GABAergic disinhibition facilitates polysynaptic excitatory transmission in rat anterior cingulate cortex

Various studies implicate the anterior cingulate cortex (ACC) in processing pain. Combining whole-cell patch clamp recordings in rat ACC slices and a formalin-induced conditioned place avoidance (F-CPA) behavioral model, the present study was to address the effect of GABA(A) receptors on excitatory transmission to ACC layer V neurons and its possible functional significance related to pain. Removal of GABA(A) inhibition by bicuculline (10 microM) induced a novel long-lasting response in layer V neurons, which could be blocked by high divalent extracellular solution and was sensitive to relatively higher rate stimuli. Co-application of NMDA receptor antagonist APV (50 microM) and non-NMDA receptor antagonist DNQX (10 microM) completely blocked the responses. Enhancement of inhibition by intra-ACC microinjection of muscimol abolished the acquisition of F-CPA without affecting formalin-induced acute nociceptive responses. These results suggest that GABA(A) inhibition may be involved in pain-related aversion by modulating glutamate-mediated excitatory transmission in the ACC.     

Matrix metalloproteinase 11 depletion inhibits cell proliferation in gastric cancer cells

Our previous study has shown that matrix metalloproteinase 11 (MMP11) is highly expressed in tumor cell lines and primary tumor of gastric cancer (GC). In order to reveal the correlation between expression of MMP11 and biological features of GC cell, we have constructed the recombinant plasmids producing hairpin small interfering RNA (siRNA) to target MMP11 mRNA using a vector-based RNA interference technology. Stable transfection of recombinants into GC cell line BGC823 specifically depleted the mRNA and protein of MMP11 as demonstrated by RT-PCR and Western blotting analysis. The siRNA-treated cells exhibited significantly decreased growth ability compared with mock transfectants and parental BGC823 cells. Furthermore, colony formation of MMP11 deficient cells was dramatically inhibited in soft agar and tumorigenicity was reduced in nude mice, respectively. These results provide new insights into the function of MMP11 and suggest that MMP11 may play an important role in the control of cell proliferation and tumor development in GC.     

Two-dimensional Cu and first Pb coordination polymers based on a flexible 1,4-cyclohexanedicarboxylate ligand displaying different conformations and coordination modes

Two novel metal-organic hybrid coordination polymers {[Cu(bpo)(chdc)(H₂O)](H₂O)_0.5}_n (1) and [Pb(chdc)(H₂O)]_n (2) have been synthesized under different conditions and structurally characterized by single-crystal X-ray diffraction technique, where H₂chdc refers to a flexible 1,4-cyclohexanedicarboxylic acid ligand and bpo is 2,5-bis(4-pyridyl)-1,3,4-oxadiazole. Complex 1 has a two-dimensional (2-D) grid-like [11.28 × 13.63 Å²] framework in which the Cu^II centers are extended via bidentate bridging ligands bpo and e,e-trans-chdc along two directions, exhibiting large porous cavities. Coordination polymer 2 represents the first Pb^II complex of H₂chdc in which the larger Pb^II centers are connected by e,a-cis-chdc anions to afford a 2-D close-knit structure.     

Selecting cases from nuclear families for case-control association analysis

We examine the efficiency of a number of schemes to select cases from nuclear families for case-control association analysis using the Genetic Analysis Workshop 14 simulated dataset. We show that with this simulated dataset comparing all affected siblings with unrelated controls is considerably more powerful than all of the other approaches considered. We find that the test statistic is increased by almost 3-fold compared to the next best sampling schemes of selecting all affected sibs only from families with affected parents (AF aff), one affected sib with most evidence of allele-sharing from each family (SF), and all affected sibs from families with evidence for linkage (AF L). We consider accounting for biological relatedness of samples in the association analysis to maintain the correct type I error. We also discuss the relative efficiencies of increasing the ratio of unrelated cases to controls, methods to confirm associations and issues to consider when applying our conclusions to other complex disease datasets.     

Modeling and simulation of the swelling behavior of pH-stimulus-responsive hydrogels

The modulation of the swelling ability of the hydrogel matrix by pH-stimulus enables the dynamic control of the swelling forces, thereby obtaining effective diffusivity and permeability of the solutes, or mechanical energy from the hydrogel. In this work, a chemo-electro-mechanical model describing hydrogel behavior, based on multi-field effects, is developed to simulate the swelling and shrinking of these fascinating bio-materials, and it is termed the multi-effect-coupling pH-stimulus (MECpH) model. This model accounts for the ionic fluxes within both the hydrogel and solution, the coupling between the electric field, ionic fluxes, and mechanical deformations of the hydrogel. The main contribution of this model is to incorporate the relationship between the concentrations of the ionized fixed-charge groups and the diffusive hydrogen ion, which follows a Langmuir isotherm, into the Poisson-Nernst-Planck system. To validate this MECpH model, one-dimensional steady-state simulations under varying pH solution are carried out via a meshless Hermite-Cloud methodology, and the numerical results are compared with available experimental data. It is shown that the presently developed MECpH model is accurate, efficient, and numerically stable.     

Proteome analysis of antiproliferative mechanism of 12-O-tetradecanoylphorbol 13-acetate on cultured nasopharyngeal carcinoma CNE2 cells

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is a plant derivative with multiple function as tumor promoter, differentiation revulsant or leukemia therapy drug. The molecular mechanism of its function is perplexing. Many studies have focused on the mechanism of TPA stimulation in tumor promotion of mouse models or terminal differentiation of leukemia cells, but the effect of TPA on nasopharyngeal carcinoma (NPC) remains unclear, while TPA was considered to be associated with NPC development. In the present study, we employed proteomics techniques to study protein changes of a poorly differentiated squamous carcinoma cell line-CNE2 of human NPCs cells induced by TPA. Six significantly and reproducibly changed proteins were identified and their functional implications were discussed in some details.     

An improved strategy for high-level production of human vasostatin120-180

We previously reported a strategy for expression and purification of human Vasostatin120-180 (VAS), a potent angiogenesis inhibitor in a GST fusion form; however, the yield of 7.2 mg per liter of culture was relatively low. The aim of this study was to develop a more efficient system to improve and facilitate the production of VAS protein in a soluble and native form in Escherichia coli. The VAS gene with optimized condons was cloned into pET28a and overexpressed as a N-terminal His-tagged fusion protein. Between His-tag and VAS, an enterokinase recognition site was introduced to release the intact VAS. Optimal expression of soluble His-VAS was achieved by examining the contribution of chaperone coexpression and lower temperature fermentation. Ammonium sulfate precipitation was first employed to remove nucleic acid and partial host proteins. When further purified by Ni2+ affinity chromatography, 40 mg of His-VAS was isolated with purity over 85% from 1 L of culture. After desalting with Sephadex G15 and digestion with His-EK, followed by the removal of the His-tag and His-EK with Ni(2+)-NTA resin, 21 mg of intact VAS was finally obtained from 1 L of bacterial culture, which was approximately 3-fold the yield we previously obtained via GST fusion expression strategy. The identity of His-VAS and VAS was confirmed by Western blot. Purified VAS displayed distinct anti-angiogenic activity, which was shown by the endothelial cell proliferation inhibition assay and chicken chorioallantoic membrane assay. In sum, we greatly improved the yield of intact and bioactive VAS protein, and using this successful example, we propose a more efficient system for the high-level production of intact functional proteins, especially for low molecule weight peptides.     

Crystal structures of HIV-1 Tat-derived nonapeptides Tat-(1-9) and Trp2-Tat-(1-9) bound to the active site of dipeptidyl-peptidase IV (CD26)

CD26 or dipeptidyl-peptidase IV (DPPIV) is engaged in immune functions by co-stimulatory effects on activation and proliferation of T lymphocytes, binding to adenosine deaminase, and regulation of various chemokines and cytokines. DPPIV peptidase activity is inhibited by both Tat protein from human immunodeficiency virus (HIV)-1 and its N-terminal nonapeptide Tat-(1-9) with amino acid sequence MDPVDPNIE, suggesting that DPPIV mediates immunosuppressive effects of Tat protein. The 2.0- and 3.15-A resolution crystal structures of the binary complex between human DPPIV and nonapeptide Tat-(1-9) and the ternary complex between the variant MWPVDPNIE, called Trp(2)-Tat-(1-9), and DPPIV bound to adenosine deaminase show that Tat-(1-9) and Trp(2)-Tat-(1-9) are located in the active site of DPPIV. The interaction pattern of DPPIV with Trp(2)-Tat-(1-9) is tighter than that with Tat-(1-9), in agreement with inhibition constants (K(i)) of 2 x 10(-6) and 250 x 10(-6) m, respectively. Both peptides cannot be cleaved by DPPIV because the binding pockets of the N-terminal 2 residues are interchanged compared with natural substrates: the N-terminal methionine occupies the hydrophobic S1 pocket of DPPIV that normally accounts for substrate specificity by binding the penultimate residue. Because the N-terminal sequence of the thromboxane A2 receptor resembles the Trp(2)-Tat-(1-9) peptide, a possible interaction with DPPIV is postulated.     

Regulation of cystic fibrosis transmembrane regulator trafficking and protein expression by a Rho family small GTPase TC10

The cystic fibrosis transmembrane conductance regulator (CFTR)-interacting protein, CFTR-associated ligand (CAL) down-regulates total and cell surface CFTR by targeting CFTR for degradation in the lysosome. Here, we report that a Rho family small GTPase TC10 interacts with CAL. This interaction specifically up-regulates CFTR protein expression. Co-expression of the constitutively active form, TC10Q75L, increases total and cell surface CFTR in a dose-dependent fashion. Moreover, co-expression of the dominant-negative mutant TC10T31N causes a dose-dependent reduction in mature CFTR. The effect of TC10 is independent of the level of CFTR expression, because a similar effect was observed in a stable cell line that expresses one-tenth of CFTR. Co-expression of TC10Q75L did not have a similar effect on the expression of plasma membrane proteins such as Frizzled-3 and Pr-cadherin or cytosolic proteins such as tubulin and green fluorescent protein. TC10Q75L also did not have a similar effect on the vesicular stomatitis virus glycoprotein. Co-expression of constitutively active and dominant-negative forms of Cdc42 or RhoA did not affect CFTR expression in a manner similar to TC10, indicating that the effect of TC10 is unique within the Rho family. Metabolic pulse-chase experiments show that TC10 did not affect CFTR maturation, suggesting that it exerts its effects on the mature CFTR. Importantly, TC10Q75L reverses CAL-mediated CFTR degradation, suggesting that TC10Q75L inhibits CAL-mediated degradation of CFTR. TC10Q75L does not operate by reducing CAL protein expression or its ability to form dimers or interact with CFTR. Interestingly, the expression of TC10Q75L causes a dramatic redistribution of CAL from the juxtanuclear region to the plasma membrane where the two molecules overlap. These data suggest that TC10 regulates both total and plasma membrane CFTR expression by interacting with CAL. The GTP-bound form of TC10 directs the trafficking of CFTR from the juxtanuclear region to the secretory pathway toward the plasma membrane, away from CAL-mediated degradation of CFTR in the lysosome.