in the prostectomy specimens made untypical by castration.

Age Factors↗
PubMed2001

The absorption edge of protein-bound mercury and a double-edge strategy for HgMAD data acquisition.

The L(III) absorption edge of protein-bound mercury (Hg) has been experimentally determined using X-ray data collection from a crystal. This absorption edge is 12 291 eV, 4 eV higher than the theoretical value of elemental Hg. Considering the possible shift of the Hg absorption edge with the chemical environment in different protein crystals, a double-edge strategy for multiwavelength anomalous diffraction (MAD) data collection has been developed. The approach provides a convenient way to optimize the dispersive signal between a remote wavelength and two edge wavelengths separated from each other by 4 eV. The dispersive signals derived from both edges are used, along with anomalous signals, in MAD phasing and phase refinement. This approach has been used in the crystal structure determination of three proteins containing one Hg atom per 186-196 amino-acid residues at 2.0, 2.6 and 2.7 A resolution. A set of four wavelengths is recommended for HgMAD data acquisition: 1.0087 A (12 291 eV, edge1), 1.0084 A (12 295 eV, edge2), 1.0064 A (12 320 eV, peak) and 0.9918 A (12 500 eV, remote). Although it is no longer necessary to determine the L(III) absorption edge of protein-bound Hg experimentally, an initial fluorescence scan on the crystal for data collection is still necessary to verify the existence of Hg in the crystal.

Crystallography, X-Ray↗
PubMed2001

C-terminal region amino acid substitutions contribute to catalytic differences between murine class alpha glutathione transferases mGSTA1-1 and mGSTA2-2 toward anti-diol epoxide isomers of benzo[c]phenanthrene.

The molecular basis for catalytic differences between structurally closely related murine class alpha glutathione (GSH) transferases mGSTA1-1 and mGSTA2-2 in the GSH conjugation of anti-diol epoxide isomers of benzo[c]phenanthrene (anti-B[c]PDE) was investigated. GSH conjugation of both (-)- and (+)-enantiomers of anti-B[c]PDE was observed in the presence of mGSTA1-1 (60 and 40% GSH conjugation, respectively), whereas mGSTA2-2 exhibited a preference for the (-)-anti-isomer (>97%). In addition, the specific activity of mGSTA2-2 toward the (-)-anti-B[c]PDE isomer was relatively higher than that of mGSTA1-1. The amino acid sequences of mGSTA1-1 and mGSTA2-2 differ at 10 positions that are distributed in three sections. Section I contains amino acid residues in positions 65 and 95; section II contains residues in positions 157, 162, and 169, and section III contains residues in positions 207, 213, 218, 221, and 222. Enzyme activity measurements with chimeras of mGSTA1-1 and mGSTA2-2 revealed that amino acid substitutions in section III account for their differential enantioselectivity and catalytic activity toward anti-B[c]PDE. Site-directed mutagenesis of amino acid residues in section III of mGSTA2-2 with corresponding residues of mGSTA1-1 followed by activity measurements of the wild type and mutated enzymes indicates that leucine 207 and phenylalanine 221 may be critical for the high catalytic activity of mGSTA2-2 toward (-)-anti-B[c]PDE. Molecular modeling studies demonstrated that the active site of mGSTA1-1 accommodates both enantiomers of anti-B[c]PDE, whereas the (-)-anti-isomer interacts more favorably with active site residues in mGSTA2-2. The results of this study clearly indicate that amino acid substitutions in the C-terminal region contribute to catalytic differences between mGSTA1-1 and mGSTA2-2 with respect to anti-B[c]PDE.

Amino Acid Substitution↗
PubMed2001

Measuring the P-odd pion-nucleon coupling h((1))(piNN) in pi(+)-photoproton production near threshold.

We show that gamma-->p-->pi(+)n in the threshold region is an excellent candidate for measuring the leading parity-violating pion-nucleon coupling h((1))(piNN) to an uncertainty of 20% if it has a natural size from dimensional analysis. The conclusion is based on a large unpolarized cross section, a new low-energy theorem for the photon polarization asymmetry at the threshold A(gamma)/(th) = square root of 2 f(pi)(mu(p)-mu(n))h((1))(piNN)/g(A)m(N) approximately h((1))(piNN)/2, and its strong dominance at forward and backward angles in the threshold region.

Journal Article↗
PubMed2001

Bisubstrate analogue inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: synthesis and biochemical and crystallographic studies.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), leading to the biosynthesis of folate cofactors. Like other enzymes in the folate pathway, HPPK is an ideal target for the development of antimicrobial agents because the enzyme is essential for microorganisms but is absent from human and animals. Three bisubstrate analogues have been synthesized for HPPK and characterized by biochemical and X-ray crystallographic analyses. All three bisubstrate analogues consist of a pterin, an adenosine moiety, and a link composed of 2-4 phosphoryl groups. P(1)-(6-Hydroxymethylpterin)-P(2)-(5'-adenosyl)diphosphate (HP(2)A, 5) shows little affinity and inhibitory activity for E. coli HPPK. P(1)-(6-Hydroxymethylpterin)-P(3)-(5'-adenosyl)triphosphate (HP(3)A, 6) shows moderate affinity and inhibitory activity with K(d) = 4.25 microM in the presence of Mg(2+) and IC(50) = 1.27 microM. P(1)-(6-Hydroxymethylpterin)-P(4)-(5'-adenosyl)tetraphosphate (HP(4)A, 7) shows the highest affinity and inhibitory activity with K(d) = 0.47 microM in the presence of Mg(2+) and IC(50) = 0.44 microM. The affinity of MgHP(4)A for HPPK is approximately 116 and 76 times higher than that of MgADP and 6-hydroxymethylpterin, respectively. The crystal structure of HPPK in complex with 7 (HPPK.MgHP(4)A) has been determined at 1.85 A resolution with a crystallographic R factor of 0.185. The crystal structure shows that 7 occupies both HP- and ATP-binding sites and induces significant conformational changes in HPPK. The biochemical and structural studies of the bisubstrate analogues indicate that the bisubstrate analogue approach can produce more potent inhibitors for HPPK and the minimum length of the link for a bisubstrate analogue is approximately 7 A.

Adenosine Triphosphate↗
PubMed2001

Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes.

The crystal structure of guanylate kinase (GK) from yeast (Saccharomyces cerevisiae) with a non-acetylated N terminus has been determined in its unligated form (apo-GK) as well as in complex with GMP (GK.GMP). The structure of apo-GK was solved with multiwavelength anomalous diffraction data and refined to an R-factor of 0.164 (R(free)=0.199) at 2.3 A resolution. The structure of GK.GMP was determined using the crystal structure of GK with an acetylated N terminus as the search model and refined to an R-factor of 0.156 (R(free)=0.245) at 1.9 A. GK belongs to the family of nucleoside monophosphate (NMP) kinases and catalyzes the reversible phosphoryl transfer from ATP to GMP. Like other NMP kinases, GK consists of three dynamic domains: the CORE, LID, and NMP-binding domains. Dramatic movements of the GMP-binding domain and smaller but significant movements of the LID domain have been revealed by comparing the structures of apo-GK and GK.GMP. apo-GK has a much more open conformation than the GK.GMP complex. Systematic analysis of the domain movements using the program DynDom shows that the large movements of the GMP-binding domain involve a rotation around an effective hinge axis approximately parallel with helix 3, which connects the GMP-binding and CORE domains. The C-terminal portion of helix 3, which connects to the CORE domain, has strikingly higher temperature factors in GK.GMP than in apo-GK, indicating that these residues become more mobile upon GMP binding. The results suggest that helix 3 plays an important role in domain movement. Unlike the GMP-binding domain, which moves toward the active center of the enzyme upon GMP binding, the LID domain moves away from the active center and makes the presumed ATP-binding site more open. Therefore, the LID domain movement may facilitate the binding of MgATP. The structure of the recombinant GK.GMP complex superimposes very well with that of the native GK.GMP complex, indicating that N-terminal acetylation does not have significant impact on the three-dimensional structure of GK.

Acetylation↗