Biomedical subjects

Zhenyu Lu

Publications and source records attributed to Zhenyu Lu.

17 recordsLinked to original sources

PubMed2006

TGF-beta2 inhibits AKT activation and FGF-2-induced corneal endothelial cell proliferation.

The corneal endothelial cells form a boundary layer between anterior chamber and cornea. This single cell layer is important to maintain cornea transparency by eliciting net fluid transport into the anterior chamber. Injuries of the corneal endothelial layer in humans lead to corneal swelling and translucence. This hindrance is thought to be due to limited proliferative capacity of the endothelial layer. Fibroblast growth factor 2 (FGF-2) and transforming growth factor-beta 2 (TGF-beta2) are both found in aqueous humor, and these two cytokines promote and inhibit cell growth, respectively. The intracellular signaling mechanisms by which TGF-beta2 suppresses the mitogenic response to FGF-2, however, remain unclear. We have addressed this question by investigating potential crosstalk between FGF-2-induced and TGF-beta2-regulated intracellular signaling events in cultured bovine corneal endothelial (BCE) cells. We found that TGF-beta2 and FGF-2 oppositely affect BCE cell proliferation and TGF-beta2 can override the stimulating effects of FGF-2 by increasing COX-2 expression in these cells. Consistent with these findings, overexpression of COX-2 significantly reduced FGF-2-induced cell proliferation whereas a COX-2 specific inhibitor NS398 reversed the effect of TGF-beta2 on FGF-2-induced cell proliferation. The COX-2 product prostaglandin E2 (PGE-2) blocks FGF-2-induced cell proliferation. Whereas FGF-2 stimulates cell proliferation by activating the AKT pathway, TGF-beta2 and PGE-2 both inhibit this pathway. In accordance with the effect of PGE-2, cAMP also inhibits FGF-2-induced AKT activation. These findings suggest that the mitogenic response to FGF-2 in vivo in the corneal endothelial layer may be inhibited by TGF-beta2-induced suppression of the PI3-kinase/AKT signaling pathway.

Animals↗

PubMed2006

Direct detection of the formation of V-amylose helix by single molecule force spectroscopy.

An important polysaccharide, amylose crystallizes as a regular single left-handed helix from a propanol, butanol, or iodine solution. However, its solution structure remains elusive because amylose does not form molecular solutions in these solvents, and standard spectroscopic techniques cannot be exploited to determine its structure. Using AFM, we forced individual amylose chains adsorbed to a surface to enter these poor solvents and carried out stretch-release measurements on them in solution. In this manner, we directly captured the formation of individual amylose helices induced by butanol and iodine. With an accuracy approaching that of X-ray diffraction on amylose crystals, we determined that the pitch of the helix in solution is 1.3 angstroms/ring. We also directly measured the force driving the formation of the helix in solution to be 50 pN. SMD simulations in explicit butanol reproduced the AFM-measured force-extension curves and revealed that the long plateau feature is caused by the rupture of O(2)n-O(6)(n+6) and O(3)n-O(6)(n+6) hydrogen bonds and by the unwinding of the helix. We also found that amylose helices formed in iodine solution are more compliant and hysteretic as compared to helices in butanol, which extend/relax reversibly. In iodine solution, the formation of the helix is inhibited by force and limited by the slow kinetics of the amylose-iodine complex. By forcing individual molecules into poor solvents and performing force spectroscopy measurements in solution, our AFM approach uniquely supplements X-ray diffraction and NMR methods for investigating solution conformations of insoluble biopolymers.

Amylose↗

PubMed2006

Simulating force-induced conformational transitions in polysaccharides with the SMD replica exchange method.

Conventional steered molecular dynamics (SMD) simulations do not readily reproduce equilibrium conditions of atomic force microscopy (AFM) stretch and release measurements of polysaccharides undergoing force-induced conformational transitions because of the gap between the timescales of computer simulations ( approximately 1 mus) and AFM measurements ( approximately 1 s). To circumvent this limitation, we propose using the replica exchange method (REM) to enhance sampling during SMD simulations. By applying REM SMD to a small polysaccharide system and comparing the results with those from AFM stretching experiments, we demonstrate that REM SMD reproduces the experimental results not only qualitatively but quantitatively, approaching near equilibrium conditions of AFM measurements. As tested in this work, hysteresis and computational time of REM SMD simulations of short polysaccharide chains are significantly reduced as compared to regular SMD simulations, making REM SMD an attractive tool for studying forced-induced conformational transitions of small biopolymer systems.

Computer Simulation↗

PubMed2006

Nuclear quantum effects on an enzyme-catalyzed reaction with reaction path potential: proton transfer in triosephosphate isomerase.

Nuclear quantum mechanical effects have been examined for the proton transfer reaction catalyzed by triosephosphate isomerase, with the normal mode centroid path integral molecular dynamics based on the potential energy surface from the recently developed reaction path potential method. In the simulation, the primary and secondary hydrogens and the C and O atoms involving bond forming and bond breaking were treated quantum mechanically, while all other atoms were dealt classical mechanically. The quantum mechanical activation free energy and the primary kinetic isotope effects were examined. Because of the quantum mechanical effects in the proton transfer, the activation free energy was reduced by 2.3 kcal/mol in comparison with the classical one, which accelerates the rate of proton transfer by a factor of 47.5. The primary kinetic isotope effects of kH/kD and kH/kT were estimated to be 4.65 and 9.97, respectively, which are in agreement with the experimental value of 4+/-0.3 and 9. The corresponding Swain-Schadd exponent was predicted to be 3.01, less than the semiclassical limit value of 3.34, indicating that the quantum mechanical effects mainly arise from quantum vibrational motion rather than tunneling. The reaction path potential, in conjunction with the normal mode centroid molecular dynamics, is shown to be an efficient computational tool for investigating the quantum effects on enzymatic reactions involving proton transfer.

Carbon↗

PubMed2006

Effect of CTCF-binding motif on regulation of PAX6 transcription.

PURPOSE: Previous studies indicate that the CCCTC binding transcription factor (CTCF) regulates homeobox PAX6 gene transcription in corneal epithelial cells. In the present study, the effect was investigated of CTCF activity on PAX6 transcription through interaction with five essential motifs located in an 80-bp region upstream from the PAX6 P0 promoter. METHODS: An electrophoretic mobility shift assay (EMSA) was used to determine the interaction between CTCF and DNA binding motifs. DNA mutagenesis was applied in identification of DNA motif functions. Immunohistochemistry and Western blot analyses were performed to detect the stress-induced effect on CTCF activity. RESULTS: The five identified CTCF-binding motifs were mutated one by one or in different combinations. Interactions of CTCF with these mutated motifs were determined by EMSA and DNA-binding competitions. All five CCCTC motifs were functional for the CTCF binding and DNA-binding activity of CTCF was proportionally decreased after increases in mutations of motif numbers. In addition, ultraviolet (UV) irradiation and epidermal growth factor (EGF) induced suppression and activation of CTCF expression, respectively. Effects of UV and EGF induction were due to alterations in CTCF expression and activity resulting in changes in CTCF DNA binding activity to the PAX6 promoter region detected by EMSA. CONCLUSIONS: These findings indicate that CTCF regulates PAX6 expression in response to stress-induced conditions and that the molecular base of CTCF controlling PAX6 expression is through five functional and specific motifs in the region upstream from the PAX6 P0 promoter in corneal epithelial cells.

Animals↗

PubMed2006

Pax6 regulation in retinal cells by CCCTC binding factor.

PURPOSE: A previous study demonstrated that CTCF (CCCTC binding factor) regulates homeobox Pax6 gene expression in early embryonic stages and plays a dominant role in eye development. The purpose of the present study was to explore further the mechanism of CTCF controlling Pax6 gene expression in human retinoblastoma (Rb) cells and in the development of chicken and mouse retinas. METHODS: Northern and Western analyses were used to detect expressions of CTCF and Pax6 in Rb cells. Pax6 transcription reporter and deletion mutants were used to study the regulatory interaction between CTCF and Pax6 in Rb cells and in the retina of chicken embryos. CTCF transgenic chicken embryos and mice were established by lipofection and microinjection of linearized cytomegalovirus (CMV)-CTCF construct into fertilized eggs and mouse oocytes, respectively. Injected oocytes were implanted in the uterus of foster mothers through microinjection into the ovarian duct. The expression of CTCF and Pax6 was determined in embryo sections by immunochemistry. RESULTS: Stimulation of Rb cells with 10% FBS resulted in an increase in CTCF expression and a decrease in Pax6 expression. To study the regulatory mechanism, the Pax6 reporter and its deletion mutant activities were determined in transfected Rb cells and chicken embryonic retinas, revealing that CTCF interacts with the Pax6 gene in Rb cells through transcription control in the 5'-flanking region upstream from the Pax6 P0 promoter. Overexpression of CTCF in Rb cells suppressed Pax6 reporter activity and downregulated endogenous Pax6 expression. In contrast, downregulation of CTCF expression by knockdown of CTCF mRNA using specific small interfering (si)RNA markedly enhanced Pax6 expression in Rb cells. Further study in CTCF transgenic mouse embryos verified that overexpression of CTCF suppressed Pax6 gene expression in the retina. CONCLUSIONS: CTCF plays an important role in regulating Pax6 expression in Rb cells and in the developmental retina, and the regulation of Pax6 gene expression by CTCF in the retina is through transcriptional regulation.

Animals↗

PubMed2005

Particle adhesion studies relevant to chemical mechanical polishing.

This study describes particle adhesion experiments carried out to elucidate interactions between particles in slurries used for polishing of wafers and disks. For this purpose the packed column technique was employed, which simulated chemical mechanical polishing of copper with silica and alumina, as well as of silicic oxide with ceria. The model systems consisted of uniform copper and glass beads as collectors, representing the wafers, and colloidal dispersions of silica, alumia, and silica coated with nanosize ceria, all of well-defined properties that are used as abrasives. It was shown that a strong correlation exists between deposition and detachment results of the adhesion studies and the polish rates measured using actual substrates with the same or similar slurries.

Journal Article↗

PubMed2005

Insulin-activated, K+-channel-sensitive Akt pathway is primary mediator of ML-1 cell proliferation.

Voltage-gated K(+) channel activities are involved in regulating growth factor-stimulated cell proliferation in a variety of cell types. Here we report that suppression of a voltage-gated K(+) channel with 4-aminopyridine (4-AP), barium, and tetraethylammonium inhibited both EGF- and insulin-stimulated myeloblastic leukemia ML-1 cell proliferation in a concentration-dependent manner. Both MAPK/ERK and Akt pathways are known to mediate cell proliferative signals of a variety of growth factors including insulin. In serum-starved ML-1 cells, insulin rapidly stimulated phosphorylation of ERK1/2 and Akt, and the phosphorylation levels peaked approximately 30 min after treatment. Pretreatment of ML-1 cells with 4-AP potently and dose-dependently prevented phosphorylation of ERK1/2 and Akt. However, insulin-induced activation of the Akt pathway also played a role in promoting ML-1 cell proliferation. Flow cytometry analysis revealed that although ML-1 cells were primarily arrested at G(1) phase by serum starvation for 36 h, they reentered the cell cycle after treatment with serum or insulin for 24 h. However, concomitant 4-AP treatment was able to attenuate cell cycle progression in synchronized ML-1 cells stimulated with growth factors. Our results strongly suggest that a 4-AP-sensitive K(+) channel activity plays an important role in controlling proliferation of ML-1 cells by affecting the activation of multiple signal transduction processes induced by insulin.

4-Aminopyridine↗

PubMed2005

Reaction path determination for quantum mechanical/molecular mechanical modeling of enzyme reactions by combining first order and second order "chain-of-replicas" methods.

A two-step procedure for the determination of reaction paths in enzyme systems is presented. This procedure combines two chain-of-states methods: a quantum mechanical/molecular mechanical (QM/MM) implementation of the nudged elastic band (NEB) method and a second order parallel path optimizer method both recently developed in our laboratory. In the first step, a reaction path determination is performed with the NEB method, along with a restrained minimization procedure for the MM environment to obtain a first approximation to the reaction path. In the second step, the calculated path is refined with the parallel path optimizer method. By combining these two methods the reaction paths are determined accurately, and in addition, the number of path optimization iterations are significantly reduced. This procedure is tested by calculating both steps of the isomerization of 2-oxo-4-hexenedioate by 4-oxalocrotonate tautomerase, which have been previously determined by our group. The calculated paths agree with the previously reported results and we obtain a reduction of 45%-55% in the number of path optimization cycles.

Binding Sites↗

PubMed2004

Elastic properties of single amylose chains in water: a quantum mechanical and AFM study.

Recent single-molecule atomic force microscopy (AFM) experiments have revealed that some polysaccharides display large deviations from force-extension relationships of other polymers which typically behave as simple entropic springs. However, the mechanism of these deviations has not been fully elucidated. Here we report the use of novel quantum mechanical methodologies, the divide-and-conquer linear scaling approach and the self-consistent charge density functional-based tight binding (SCC-DFTB) method, to unravel the mechanism of the extensibility of the polysaccharide amylose, which in water displays particularly large deviations from the simple entropic elasticity. We studied the deformations of maltose, a building block of amylose, both in a vacuum and in solution. To simulate the deformations in solution, the TIP3P molecular mechanical model is used to model the solvent water, and the SCC-DFTB method is used to model the solute. The interactions between the solute and water are treated by the combined quantum mechanical and molecular mechanical approach. We find that water significantly affects the mechanical properties of maltose. Furthermore, we performed two nanosecond-scale steered molecular dynamics simulations for single amylose chains composed of 10 glucopyranose rings in solution. Our SCC-DFTB/MM simulations reproduce the experimentally measured force-extension curve, and we find that the force-induced chair-to-boat transitions of glucopyranose rings are responsible for the characteristic plateau in the force-extension curve of amylose. In addition, we performed single-molecule AFM measurements on carboxymethyl amylose, and we found that, in contrast to the results of an earlier work by others, these side groups do not significantly affect amylose elasticity. By combining our experimental and modeling results, we conclude that the nonentropic elastic behavior of amylose is governed by the mechanics of pyranose rings themselves and their force-induced conformational transitions.

Amylose↗

PubMed2004

K(+) channel activity and redox status are differentially required for JNK activation by UV and reactive oxygen species.

Upon exposure to ultraviolet (UV) radiation, osmotic changes or the presence of reactive oxygen species (ROS) c-Jun N-terminal kinases (JNKs) are rapidly activated. Extensive studies have elucidated molecular components that mediate the activation of JNKs. However, it remains unclear whether activation of JNKs by various stress signals involves different pathways. Here we show that K(+) channel activity is involved in mediating apoptosis induced by UV but not by H(2)O(2) in myelocytic leukemic ML-1 cells. Specifically, JNKs were rapidly phosphorylated upon treatment of ML-1 cells with UV and H(2)O(2). UV-induced, but not H(2)O(2)-induced, JNK-1 phosphorylation was inhibited by pretreatment with 4-aminopyridine (4-AP), a K(+) channel blocker. 4-AP also blocked UV-induced increase in JNK activity as well as p38 phosphorylation. Immunofluorescent microscopy revealed that phosphorylated JNKs were concentrated at centrosomes in ML-1 cells and that these proteins underwent rapid subcellular translocation upon UV treatment. Consistently, the subcellular translocation of JNKs induced by UV was largely blocked by 4-AP. Furthermore, UV-induced JNK activation was blocked by NEM, a sulfhydryl alkylating agent also affecting K(+) current. Both UV- and H(2)O(2)-induced JNK activities were inhibited by glutathione, suggesting that the redox status does play an important role in the activation of JNKs. Taken together, our findings suggest that JNK activation by UV and H(2)O(2) is mediated by distinct yet overlapping pathways and that K(+) channel activity and redox status are differentially required for UV- and H(2)O(2)-induced activation of JNKs.

4-Aminopyridine↗

PubMed2004

Parallel iterative reaction path optimization in ab initio quantum mechanical/molecular mechanical modeling of enzyme reactions.

The determination of reaction paths for enzyme systems remains a great challenge for current computational methods. In this paper we present an efficient method for the determination of minimum energy reaction paths with the ab initio quantum mechanical/molecular mechanical approach. Our method is based on an adaptation of the path optimization procedure by Ayala and Schlegel for small molecules in gas phase, the iterative quantum mechanical/molecular mechanical (QM/MM) optimization method developed earlier in our laboratory and the introduction of a new metric defining the distance between different structures in the configuration space. In this method we represent the reaction path by a discrete set of structures. For each structure we partition the atoms into a core set that usually includes the QM subsystem and an environment set that usually includes the MM subsystem. These two sets are optimized iteratively: the core set is optimized to approximate the reaction path while the environment set is optimized to the corresponding energy minimum. In the optimization of the core set of atoms for the reaction path, we introduce a new metric to define the distances between the points on the reaction path, which excludes the soft degrees of freedom from the environment set and includes extra weights on coordinates describing chemical changes. Because the reaction path is represented by discrete structures and the optimization for each can be performed individually with very limited coupling, our method can be executed in a natural and efficient parallelization, with each processor handling one of the structures. We demonstrate the applicability and efficiency of our method by testing it on two systems previously studied by our group, triosephosphate isomerase and 4-oxalocrotonate tautomerase. In both cases the minimum energy paths for both enzymes agree with the previously reported paths.

Algorithms↗

PubMed2004

Reaction path potential for complex systems derived from combined ab initio quantum mechanical and molecular mechanical calculations.

Combined ab initio quantum mechanical and molecular mechanical calculations have been widely used for modeling chemical reactions in complex systems such as enzymes, with most applications being based on the determination of a minimum energy path connecting the reactant through the transition state to the product in the enzyme environment. However, statistical mechanics sampling and reaction dynamics calculations with a combined ab initio quantum mechanical (QM) and molecular mechanical (MM) potential are still not feasible because of the computational costs associated mainly with the ab initio quantum mechanical calculations for the QM subsystem. To address this issue, a reaction path potential energy surface is developed here for statistical mechanics and dynamics simulation of chemical reactions in enzymes and other complex systems. The reaction path potential follows the ideas from the reaction path Hamiltonian of Miller, Handy and Adams for gas phase chemical reactions but is designed specifically for large systems that are described with combined ab initio quantum mechanical and molecular mechanical methods. The reaction path potential is an analytical energy expression of the combined quantum mechanical and molecular mechanical potential energy along the minimum energy path. An expansion around the minimum energy path is made in both the nuclear and the electronic degrees of freedom for the QM subsystem internal energy, while the energy of the subsystem described with MM remains unchanged from that in the combined quantum mechanical and molecular mechanical expression and the electrostatic interaction between the QM and MM subsystems is described as the interaction of the MM charges with the QM charges. The QM charges are polarizable in response to the changes in both the MM and the QM degrees of freedom through a new response kernel developed in the present work. The input data for constructing the reaction path potential are energies, vibrational frequencies, and electron density response properties of the QM subsystem along the minimum energy path, all of which can be obtained from the combined quantum mechanical and molecular mechanical calculations. Once constructed, it costs much less for its evaluation. Thus, the reaction path potential provides a potential energy surface for rigorous statistical mechanics and reaction dynamics calculations of complex systems. As an example, the method is applied to the statistical mechanical calculations for the potential of mean force of the chemical reaction in triosephosphate isomerase.

Binding Sites↗

PubMed2004

Transmission coefficient calculation for proton transfer in triosephosphate isomerase based on the reaction path potential method.

A global potential energy surface has been constructed through interpolation of our recently developed reaction path potential for chemical reactions in enzymes which is derived from combined ab initio quantum mechanical and molecular mechanical calculations. It has been implemented for the activated molecular dynamics simulations of the initial proton transfer reaction catalyzed by triosephosphate isomerase. To examine the dynamical effects on the rate constants of the enzymatic reaction, the classical transmission coefficient kappa(t) is evaluated to be 0.47 with the reactive flux approach, demonstrating considerable deviations from transition state theory. In addition, the fluctuations of protein environments have small effects on the barrier recrossing, and the transmission coefficient kappa(t) strongly depends on the fluctuations of atoms near the active site of the enzyme.

Algorithms↗

PubMed2004

Regulation of eye development by transcription control of CCCTC binding factor (CTCF).

CCCTC binding factor (CTCF), a transcriptional regulator, plays important roles in epigenetics and development. In the present study, we report that overexpression of CTCF in transgenic mice during embryonic development suppresses Pax6 gene expression. This effect causes defects in ocular development that result in microophthalmia. In eye-derived cells transfected with a tetracycline turn-on CTCF system, up-regulation of CTCF expression significantly suppressed Pax6 expression. In contrast, the knockdown of CTCF mRNA resulted in the down-regulation of CTCF protein expression, which in turn enhanced the Pax6 expression. CTCF controls Pax6 transcription by interacting with a repressor element located in the 5'-flanking region upstream of the Pax6 P0 promoter. This interaction suppressed Pax6 gene transcription by blocking the effect of an ectoderm enhancer located 3.5 kb upstream from the P0 promoter. We also found an 80-bp sequence in a region -1.2 kbp upstream from the P0 promoter that contained multiple CTCF binding sites and interacted with nuclear proteins obtained from eye-derived cells forming electrophoretic mobility shift assay complexes with CTCF. We conclude that a novel function of CTCF is to regulate Pax6 transcription by binding to the repressor element, which in turn blocks the effect of the ectoderm enhancer resulting in the inhibition of P0 promoter activity.

Animals↗

PubMed2003

The use of monodispersed colloids in the polishing of copper and tantalum.

The properties of abrasive particles play a significant role in chemical mechanical polishing (CMP) of metal and dielectric films in semiconductor device manufacturing. This study investigates the effects of the particle size, shape, and hardness of abrasives on the polishing of copper and tantalum films in the presence of different slurry chemistries. Well-defined dispersions of uniform particles, including spherical silica of varying diameters, hematite of different shapes, and hematite cores coated with silica of different thicknesses, were prepared and used to polish blanket films of Cu and Ta. It was shown that the total surface area of the solids in the slurry controlled the rate of material removal by pure silica for both Cu and Ta, while the surface quality of the polished films was better when higher silica content was used. The shape or the aspect ratio of hematite particles had a minor effect on the removal rate. In contrast, when hematite particles coated with silica were employed in the polishing of Cu and Ta, the polish rate decreased with increasing thickness of the shell.

Journal Article↗

PubMed2002

[Construction and tumorigenic study on a novel fusion gene AML1-MTG16].

OBJECTIVE: To test whether splicing overlapping extension(SOE) method can be a tool for obtaining rare fusion gene's transcripts and to study the tumorigenic capacity of a novel fusion gene AML1-MTG16. METHODS: SOE method was used to obtain AML1- MTG16 fusion gene's transcripts. MTG16, AML1-MTG16 and AML1-MTG16 without III,VI conserved domains of MTG16 segment were inserted into pEGFP- C1,pDsRed-N1 vector respectively,then transfected NIH3T3 cell line by lipofection. Forty-eight hours later, the transfected cells were examined by laser-scanning confocal microscopy. Stable transfected cells were obtained by G418 500ug/ul selection for one month. Growth curve, soft agar colonies formation tumorigenesis in nude mice were done to compare the difference between stable transfected cells. RESULTS: Recombined AML1-MTG16 by SOE contained its CDS. NIH3T3 expressing AML1-MTG16 had a faster proliferation in medium, colony growth in soft agar. AML1-MTG16 expression cells also induced tumors formation following injection into nude mouse. MTG16,AML1-MTG16 and AML1-MTG16 without III,VI conserved domains of MTG16 were colocalized in the nucleus of cotransfected NIH3T3 cells under the examination of laser-scanning confocal microscope. CONCLUSION: SOE is an effective method to get rare fusion gene's transcripts. AML1-MTG16 plays an important role in leukemogenesis. MTG16 may also have a carcinogenic property within the AML1-MTG16 fusion gene. Carcinogenic property of AML1-MTG16 is restricted to its localization in the nuclear matrix. N terminal of MTG16 may play an important part in the carcinogenic activity of AML1-MTG16.

3T3 Cells↗