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Biomedical subjects

Cheng Zhu

Publications and source records attributed to Cheng Zhu.

At least 19 recordsLinked to original sources

Evaluating the effects of CD8/CD4 on T cell function in terms of TCR-pMHC-coreceptor catch and slip bonds.

BACKGROUND: T cells recognize antigens through T cell receptor (TCR) interactions with peptide-major histocompatibility complex (pMHC) assisted by the CD4 or CD8 coreceptors depending on the MHC class. Under force, TCR-pMHC interactions form catch or slip bonds whose profiles have been proposed to shape TCR triggering and ligand discrimination, but their predictive value, particularly in the presence of coreceptor engagement, remains incompletely characterized. METHODS: Built upon our recent correlative analysis of TCR-pMHC bimolecular catch bonds with T cell function, we analyzed 27 paired TCR-pMHC datasets measured under conditions that either prevented or permitted CD8 or CD4 co-engagement, drawn from multiple laboratories, cell types and measurement techniques, supplemented with a set of new experiments, to correlate 2D and 3D biophysical parameters with antigen-induced T cell responses. RESULTS: We found that metrics extracted from force-dependent bond lifetime curves (e.g. the optimal force and catch bond intensity) predict T cell activation and thymocyte selection better than force-free parameters measured either in situ at the T cell membrane or in fluid phase using purified ectodomain proteins, both when coreceptor contributions are absent and present. Moreover, CD8 co-engagement with pMHC systematically increases these metrics and improves TCR ligand discrimination, indicating coreceptor-mediated amplification of, or conversion to, catch-bonds. CONCLUSION: Our findings highlight the importance of force in antigen recognition by the TCR and reveal that parameters derived from the bond profile, especially in the presence of coreceptor, are more informative predictors of T cell activation compared to conventional affinity-based measurements. These results offer insights into the relation between catch bonds and CD8 co-engagement in TCR antigen recognition.

Receptors, Antigen, T-Cell↗

Affinity and kinetic analysis of Fcgamma receptor IIIa (CD16a) binding to IgG ligands.

Binding of pathogen-bound immunoglobulin G (IgG) to cell surface Fc gamma receptors (FcgammaRs) triggers a wide variety of effector functions. The binding kinetics and affinities of IgG-FcgammaR interactions are hence important parameters for understanding FcgammaR-mediated immune functions. We have measured the kinetic rates and equilibrium dissociation constants of IgG binding to a soluble FcgammaRIIIa fused with Ig Fc (sCD16a) using the surface plasmon resonance technique. sCD16a interacted with monomeric human IgG and its subtypes IgG1 and IgG3 as well as rabbit IgG with on-rates of 6.5 x 10(3), 8.2 x 10(3), 1.1 x 10(4) and 1.8 x 10(4) m(-1) s(-1), off-rates of 4.7 x 10(-3), 5.7 x 10(-3), 5.9 x 10(-3), and 1.9 x 10(-2) s(-1), and equilibrium dissociation constants of 0.72, 0.71, 0.56, and 1.1 mum, respectively. The kinetics and affinities measured by surface plasmon resonance agreed with those obtained from real time flow cytometry and competition inhibition binding experiments using cell surface CD16a. These data add to our understanding of IgG-FcgammaR interactions.

Animals↗

Expression of Smad ubiquitin regulatory factor 2 (Smurf2) in rhesus monkey endometrium and placenta during early pregnancy.

Smad ubiquitin regulatory factor 2 (Smurf2) is an E3 ubiquitin ligase that is involved in the Smad-mediated TGF-beta signaling. TGF-beta has been shown to play an important role during normal embryo implantation, but whether Smurf2 is involved in this process has not been reported. This study was first conducted to investigate the expression of Smurf2 transcript and protein in different compartments of the rhesus monkey uteri and placenta during early pregnancy. The results showed that both the cloned partial sequence of Smurf2 gene and the corresponding amino acid residues shared 99% identity with those of human homologs. On day 12 (D12) of pregnancy, strong signals of Smurf2 mRNA were found in basalis glandular epithelium and luminal epithelium, and moderate expressions were detected in functionalis glandular epithelium. During early villi stage and villi placental stage, Smurf2 mRNAs were mainly localized in the placenta villi, trophoblastic column, trophoblastic shell, and basalis glandular epithelium. There appeared strong staining signals in the arterioles on D26 of pregnancy, but faint staining signals on D18 of pregnancy. No specific staining of Smurf2 mRNA was observed in stromal cells and myometrium. The expression pattern of Smurf2 protein was generally similar to that of its mRNA. These results provide the first evidence that Smurf2 may play specific roles in glandular secretion, trophoblastic cell invasion, and placentation through mediating the expression of the related proteins of TGF-beta signaling pathway during early pregnancy.

Amino Acid Sequence↗

A structure-based sliding-rebinding mechanism for catch bonds.

Catch bonds, whose lifetimes are prolonged by force, have been observed in selectin-ligand interactions and other systems. Several biophysical models have been proposed to explain this counterintuitive phenomenon, but none was based on the structure of the interacting molecules and the noncovalent interactions at the binding interface. Here we used molecular dynamics simulations to study changes in structure and atomic-level interactions during forced unbinding of P-selectin from P-selectin glycoprotein ligand-1. A mechanistic model for catch bonds was developed based on these observations. In the model, "catch" results from forced opening of an interdomain hinge that tilts the binding interface to allow two sides of the contact to slide against each other. Sliding promotes formation of new interactions and even rebinding to the original state, thereby slowing dissociation and prolonging bond lifetimes. Properties of this sliding-rebinding mechanism were explored using a pseudoatom representation and Monte Carlo simulations. The model has been supported by its ability to fit experimental data and can be related to previously proposed two-pathway models.

Computer Simulation↗

Transport governs flow-enhanced cell tethering through L-selectin at threshold shear.

Flow-enhanced cell adhesion is a counterintuitive phenomenon that has been observed in several biological systems. Flow augments L-selectin-dependent adhesion by increasing the initial tethering of leukocytes to vascular surfaces and by strengthening their subsequent rolling interactions. Tethering or rolling might be influenced by physical factors that affect the formation or dissociation of selectin-ligand bonds. We recently demonstrated that flow enhanced rolling of L-selectin-bearing microspheres or neutrophils on P-selectin glycoprotein ligand-1 by force decreased bond dissociation. Here, we show that flow augmented tethering of these microspheres or cells to P-selectin glycoprotein ligand-1 by three transport mechanisms that increased bond formation: sliding of the sphere bottom on the surface, Brownian motion, and molecular diffusion. These results elucidate the mechanisms for flow-enhanced tethering through L-selectin.

Biological Transport↗

Flow-enhanced adhesion regulated by a selectin interdomain hinge.

L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin-ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.

Cell Adhesion↗

[Liposome-mediated glial growth factor 2 gene therapy in brain injury: an experimental study with rats].

OBJECTIVE: To explore the protective effect of glial growth factor-2 (GGF2) on brain injury. METHODS: Thirty-four SD rats underwent lateral fluid percussion to establish brain injury models and then were randomly divided into 4 groups: treatment group (n = 10, the plasmid pEGFP-N1-GGF2 mixed with liposome was injected into the brain tissue directly), vector control group (n = 10, the vector pEGFP-N1 mixed with liposome was injected into the brain tissue directly), liposome control group (n = 10, liposome was injected), and sham operation group (n = 4). Three assessment tasks were performed for neurobehavioral evaluation: Clivas Test, Beam Balance Test and Beam Walking Test. 10 days after brain injury, the rats were sacrificed and their brains were embedded in paraffin for HE staining, Nissle staining and immunohistochemical examination of MBP, NSE, and GFAP. RESULTS: The Clivas test score of the treatment group was 66.25 +/- 3.54, significantly higher than those of the vector control group and. liposome control group (58.31 +/- 3.72 and 57.21 +/- 3.93 respectively, both P < 0.05). The beam test score of the treatment group was 2.59 +/- 0.21, significantly lower than those the vector control group and liposome control group (3.41 +/- 0.25 and 3.24 +/- 0.22 respectively, both P < 0.05). The walking test score of the treatment group was 20.15 +/- 2.59, significantly lower than those of control group and liposome control group (27.00 +/- 3.47 and 27.80 +/- 3.00 respectively, both P < 0.05). The improvement in beam walking test was the greatest. The neuron number in the external granular layer and external pyramidal layer in cortex of the treatment group was 98 +/- 10, significantly more than those of the vector control group and liposome group (75 +/- 7 and 67 +/- 8, both P < 0.05). The neuron number in the internal pyramidal layer in cortex of the treatment group was 37 +/- 4, significantly more than those of the vector control group and liposome group (19 +/- 3 and 23 +/- 4 respectively, both P < 0.05). The neuron number in the CA1 region in hippocampus of the treatment group was 102 +/- 11, significantly more than those of the vector control group and liposome group (67 +/- 8 and 58 +/- 9 respectively, both P < 0.01). Higher level of immunoreactivity with MBP was also detected in the cortex in the rats of the treatment group. CONCLUSION: Cationic liposome-mediated GGF2 gene therapy effectively promotes the recovery of brain injury.

Animals↗

Accumulation of copper in brown rice and effect of copper on rice growth and grain yield in different rice cultivars.

A pot experiment with 38 commonly cultured rice cultivars showed that the effect of Cu (100 mg kg(-1)) on rice growth, grain yield and accumulation of Cu in brown rice varied greatly with different cultivars. Although the average Cu concentration in brown rice of the 38 cultivars was significantly increased (P<0.01) compared with the control, in none of the cultivars did Cu concentration in brown rice exceed the maximum permissible limit of 10 mg Cu kg(-1). This suggests that rice grown in Cu-contaminated paddy soil (100 mg Cu kg(-1)) will not adversely affect human health through the food chain. Because of the significant negative correlation between grain weight and Cu concentration in brown rice with the soil Cu treatment, screening for cultivars with low Cu accumulation in brown rice and high grain yield for Cu-contaminated areas is feasible. The present research led to the recommendation of three such cultivars: Jiahua, Zhenxian 866, Zhe 733. The average grain yield under Cu treatment (100 mg Cu kg(-1) soil) was significantly (P<0.01) reduced compared with the control. The decreases or increases of grain yields mainly resulted from the combined effects of the panicles per pot, spikelets per panicle and filled spikelets per panicle under the soil Cu treatment. Furthermore, there were significant (r=0.869, P<0.01) positive correlations between the RC (relative changes) of spikelets per panicle and filled spikelets per panicle under the soil Cu treatment.

Copper↗

Expression of prostasin and protease nexin-1 in rhesus monkey (Macaca mulatta) endometrium and placenta during early pregnancy.

Serine proteases have been documented to play key roles in uterine matrix turnover and trophoblastic invasion during implantation. Roles of prostasin serine protease in these processes, however, are currently unclear. The present study was first conducted to investigate the colocalization of prostasin and its cognate serpin, protease nexin-1 (PN-1), in rhesus monkey endometrium and placenta on days 12, 18, and 26 of pregnancy by using in situ hybridization (ISH) and immunohistochemistry. With ISH, expression of prostasin mRNA was intensely localized in the glandular epithelium on days 12 and 18 and in the placental villi, trophoblastic column, trophoblastic shell, and fetal-maternal border on days 18 and 26. With the progress of pregnancy, expression level in the glandular epithelium was significantly decreased, and the accumulation in the placental compartments was further increased. In addition, the stroma and arterioles exhibited modest levels of prostasin signals. However, expression level of PN-1 in these compartments on adjacent sections in the three stages of early pregnancy was weak or below the level of detection. Prostasin protein expression in the endometrium was found to be consistent with the distribution patterns revealed in the ISH experiments. It may be suggested from these results that prostasin is involved in endometrial epithelial morphology establishment, tissue remodeling, and trophoblastic invasion during early pregnancy. The cognate serpin PN-1 was not coordinately expressed along with prostasin, creating a tissue environment favorable for proteolytic activities of prostasin during early pregnancy events.

Amyloid beta-Protein Precursor↗

Catalytic asymmetric assembly of stereodefined propionate units: an enantioselective total synthesis of (-)-pironetin.

Double diastereoselection in alkaloid-catalyzed acyl halide-aldehyde cyclocondensation (AAC) reactions provides a strategy for realizing syn- or anti-selective propionate aldol additions from a common reaction manifold. Matched AAC homologation of enantioenriched aldehydes afford cis-disubstituted beta-lactones as surrogates for syn aldols; the mismatched AAC reactions provide anti-selective aldols in the form of trans-disubstituted 2-oxetanones. The utility of this reaction technology in synthesis activities is exemplified in a catalytic asymmetric total synthesis of (-)-pironetin.

Antineoplastic Agents↗

Role and regulation of nodal/activin receptor-like kinase 7 signaling pathway in the control of ovarian follicular atresia.

Although the role of the TGF beta superfamily members in the regulation of ovarian folliculogenesis has been extensively studied, their involvement in follicular atresia is not well understood. In the present study, we have demonstrated for the first time that Nodal, a member of the TGF beta superfamily, is involved in promoting follicular atresia as evidenced by the following: 1) colocalization of Nodal and its type I receptor Activin receptor-like kinase 7 (ALK7) proteins in the granulosa cells was only observed in atretic antral follicles, whereas they were present in theca cells and granulosa cells of healthy follicles, respectively; 2) addition of recombinant Nodal or overexpression of Nodal by adenoviral infection induced apoptosis of otherwise healthy granulosa cells; 3) constitutively active ALK7 (ALK7-ca) overexpression mimicked the function of Nodal in the induction of granulosa cell apoptosis. Furthermore, overexpression of Nodal or ALK7-ca increased phosphorylation and nuclear translocation of Smad2, decreased X-linked inhibitor of apoptotic proteins (Xiap) expression at both mRNA and protein level and phospho-Akt content, as well as triggered mitochondrial release of death proteins Smac/DIABLO, Omi/HtrA2, and cytochrome c in the granulosa cells. Dominant-negative Smad2 significantly attenuated ALK7-ca-induced down-regulation of Xiap and thus rescued granulosa cells from undergoing apoptosis. In addition, whereas up-regulation of Xiap significantly attenuated ALK7-ca-induced apoptosis, down-regulation of Xiap sensitized granulosa cells to ALK7-ca-induced apoptosis. Furthermore, ALK7-ca-induced apoptosis was significantly attenuated by forced expression of activated Akt, and Akt rescued granulosa cells from undergoing apoptosis via proteasome-mediated ALK7 degradation. Taken together, Nodal plays an atretogenic role in the ovary where it induces granulosa cell apoptosis through activation of Smad2, down-regulation of the key survival molecules Xiap and phospho-Akt, as well as the activation of mitochondrial death pathway.

Activin Receptors, Type I↗

Probabilistic modeling of rosette formation.

Rosetting, or forming a cell aggregate between a single target nucleated cell and a number of red blood cells (RBCs), is a simple assay for cell adhesion mediated by specific receptor-ligand interaction. For example, rosette formation between sheep RBC and human lymphocytes has been used to differentiate T cells from B cells. Rosetting assay is commonly used to determine the interaction of Fc gamma-receptors (FcgammaR) expressed on inflammatory cells and IgG coated on RBCs. Despite its wide use in measuring cell adhesion, the biophysical parameters of rosette formation have not been well characterized. Here we developed a probabilistic model to describe the distribution of rosette sizes, which is Poissonian. The average rosette size is predicted to be proportional to the apparent two-dimensional binding affinity of the interacting receptor-ligand pair and their site densities. The model has been supported by experiments of rosettes mediated by four molecular interactions: FcgammaRIII interacting with IgG, T cell receptor and coreceptor CD8 interacting with antigen peptide presented by major histocompatibility molecule, P-selectin interacting with P-selectin glycoprotein ligand 1 (PSGL-1), and L-selectin interacting with PSGL-1. The latter two are structurally similar and are different from the former two. Fitting the model to data enabled us to evaluate the apparent effective two-dimensional binding affinity of the interacting molecular pairs: 7.19x10(-5) microm4 for FcgammaRIII-IgG interaction, 4.66x10(-3) microm4 for P-selectin-PSGL-1 interaction, and 0.94x10(-3) microm4 for L-selectin-PSGL-1 interaction. These results elucidate the biophysical mechanism of rosette formation and enable it to become a semiquantitative assay that relates the rosette size to the effective affinity for receptor-ligand binding.

Animals↗

Effects of E-cadherin on mouse embryo implantation and expression of matrix metalloproteinase-2 and -9.

E-cadherin is a cell surface glycoprotein, which is responsible for adhesion between epithelial cells. Whether it is involved in embryo implantation is still unknown. In a mouse intrauterine horn injection model, one uterine horn in each mouse was injected with different doses of E-cadherin antibody on day 3 of pregnancy. The results showed that embryo implantation was significantly inhibited in the mice injected with 3 microg E-cadherin antibody. The mouse uteri in this group were collected on days 5, 6, and 7 of pregnancy and expressions of MMP-2 and -9 were studied. In situ hybridization and RT-PCR results showed that the expression of MMP-2 and -9 mRNAs in uteri of E-cadherin antibody treated group was increased on days 5-7. The results of gelatin zymography of MMPs showed that the activities of pro-MMP-2, MMP-2, and pro-MMP-9 were increased significantly on days 5 and 6, and pro-MMP-9 activity was increased on day 7. The present study suggested that E-cadherin was involved in embryo implantation through decreasing the expressions and activities of MMP-2 and -9.

Animals↗

Gene expression of transforming growth factor-beta receptors types I and II in rat endometrium during the estrous cycle and early pregnancy.

Roles of transforming growth factor-beta (TGF-beta) receptor types I (TbetaRI) and II (TbetaRII) during the estrous cycle and implantation of rodents are currently unclear. In the present study, the spatial and temporal expressions of TbetaRI and TbetaRII in rat endometrium during the estrous cycle, pre-, and peri-implantation were examined using in situ hybridization and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). With in situ hybridization, TbetaRI and TbetaRII were expressed at weak levels in rat endometrium during the estrous cycle. During pre-implantation, both receptors were expressed in the luminal epithelium and glandular epithelium on Days 0.5 and 1.5 of pregnancy, but were down-regulated on Days 2.5 and 3.5. During peri-implantation, both TGF-beta receptors were localized in the luminal epithelium and subepithelial stroma to facilitate attachment reaction and trophoblast invasion. They were highly expressed on Day 4.5, whereas were down-regulated on Days 5.5 and 6.5. Semi-quantitative RT-PCR analysis confirmed the data obtained by in situ hybridization. These results suggest that during pre-implantation, both TGF-beta receptors are functional in the proliferation of endometrial epithelial cells. During peri-implantation, both TGF-beta receptors play important roles during the onset of the uterine receptivity and the attachment reaction. TGF-beta signaling is down-regulated when trophoblast invasion begins.

Activin Receptors, Type I↗

Involvement of SMAD4, but not of SMAD2, in transforming growth factor-beta1-induced trophoblast expression of matrix metalloproteinase-2.

Matrix metalloproteinases (MMPs) play crucial roles in extravillous trophoblast invasion. In the present study, we examined the possible role of Smad4 and Smad2 in transforming growth factor (TGF)-beta1-induced MMP-2 expression, using the well-established invasive extravillous trophoblast cell line HTR-8/SVneo. Recombinant sense Smad4 or Smad2 retroviral vectors were constructed by inserting full-length Smad4 or Smad2 cDNA into pLXSN retroviral vector. Stable PT67 packaging cell clones were isolated and viral supernatants were used to infect HTR-8/SVneo cells. Effects of retroviral expression of Smad4 and Smad2 on TGF-beta1-regulated MMP-2 expression were assessed by semi-quantitative reverse transcription-polymerase chain reaction and gelatin zymography. The results showed that over-expression of Smad4 augmented MMP-2 mRNA abundance and the secretion of pro-MMP-2, and mimicked the inductive effect of TGF-beta1 on the production of MMP-2. However, retrovirus-mediated sense Smad2 gene transfer had no effect. These findings suggest that Smad4, but not Smad2, mediates TGF-beta1-induced MMP-2 expression in invasive extravillous trophoblasts.

Blotting, Western↗

Proteasome subunit LMP2 is required for matrix metalloproteinase-2 and -9 expression and activities in human invasive extravillous trophoblast cell line.

The ubiquitin-proteasome pathway (UPP) is involved in the degradation of the extracellular matrix (ECM) and trophoblastic invasion during early pregnancy. Our previous studies demonstrated that inhibition of UPP suppresses expression of matrix metalloproteinase (MMP)-2 and -9. LMP2 is an important proteasome subunit that is critical for proteasome activity. This study investigated the regulatory mechanism of LMP2 on the expression and activities of MMP-2 and MMP-9. Our results showed that transfection of LMP2 siRNA plasmid into the human invasive extravillous trophoblast cell line (HTR8/Svneo) could significantly suppress expression of LMP2 mRNA and protein. The mRNA expression of MMP-2 and MMP-9 and their activities were markedly decreased in the LMP2-inhibited cells. Inhibition of LMP2 could also reduce IkappaBalpha mRNA level, although the expression of phosphorylated IkappaBalpha was increased. In the LMP2-inhibited cells, expression of mRNA encoding NF-kappaB subunits p50 and p65 remained normal, but the p50 protein level was significantly decreased in the cytosolic and nuclear extracts, while p65 protein was markedly reduced only in the nuclear extract. We also demonstrated that blockage of the NF-kappaB pathway by the NF-kappaB translocation inhibitor SN50 markedly reduced the expression of MMP-2 and MMP-9 in HTR8/Svneo cells, a result that is fully consistent with the results from the LMP2-inhibited HTR8/Svneo cells. These data suggest that LMP2 contributes to IkappaBalpha degradation and p50 generation, and that inhibition of LMP2 suppresses expression and activities of MMP-2 and MMP-9 by blocking the transfer of active NF-kappaB heterodimers into the nucleus.

Cell Line↗