Biomedical subjects

Lei Zheng

Publications and source records attributed to Lei Zheng.

At least 37 records · Page 2Linked to original sources

PubMed2005

[Fe(bpb)(CN)2]- as a versatile building block for the design of novel low-dimensional heterobimetallic systems: synthesis, crystal structures, and magnetic properties of cyano-bridged Fe(III)-Ni(II) complexes [(bpb)(2-) = 1,2-bis(pyridine-2-carboxamido)benzenate].

A dicyano-containing [Fe(bpb)(CN)2]- building block has been employed for the synthesis of cyano-bridged heterometallic Ni(II)-Fe(III) complexes. The presence of steric bpb(2-) ligand around the iron ion results in the formation of low-dimensional species: five are neutral NiFe2 trimers and three are one-dimensional (1D). The structure of the 1D complexes consists of alternating [NiL]2+ and [Fe(bpb)(CN)2]- generating a cyano-bridged cationic polymeric chain and the perchlorate as the counteranion. In all complexes, the coordination geometry of the nickel ions is approximately octahedral with the cyano nitrogen atoms at the trans positions. Magnetic studies of seven complexes show the presence of ferromagnetic interaction between the metal ions through the cyano bridges. Variable temperature magnetic susceptibility investigations of the trimeric complexes yield the following J(NiFe) values (based on the spin exchange Hamiltonian H = -2J(NiFe) S(Ni) (S(Fe(1)) + S(Fe(2))): J(NiFe) = 6.40(5), 7.8(1), 8.9(2), and 6.03(4) cm(-1), respectively. The study of the magneto-structural correlation reveals that the cyanide-bridging bond angle is related to the strength of magnetic exchange coupling: the larger the Ni-N[triple bond]C bond angle, the stronger the Ni- - -Fe magnetic interaction. One 1D complex exhibits long-range antiferromagnetic ordering with T(N) = 3.5 K. Below T(N) (1.82 K), a metamagnetic behavior was observed with the critical field of approximately 6 kOe. The present research shows that the [Fe(bpb)(CN)2]- building block is a good candidate for the construction of low-dimensional magnetic materials.

Journal Article↗

PubMed2005

Mechanistic study of proton transfer and hysteresis in catalytic antibody 16E7 by site-directed mutagenesis and homology modeling.

Antibody 16E7 catalyzes the carbon protonation of enol ether 2 to hemiacetal 3, and the carbon deprotonation of benzisoxazole 7 to phenol 8. This antibody shows an extreme case of hysteresis, requiring several hours to reach full activity. Antibody 16E7 was expressed as recombinant chimeric Fab in Escherichia coli. A model for the three-dimensional structure was produced by homology modeling and used for a docking procedure to obtain models for antibody-ligand complexes. Site-direct mutagenesis of GluL39, identified as a possible catalytic residue by the model, to either glutamine or alanine abolished catalysis, showing that both the protonation reaction of enol ether 2 and the deprotonation of benzisoxazole 7 are promoted by the same residue. The model furthermore suggested that substrate access to the catalytic site might be hindered by a flexible HCDR3 loop held in closed position by a hydrogen bond between SerH99 and GluL39, which could explain the observed hysteresis effect. In agreement with this model, mutagenesis of SerH99 to alanine, or deletion of this residue, was found to reduce hysteresis by approximately 50%.

Antibodies, Catalytic↗

PubMed2005

Effect of nano-TiO(2) on strength of naturally aged seeds and growth of spinach.

The effects of nano-TiO(2) (rutile) and non-nano-TiO(2) on the germination and growth of naturally aged spinach seeds were studied by measuring the germination rate and the germination and vigor indexes of aged spinach seeds. An increase of these factors was observed at 0.25-4% nano-TiO(2) treatment. During the growth stage, the plant dry weight was increased, as was the chlorophyll formation, the ribulosebisphosphate carboxylase/oxygenase activity, and the photosynthetic rate. The best results were found at 2.5% nano-TiO(2). The effects of non-nano-TiO(2) are not significant. It is shown that the physiological effects are related to the nanometer-size particles, but the mechanism by which nano-TiO(2) improves the growth of spinach seeds still needs further study.

Chlorophyll↗

PubMed2005

Effect of nano-TiO2 on photochemical reaction of chloroplasts of spinach.

The effects of nano-TiO2 (rutile) on the photochemical reaction of chloroplasts of spinach were studied. The results showed that when spinach was treated with 0.25% nano-TiO2, the Hill reaction, such as the reduction rate of FeCy, and the rate of evolution oxygen of chloroplasts was accelerated and noncyclic photophosphorylation (nc-PSP) activity of chloroplasts was higher than cyclic photophosphorylation (c-PSP) activity, the chloroplast coupling was improved and activities of Mg2+-ATPase and chloroplast coupling factor I (CF1)-ATPase on the thylakoid membranes were obviously activated. It suggested that photosynthesis promoted by nano-TiO2 might be related to activation of photochemical reaction of chloroplasts of spinach.

Biochemistry↗

PubMed2005

Hardness and Young's modulus of transparent dentin associated with aging and carious disease.

This study investigated the changes in hardness and Young's modulus of the transparent layer of dentin associated with aging and the carious process. Eighteen extracted human molars with or without coronal caries were used in this study. The normal teeth were divided into two groups by age, and the carious teeth were divided into two groups of active or arrested caries. After polishing the specimens parallel to the long axis of the tooth, both hardness and Young's modulus were measured using a nanoindentation tester. The hardness and Young's modulus of the transparent layer in aged dentin were higher than the other portions of aged dentin. The transparent layer under carious lesions had a significantly lower hardness than the underlying normal dentin, whereas its Young's modulus was not significantly reduced. The hardness and Young's modulus of the transparent layer in active carious lesions were lower than those in arrested carious lesions.

Aging↗

PubMed2005

[Study of antithrombotic function of endothelium in vascular tissue engineering].

OBJECTIVE: To investigate the current situation and developing trend of antithrombotic function study of endothelium in vascular tissue engineering. METHODS: The effect of several elements on the antithrombotic ability of endothelium, including the source of endothelium, the characteristic of the matrix materials, the cell culture methods, and the endothelium's gene modification were analyzed. RESULTS: The normal antithrombotic function of tissue engineered vascular relied on the source of endothelium, gene modification of seeding cells, the cell culture methods in vitro, and the characteristic of the scaffolds. CONCLUSION: The establishment of an ideal antithrombotic functional tissue engineering vascular still requires further studies in various aspects including seeding cells, matrix materials, and cell culture methods. Gene modification of vascular endothelium, which improves the antithrombotic ability, deserves more attention.

Animals↗

PubMed2005

[Effect of Pb2+ on spectral characteristics of DNA from fish intestines].

The interaction of Pb2+ and DNA of fish intestines was investigated using absorption spectrum, fluorescence emission spectrum, and electrophoresis. Ultraviolet absorption spectra indicated that with the addition of Pb2+, the DNA generated obvious hypochromic effect, and the absorption peak at 207 nm of DNA was blue-shifted. The fluorescence emission spectra showed that with the addition of Pb2+ , the emission peak of DNA at about 419 nm did not have obvious changes, but the intensity of fluorescence reduced gradually, showing that Pb2+ has quenching effect on the DNA endogenous fluorescence. Pb2+ has 0.8 binding sites to DNA, and the fluorescence quenching of DNA caused by Pb2+ belongs to static quenching. The binding constants of binding sites are 6.08 x 10(4) L x mol(-1) and 2.82 x 10(4) L x mol(-1) from the Lineweaver-Burk plot. The experiments of ultraviolet and fluorescence spectra showed that exogenous Pb2+ could cause some changes to the conformation of DNA, but analysis of electrophoresis proved that exogenous Pb2+ at different concentration did not result in DNA breakage.

Algorithms↗

PubMed2005

[In vitro culture and identification of tolerogenic dendritic cells from mouse bone marrow].

OBJECTIVE: To establish a simple and economic method for in vitro culture of tolerogenic dendritic cells (Tol-DC) from mouse bone marrow to facilitate further research of the application of Tol-DC in autoimmune disease. METHODS: The dendritic cells were derived from in vitro culture of the precursor cells from mouse bone marrow with recombinant mouse granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and interleukin (IL)-4, respectively. Eighteen hours before completion of cell culture, lipopolysaccharide (LPS) was added in the medium for stimulating rmGM-CSF-treated cells, but not IL-4-treated cells. The cells were collected on day 6, and the cell morphology, phenotype and immunofunction were analyzed. RESULTS: DC cultured in vitro had a CD11c(+) expression rate of 76.67% with typical morphology. The DCs without LPS treatment were characterized by moderate expression of class II major histocompatibility complex (MHCII) and low expression of costimulatory molecules, while those with LPS treatment had high expressions of MHCIIand costimulatory molecules. CONCLUSION: Immature tolerogenic DCs can be derived in large quantity using this method with the potential for use in the treatment of autoimmune diseases.

Animals↗

PubMed2004

The mechanism of ammonia transport based on the crystal structure of AmtB of Escherichia coli.

Ammonium is one of the most important nitrogen sources for bacteria, fungi, and plants, but it is toxic to animals. The ammonium transport proteins (methylamine permeases/ammonium transporters/rhesus) are present in all domains of life; however, functional studies with members of this family have yielded controversial results with respect to the chemical identity (NH(4)(+) or NH(3)) of the transported species. We have solved the structure of wild-type AmtB from Escherichia coli in two crystal forms at 1.8- and 2.1-A resolution, respectively. Substrate transport occurs through a narrow mainly hydrophobic pore located at the center of each monomer of the trimeric AmtB. At the periplasmic entry, a binding site for NH(4)(+) is observed. Two phenylalanine side chains (F107 and F215) block access into the pore from the periplasmic side. Further into the pore, the side chains of two highly conserved histidine residues (H168 and H318) bridged by a H-bond lie adjacent, with their edges pointing into the cavity. These histidine residues may facilitate the deprotonation of an ammonium ion entering the pore. Adiabatic free energy calculations support the hypothesis that an electrostatic barrier between H168 and H318 hinders the permeation of cations but not that of the uncharged NH(3.) The structural data and energetic considerations strongly indicate that the methylamine permeases/ammonium transporters/rhesus proteins are ammonia gas channels. Interestingly, at the cytoplasmic exit of the pore, two different conformational states are observed that might be related to the inactivation mechanism by its regulatory partner.

Amino Acid Sequence↗

PubMed2004

Fibroblast growth factor receptors as molecular targets in thyroid carcinoma.

Several molecular abnormalities of potential therapeutic target value have been described in thyroid neoplastic transition. We report the expression of the fibroblast growth factor receptor family (FGFR-1-4) in normal thyroid tissues, human thyroid cancers of various types and behaviors, and cell lines representative of the spectrum of differentiation of tumors derived from follicular epithelial cells. FGFR-2 was the only receptor consistently detected in normal human thyroid tissue, and its expression diminished in all thyroid cancers and carcinoma cell lines, suggesting that it may have a protective role. FGFR-1 and FGFR-3 were expressed in most well-differentiated tumor types. FGFR-4, however, was expressed predominantly in aggressive tumor types and the most rapidly proliferative cell lines, indicating that it may promote the progression of these tumors. To specifically determine the function of FGFR-4 in thyroid carcinoma, gain- or loss-of-function studies were performed in cell lines representative of the spectrum of thyroid cancer behavior. Introduction of FGFR-4 resulted in enhanced cell proliferation, an effect that was more pronounced in cell lines derived from aggressive tumors than in those derived from more indolent neoplasms. Moreover, transduction of a dominant-negative FGFR attenuated cell proliferation in the aggressive poorly differentiated cell lines with no appreciable effect in well-differentiated cells. Pharmacologic FGFR-4 tyrosine kinase inhibition resulted in significant proliferation arrest in an aggressive cell line endogenously expressing the receptor. Furthermore, systemic administration of the FGFR tyrosine kinase inhibitor PD173074 resulted in significant inhibition of follicular thyroid carcinoma-derived cell growth in xenografted severe combined immunodeficient mice. These data indicate a role for FGFR-4 in human thyroid cancer cell progression and provide a rationale for FGFR manipulation as a potentially novel therapeutic approach.

Adenoviridae↗

PubMed2004

Sp1-mediated transcriptional control of fibroblast growth factor receptor 4 in sarcomas of skeletal muscle lineage.

Fibroblast growth factor receptors (FGFRs) have been implicated in a multitude of differentiating and proliferative actions. FGFR4 is expressed mainly in lung, kidney, pancreas, spleen, and developing muscle. FGFR4 was found to be overexpressed in some human malignancies, where it has been implicated in their pathogenesis. Recently, FGFR4 was found to be overexpressed in pediatric rhabdomyosarcomas, based on cDNA microarray analysis. Using Northern blotting, reverse transcription-polymerase chain reaction, and Western blotting, we classified four human rhabdomyosarcoma-derived cell lines based on their relative expression of FGFR4. We defined a 214 bp (-115/+99) promoter that functioned as a minimal promoter and examined cis-DNA elements implicated in the control of expression of the FGFR4 gene in these cells. Overlapping 40- to 50-bp fragments of the minimal promoter were examined by electrophoretic mobility shift assay using nuclear extracts from cell lines with high (HS729-1015) or low (HS729-1016) FGFR4 expression. Fragment C (-65/-26) formed specific complexes with nuclear extracts from both cell lines. Fragment B (-95/-56), however, formed distinct complexes mainly with the high FGFR4-expressing HS729-1015 cells. Both fragments yielded complexes that were competed by an Sp oligonucleotide and supershifted by Sp1 and by Sp3 antibodies. Transfection of Sp1 but not Sp3 efficiently activated FGFR4 promoter activity, an effect that was significantly more pronounced in the HS729-1015 cell line than in the low FGFR4-expressing HS729-1016 cell line. Deletion of each of the two Sp-binding sites in fragments B and C resulted in loss of promoter activity. In particular, deletion of the 5' Sp-binding site in fragment B was associated with the greatest loss of activity. Sp1 protein expression correlated with FGFR4 expression in cell lines and primary human rhabdomyosarcomas. Furthermore, transfection of Sp1 and methylation inhibition was effective in inducing the endogenous FGFR4 gene in HS729-1015 cells. Our findings point to Sp1 as an important contributor to FGFR4 transcriptional control and elucidate a potential mechanism for the heterogeneous expression of FGFR4 in neoplasms derived from the same cell lineage.

Azacitidine↗

PubMed2004

Expression improvement and mechanistic study of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis.

Antibody 10F11 catalyzes the retro-Diels-Alder reaction of the bicyclic prodrug 1 releasing HNO and anthracene 4 (kcat/kuncat=2500). Earlier X-ray crystal structures of Fab 10F11 showed that tryptophan H104 at the bottom of the binding pocket interacts by pi-stacking with the aromatic ring of the substrate. Antibody 10F11 was expressed as a chimeric Fab and subjected to site-directed mutagenesis. Expression was improved by substituting a serine for a phenylalanine residue on the Fv-domain surface. Nine active-site mutants were then prepared including replacements at TrpH104, PheH101 and SerH100. Catalysis depends mainly on TrpH104 and PheH101. Catalysis is most likely caused by a combination of shape complementarity and specific electronic interactions between transition state and the aromatic residue H104. Medium and de-solvation effects have no effect on the reaction rate. Catalysis was improved to (kcat/kuncat=6300) by substituting phenylalanine for LeuL101 to indirectly enhance pi-stacking between transition state and TrpH104.

Antibodies, Catalytic↗

PubMed2004

An efficient one-step site-directed and site-saturation mutagenesis protocol.

We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation libraries.

DNA Primers↗

PubMed2004

Pituitary tumor-derived fibroblast growth factor receptor 4 isoform disrupts neural cell-adhesion molecule/N-cadherin signaling to diminish cell adhesiveness: a mechanism underlying pituitary neoplasia.

We previously identified pituitary tumor-derived fibroblast growth factor receptor 4 (ptd-FGFR4), an alternatively transcribed N-terminally truncated cytoplasmic receptor isoform. Unlike wild-type FGFR4, ptd-FGFR4 facilitates cell transformation and results in pituitary tumor formation in transgenic mice. To investigate differences in the tumorigenic properties of FGFR4 and ptd-FGFR4, we examined their abilities to modulate cell adhesiveness. Introduction of ptd-FGFR4 into GH4 pituitary cells or NIH 3T3 fibroblasts resulted in significant reduction in cell adhesion to a collagen IV matrix compared with FGFR4- or empty vector-transfected cells. This adhesive difference was evident in the absence or presence of FGF stimulation. Furthermore, treatment with beta1-integrin neutralizing antibody markedly reduced adhesiveness in FGFR4-transfected cells but had little effect on the depressed adhesiveness of ptd-FGFR4-transfected cells. Unlike wild-type FGFR4, ptd-FGFR4 does not associate with neural cell-adhesion molecule (NCAM). Cells expressing FGFR4 demonstrate membranous N-cadherin with a noninvasive growth pattern identical to control GH4 cells when injected into immunodeficient mice. In contrast, ptd-FGFR4-expressing cells develop invasive tumors in vivo with marked loss of N-cadherin that localizes to the cytoplasm. Consistent with these changes, beta-catenin expression was diminished and its interaction with N-cadherin was disrupted in the presence of ptd-FGFR4, but both were intact in the presence of wild-type FGFR4. These data highlight the importance of membrane-anchored FGFR4 in assembling a multiprotein FGFR4 complex with NCAM and N-cadherin playing pivotal functions in maintaining normal cell adhesion. Disruption of distinct NCAM/N-cadherin proadhesive complexes by a tumor-derived FGFR4 isoform provides a novel mechanism beyond ligand independence that explains the pathobiology of proliferative and infiltrative but nonmetastatic neoplasms.

3T3 Cells↗

PubMed2004

A catalytic antibody against a tocopherol cyclase inhibitor.

The cyclic ammonium cation 5 and its guanidinium analogue 4 are inhibitors of tocopherol cyclase. Monoclonal antibodies were raised against protein conjugates of the haptens 1-3 and screened for catalytic reactions with alkene 8, a short chain analogue of the natural substrate phytyl-hydroquinone 6, and its enol ether analogues 10a,b. Antibody 16E7 raised against hapten 3 was found to catalyze the hydrolysis of Z enol ether 10a to form hemiacetal 12 with an apparent rate acceleration of k(cat)/k(uncat)=1400. Antibody 16E7 also catalyzed the elimination of Kemp's benzisoxazole 59. The absence of cyclization in the reaction of enol ether 10a was attributed to the competition of water molecules for the oxocarbonium cation intermediate within the antibody binding pocket. Hapten and reaction design features contributing to this outcome are discussed. Antibody 16E7 provides the first example of a carboxyl group acting both as an acid in an intrinsically acid-catalyzed process and as a base in an intrinsically base-catalyzed process, as expected from first principles. In contrast to the many examples of general-acid-catalyzed processes known to be catalyzed by catalytic antibodies, the specific-acid-catalyzed cyclization of phytyl-hydroquinone 6 or its analogue 8 still eludes antibody catalysis.

Adjuvants, Immunologic↗

PubMed2004

Molecular mechanism of enantioselective proton transfer to carbon in catalytic antibody 14D9.

Catalytic antibody 14D9 catalyzes the enantioselective protonation of prochiral enol ethers with high enantioselectivity (>99% ee) and a practical turnover (k(cat) = 0.4 s(-1)), allowing for preparative scale applications. This antibody represents one of the rare examples of catalytic antibodies promoting acid-catalyzed processes. Antibody 14D9 was cloned and expressed as a chimeric Fab fragment in Escherichia coli. Crystal structures of Fab 14D9 as apo form and of its close analog 19C9 in complex with the transition state analog were determined at 2.8-A resolution. A series of site-directed mutagenesis experiments was carried out to probe the role of individual active-site amino acids. Proton transfer to carbon is catalyzed by a hydrogen bond network formed by the side chains of Asp(H101) and Tyr(L36) with a water molecule serving as a relay. The intermediate oxocarbonium ion formed during the protonation step is trapped by the same water molecule, resulting in an overall syn-addition of water to the enol ether's double bond. The enantioselectivity is caused by steric crowding at the active site, mainly because of the side chain of Phe(H84). The 20-fold lower activity of 19C9 compared with 14D9 was traced down to residue Thr(L46), which forms a nonproductive hydrogen bond with the catalytic residue Asp(H101), which competes with the critical Asp(H101)-Tyr(L36) hydrogen bond and therefore reduces catalytic efficiency. The catalytic activity of 19C9 was restored to that of 14D9 by using either site-directed mutagenesis (Thr(L46)Ala) or chain shuffling.

Amino Acid Sequence↗

PubMed2004

Oxygen tension in the rabbit lens and vitreous before and after vitrectomy.

Oxygen is believed to be one of the potential causative agents for the development of nuclear cataract following vitrectomy. The aim of this study was to determine the partial pressure of oxygen (pO2) in different compartments of the rabbit eye, and to describe the changes following vitrectomy. Twenty-six rabbits (3.5-5.3 kg) were anesthetized and oxygen tension was probed using a fiber-optic oxygen sensor system (optode). A micromanipulator was employed to ascertain the exact position of the probe within the eye. Measurements were taken pre- and post-vitrectomy at several defined positions within the vitreous, the lens and the anterior chamber. Follow-up measurements were performed 2 and 8 weeks after vitrectomy. The contralateral eye served as a control. Measurements in the normal rabbit eye showed that oxygen tension in the globe is asymmetrical with the lowest pO2 in the nucleus of the lens (10.4 mmHg+/-3.0). The region of the lens near the posterior capsule has an oxygen tension close to the values of the vitreous directly behind the posterior capsule (12.4 mmHg+/-3.1). The highest pO2 within the posterior compartment of the eye was measured close to the retinal surface (40-l60 mmHg) depending on neighboring large vessels. The tension drops off rapidly to 20 mmHg some 0.5 mm from the retina. From that position to the posterior surface of the lens there is a shallow gradient of decreasing pO2. Immediately following vitrectomy the pO2 in the BSS replacement varied from ca. 90-140 mmHg, and decreased over approximately 30 min. to levels that were 2-3 times that of normal vitreous. Two weeks after vitrectomy the pO2 values in the lens were 2-3 times as high as in the control eye (p < 0.05). In addition there is no longer a gradient in the vitreous cavity, except close to the retina. Eight weeks after vitrectomy, pO2 levels in the lens were decreased but still remained higher than in the normal eye (13.83 mmHg+/-0.02). The pO2 gradient in the vitreous was not detectable anymore. Overall the results provide evidence that oxygen levels in the lens increase significantly after vitrectomy in rabbits. If this occurs in humans it may contribute to cataract formation following surgery.

Animals↗

PubMed2004

Transmission of light to the aging human retina: possible implications for age related macular degeneration.

The purpose of this study is to determine the transmission properties of the anterior segment of the human eye as a function of age and relate those changes to possible consequences for retinal disorders. For this a new method has been developed. This consists of a probe which is inserted into the posterior sclera and detects light passing through the anterior segment. The probe is connected to a CCD spectrophotometer via a fibre optic bundle. Using this, the transmission properties of human cadaver eyes were determined. A young primate anterior segment has a maximum absorption of 365 nm due to the O-beta-glucoside of 3-hydroxykynurenine (3-HKG) in the lens. There is a steep increase in transmission of the human anterior segment at wavelengths longer than 400 nm. With aging there is an increase in absorption throughout the visible such that by the sixth decade only 20% of blue light is transmitted to the retina compared to the young primate eye. The rate of decrease of blue light was similar to the age related change of the ratio of absorbance at 365/320 nm of the lens. (IOVS 41:1454;1999). The age related rate of decrease in the transmission of blue light to the retina was similar to the rate of increase of lipofuscin formation in the retina, and the amount of light absorbed by A2E in the RPE is constant from the second to seventh decade. Although this yellowing is thought to be detrimental to the lens, it would appear to be beneficial to the retina. It was determined that the implantation of a standard IOL after cataract surgery increased the amount of light absorbed by A2-E by approximately a factor of five.

Aged↗