Cell-based biosensors based on light-addressable potentiometric sensors for single cell monitoring

Cell-based biosensors incorporate cells as sensing elements that convert changes in immediate environment to signals for processing. This paper reports an investigation on light-addressable potentiometric sensor (LAPS) to be used as a possible cell-base biosensor that will enable us to monitor extracellular action potential of single living cell under stimulant. In order to modify chip surface and immobilize cells, we coat a layer of poly-L-ornithine and laminin on surface of LAPS chip on which rat cortical cells are grown well. When 10 microg/ml acetylcholine solution is administrated, the light pointer is focused on a single neuronal cell and the extracellular action potential of the targeted cell is recorded with cell-based biosensor based on LAPS. The results demonstrate that this kind of biosensor has potential to monitor electrophysiology of living cell non-invasive for a long term, and to evaluate drugs primarily.     

Synthesis of linked berberine dimers and their remarkably enhanced DNA-binding affinities

This communication describes the facile synthesis of five novel berberine dimers and their strong affinities toward double-stranded DNA. These berberine dimers were synthesized in 37-84% yields from the reaction of berberrubine with dihaloalkanes of varying lengths, and fully characterized by HRMS and 1H NMR. Compared with the monomeric parent berberine, these dimers showed greatly enhanced binding affinities up to approximately 100-fold, with two double helical oligodeoxynucleotides, d(AAGAATTCTT)2 and d(TAAGAATTCTTA)2, which was investigated by means of fluorescence spectrometry.     

Synthesis and bioactivities of two multiple antigen peptides as potential vaccine against schistosoma

Based on the two antigenic peptides, 26-43 (P26) and 116-131 (P116), derived from 28 kDa glutathione S-transferase of Schistosoma mansoni (Sm28GST), two multiple antigenic peptides (MAPs), (P26)4-MAP and (P116)4-MAP with the same oligomeric lysine core, were synthesized by stepwise solid-phase peptide synthesis method. The antigenicities and protective effects of these two MAPs were examined on experimental animals. As shown in the dot-ELISA result, the synthetic MAPs could be recognized and bound by immunoglobins in both patient's and infected-rabbit's sera. After Kunming mice were immunized with (P26)4-MAP, the worm burden reduction rate and the liver egg reduction rate were 59.9% and 61.1%. In (P26)4-MAP or (P116)4-MAP immunized BALB/c mice, the worm burden reduction rates were 37.5% and 62.5%, respectively, and the liver egg reduction rates were 35.1% and 54.0%, respectively.     

Hydrogen sulfide may improve the hippocampal damage induced by recurrent febrile seizures in rats

The aim of the present study was to investigate the possible role of hydrogen sulfide (H(2)S) in the pathogenesis of recurrent febrile seizures (FS) in rats. On a rat model of recurrent FS, the ultrastructure of hippocampal neurons, the plasma level of H(2)S, the expressions of cystathionine b-synthase (CBS) and c-fos, and the development of mossy fiber sprouting (MFS) in hippocampus were examined after treatment with NaHS, a donor of H(2)S, or hydroxylamine (HA), an inhibitor of CBS. We found that the plasma level of H(2)S increased significantly, the expressions of CBS and c-fos increased markedly, and MFS was evident in hippocampus in FS group. NaHS alleviated the neuronal damage of recurrent FS rats, decreased the expression of c-fos, and inhibited MFS obviously. HA aggravated the neuronal damage of recurrent FS rats, further increased the expression of c-fos, and enhanced the mossy fiber outgrowth. The results showed that endogenous H(2)S system was involved in the development of FS. Exogenous H(2)S may exert beneficial effect on the pathogenesis of FS-related brain damage.     

3D coordination polymers of [2.2]paracyclophane and in situ silver(I) perfluoro-dicarboxylates: effects of the dicarboxylate spacers and conformations on the formation of complexes

Coordination polymers of [2.2]paracyclophane (pcp) with in situ silver(I) perfluoro-dicarboxylates characterized by single crystal X-ray analysis are described. Structures are found to strongly depend on the dicarboxylate spacer (n). With disilver(I) tetrafluorosuccinate ((CF₂)_n(COOAg)₂, n = 2), 3D network with composition of [Ag₄(pcp)(C₂F₄(CO₂)₂)₂] (1) forms in which silver salts afford infinite double chains and pcp act as linkages between chains. Changing the silver salt to disilver hexafluoroglutarate ((CF₂)_n(COOAg)₂, n = 3) produces 3D pillared-layer structure of composition of [Ag₄(pcp)(C₃F₆(CO₂)₂)₂] · THF (2) (THF = tetrahydrofuran), in which silver salts form 2D sheets and pcp act as pillars between the sheets. With silver octafluoroadipate (HO₂C(CF₂)_nCO₂Ag, n = 4), 2-fold interpenetrated diamond structure, [Ag₂(pcp)₂(HO₂CC₄F₈CO₂)₂]₂ · 2toluene (3), is obtained in which silver-anion chains and silver-pcp chains are connected with each other in the perpendicular manner. The three complexes represent unprecedented metal-organic networks of silver(I) multicarboxylates and polycyclic aromatic compounds. Additionally, the effects of the dicarboxylate conformations as well as the solvents on the resulting structures were discussed.     

Relaxin's induction of metalloproteinases is associated with the loss of collagen and glycosaminoglycans in synovial joint fibrocartilaginous explants

Diseases of specific fibrocartilaginous joints are especially common in women of reproductive age, suggesting that female hormones contribute to their etiopathogenesis. Previously, we showed that relaxin dose-dependently induces matrix metalloproteinase (MMP) expression in isolated joint fibrocartilaginous cells. Here we determined the effects of relaxin with or without beta-estradiol on the modulation of MMPs in joint fibrocartilaginous explants, and assessed the contribution of these proteinases to the loss of collagen and glycosaminoglycan (GAG) in this tissue. Fibrocartilaginous discs from temporomandibular joints of female rabbits were cultured in medium alone or in medium containing relaxin (0.1 ng/ml) or beta-estradiol (20 ng/ml) or relaxin plus beta-estradiol. Additional experiments were done in the presence of the MMP inhibitor GM6001 or its control analog. After 48 hours of culture, the medium was assayed for MMPs and the discs were analyzed for collagen and GAG concentrations. Relaxin and beta-estradiol plus relaxin induced the MMPs collagenase-1 and stromelysin-1 in fibrocartilaginous explants--a finding similar to that which we observed in pubic symphysis fibrocartilage, but not in articular cartilage explants. The induction of these proteinases by relaxin or beta-estradiol plus relaxin was accompanied by a loss of GAGs and collagen in joint fibrocartilage. None of the hormone treatments altered the synthesis of GAGs, suggesting that the loss of this matrix molecule probably resulted from increased matrix degradation. Indeed, fibrocartilaginous explants cultured in the presence of GM6001 showed an inhibition of relaxin-induced and beta-estradiol plus relaxin-induced collagenase and stromelysin activities to control baseline levels that were accompanied by the maintenance of collagen or GAG content at control levels. These findings show for the first time that relaxin has degradative effects on non-reproductive synovial joint fibrocartilaginous tissue and provide evidence for a link between relaxin, MMPs, and matrix degradation.     

The HNF-1 target collectrin controls insulin exocytosis by SNARE complex formation

Defective glucose-stimulated insulin secretion is the main cause of hyperglycemia in type 2 diabetes mellitus. Mutations in HNF-1 cause a monogenic form of type 2 diabetes, maturity-onset diabetes of the young (MODY), characterized by impaired insulin secretion. Here we report that collectrin, a recently cloned kidney-specific gene of unknown function, is a target of HNF-1 in pancreatic β cells. Expression of collectrin was decreased in the islets of HNF-1 (−/−) mice, but was increased in obese hyperglycemic mice. Overexpression of collectrin in rat insulinoma INS-1 cells or in the β cells of transgenic mice enhanced glucose-stimulated insulin exocytosis, without affecting Ca²⁺ influx. Conversely, suppression of collectrin attenuated insulin secretion. Collectrin bound to SNARE complexes by interacting with snapin, a SNAP-25 binding protein, and facilitated SNARE complex formation. Therefore, collectrin is a regulator of SNARE complex function, which thereby controls insulin exocytosis.     

An extended aqueous solvation model based on atom-weighted solvent accessible surface areas: SAWSA v2.0 model

A new method is proposed for calculating aqueous solvation free energy based on atom-weighted solvent accessible surface areas. The method, SAWSA v2.0, gives the aqueous solvation free energy by summing the contributions of component atoms and a correction factor. We applied two different sets of atom typing rules and fitting processes for small organic molecules and proteins, respectively. For small organic molecules, the model classified the atoms in organic molecules into 65 basic types and additionally. For small organic molecules we proposed a correction factor of hydrophobic carbon to account for the aggregation of hydrocarbons and compounds with long hydrophobic aliphatic chains. The contributions for each atom type and correction factor were derived by multivariate regression analysis of 379 neutral molecules and 39 ions with known experimental aqueous solvation free energies. Based on the new atom typing rules, the correlation coefficient (r) for fitting the whole neutral organic molecules is 0.984, and the absolute mean error is 0.40 kcal mol^–1, which is much better than those of the model proposed by Wang et al. and the SAWSA model previously proposed by us. Furthermore, the SAWSA v2.0 model was compared with the simple atom-additive model based on the number of atom types (NA). The calculated results show that for small organic molecules, the predictions from the SAWSA v2.0 model are slightly better than those from the atom-additive model based on NA. However, for macromolecules such as proteins, due to the connection between their molecular conformation and their molecular surface area, the atom-additive model based on the number of atom types has little predictive power. In order to investigate the predictive power of our model, a systematic comparison was performed on seven solvation models including SAWSA v2.0, GB/SA_1, GB/SA_2, PB/SA_1, PB/SA_2, AM1/SM5.2R and SM5.0R. The results showed that for organic molecules the SAWSA v2.0 model is better than the other six solvation models. For proteins, the model classified the atoms into 20 basic types and the predicted aqueous free energies of solvation by PB/SA were used for fitting. The solvation model based on the new parameters was employed to predict the solvation free energies of 38 proteins. The predicted values from our model were in good agreement with those from the PB/SA model and were much better than those given by the other four models developed for proteins.Figure The definition of hydrophobic carbons. Here CA, CB and CD are three carbon atoms; X represents a heteroatom. According to our definition, CB is a hydrophobic carbon, CA is not a hydrophobic carbon because a heteroatom is within four atoms and CD is not a hydrophobic carbon because CD is sp²- hydridized and in a six-member ring.Electronic Supplementary Material Supplementary material is available for this article at     

New class of competitive inhibitor of bacterial histidine kinases

Bacterial histidine kinases have been proposed as targets for the discovery of new antibiotics, yet few specific inhibitors of bacterial histidine kinases have been reported. We report here a novel thienopyridine (TEP) compound that inhibits bacterial histidine kinases competitively with respect to ATP but does not comparably inhibit mammalian serine/threonine kinases. Although it partitions into membranes and does not inhibit the growth of bacterial or mammalian cells, TEP could serve as a starting compound for a new class of histidine kinase inhibitors with antibacterial activity.     

Roles of 3-phosphoinositide-dependent kinase 1 in the regulation of endothelial nitric-oxide synthase phosphorylation and function by heat shock protein 90

The 90-kDa heat shock protein (Hsp90) plays an important role in endothelial nitric-oxide synthase (eNOS) regulation. Besides acting as an allosteric enhancer, Hsp90 was shown to serve as a module recruiting Akt to phosphorylate the serine 1179/1177 (bovine/human) residue of eNOS. Akt is activated by the phosphorylation of 3-phosphoinositide-dependent kinase 1 (PDK1). Whether PDK1 is involved in the actions of Hsp90 on eNOS phosphorylation and function remains unknown. To address this issue, we treated bovine eNOS stably transfected human embryonic kidney 293 cells with Hsp90 inhibitors and determined the alterations of phospho-eNOS, Akt, and PDK1. Both geldanamycin and radicicol, two structurally different Hsp90 inhibitors, selectively reduced serine 1179-phosphorylated eNOS, leading to decreased enzyme activity. In Hsp90-inhibited cells, eNOS-associated phospho-Akt was decreased, but the total amount of Akt associated with eNOS remained the same. Further studies showed that Hsp90 inhibition dramatically depleted intracellular PDK1. Proteasome but not caspase blockade prevented the loss of PDK1 caused by Hsp90 inhibition. Silencing the PDK1 gene by small interfering RNA was sufficient to induce reduction of phospho-Akt and consequent loss of serine 1179-phosphorylated eNOS. Moreover, overexpression of PDK1, but not Akt, reversed Hsp90 inhibition-induced loss of eNOS serine 1179 phosphorylation and salvaged enzymatic activity. Thus, in addition to functioning as a module to recruit Akt to eNOS, Hsp90 also critically stabilized PDK1 by preventing it from proteasomal degradation. Inhibition of Hsp90 function resulted in PDK1 depletion and thus triggered a cascade of Akt deactivation, loss of eNOS serine 1179 phosphorylation, and decrease of enzyme function.