Endothelin receptors in gastrointestinal smooth muscle

Endothelins (ETs) are a family of peptides with 21-amino-acid residues. ET-1 was identified as a potent vasoconstrictor produced by vascular endothelial cells. Three distinct isoforms of ET, i.e. ET-1, ET-2 and ET-3, have been found to exist in a variety of tissues. ET was later found to cause contraction as well as relaxation of smooth muscle in many physiologic systems. In the gastrointestinal tract, ET causes contraction and/or relaxation of the esophagus, stomach, ileum and colon. In the hepatobiliary system, ET causes contraction of the portal vein, hepatic stellate cells, gallbladder and common bile duct. In mammalian species, two classes of ET receptors, ET(A) and ET(B), have been cloned. ET(A) receptors have higher affinities for ET-1 and ET-2 than ET-3, while ET(B) receptors have the same affinities for ET-1, ET-2 and ET-3. In the gastrointestinal system, ET causes smooth muscle contraction through interaction with ET(A) receptors, ET(B) receptors or both ET(A) and ET(B) receptors, depending on the tissues and species. In addition to contraction, ET causes smooth muscle relaxation through interaction with ET(A) receptors or ET(B) receptors. At the present time, there are no studies showing that ET causes smooth muscle relaxation through interaction with both ET(A) and ET(B) subtypes. ET induces contraction in most of the non-sphincter muscle except the fundus of the stomach. On the other hand, ET causes relaxation and contraction in the lower esophageal and internal anal sphincters. ET may play an important role in the control of human gastrointestinal motility and portal vein pressure.     

The phenomenon of cell membrane tensile stress accumulation and its effect on endothelin-1 secretion by vascular endothelial cells

Theoretical analysis has shown that that the tensile stress in the upper cell membrane of the vascular endothelium could accumulate upstream to a very high level despite of the identical shear environments. This phenomenon is called cell membrane tension accumulation (CMTA). To verify the theoretical analysis, the secretion of endothelin-1 (ET-1) by a paired human umbilical vein segments with different lengths (10 and 15 cm, respectively) were measured. The results clearly showed highly significant differences in the secretion rates of ET-1 between the 10 cm-long vein (segment A) and the 15 cm-long vein (segment B) under the same shear stress level of 0.48 N/m². When exposed to a shear stress of 0.48 N/m² for 24 h, segment B secreted ET-1 at an average rate of 34.9154±0.9830 pg/cm² h, almost 14% higher than the average rate of 30.6274±0.4912 pg/cm² h recorded by segment A (P<0.01). The present study, therefore, confirms that CMTA does in fact occur in the blood vessel. This phenomenon affects the secretion of ET-1 by vascular endothelial cells, and may be more important than shear stress in its effect on the metabolism and biological function of endothelial cells.     

A functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells

Overexpression of HER2 correlates with more aggressive tumors and increased resistance to cancer chemotherapy. However, a functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on tumor metastasis has not been conducted. Herein we examined the regulation mechanism of heregulin- β1 (HRG)-induced MMP-1 and -9 expression in breast cancer cell lines. Our results showed that the basal levels of MMP-1 and -9 mRNA and protein expression were increased by HRG treatment. In addition, HRG-induced MMP-1 and -9 expression was significantly decreased by MEK1/2 inhibitor, U0126 but not by phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002. To confirm the role of MEK/ERK pathway on HRG-induced MMP-1 and -9 expression, MCF7 cells were transfected with constitutively active adenoviral- MEK (CA-MEK). The level of MMP-1 and -9 expressions was increased by CA-MEK. MMP-1 and -9 mRNA and protein expressions in response to HRG were higher in HER2 overexpressed cells than in vector alone. The phosphorylation of HER2, HER3, ERK, Akt, and JNK were also significantly increased in HER2 overexpressed MCF7 cells compared with vector alone. HRG-induced MMP-1 and -9 expressions were significantly decreased by lapatinib, which inhibits HER1 and HER2 activity, in both vector alone and HER2 overexpressed MCF7 cells. Finally, HRG-induced MMP-1 and MMP-9 expression was decreased by HER3 siRNA overexpression. Taken together, we suggested that HRG-induced MMP-1 and MMP-9 expression is mediated through HER3 dependent pathway and highly expressed HER2 may be associated with more aggressive metastasis than the low expressed HER2 in breast cancer cells.     

Endothelin-1 and angiotensin II secretion at different lengths of endothelial cell monolayer in the view of tensile stress accumulation in the upper endothelial cell membrane

Theoretical analysis and experimental observations have shown that tensile stress inside an endothelial cell membrane is capable of growing in the direction opposite to blood flow and can accumulate to a level that is three or more orders of magnitude higher than flow-induced shear stress on the membrane surface. This phenomenon is called cell membrane tension accumulation (CMTA). We hypothesize that correlation may exist between the endothelial cell monolayer length or CMTA and secretory function of endothelial cells. To verify this hypothesis, a paired experimental study was devised to measure the secretion of endothelin (ET-1) and angiotensin II (Ang II) by two monolayers of cultured human glomerular vascular endothelial cell (HGVEC) monolayers subjected an identical steady shear stress. After replicate cultured HGVEC monolayer with two kinds of length of 6 cm and 10 cm were subjected to the same steady laminar shear stress of 0.45 N/m² for 24 h, the average secretion rates of ET-1 and Ang II in 6 cm long increased l.7- and 0.5-fold (n=26, P<0.00l) over 10 cm long, respectively. Over 10 h of exposure to 0.65 N/m², the average secretion rate of both ET-1 and Ang II by HGVEC monolayer of 6 cm in length exceeded 0.5-fold (n=26, P<0.0001) over 10 cm in length. All these demonstrated that the close relationship may exist between length of endothelial cell monolayer and secretion of ET-1 and Ang II by endothelial cells, indicating the possible existence of the cumulative effect of the tensile stress in the upper endothelial cell membrane under the shear flow field.     

Platelet-rich plasma inhibits the apoptosis of highly adipogenic homogeneous preadipocytes in an in vitro culture system

Auto-transplantation of adipose tissue is commonly used for the treatment of tissue defects in plastic surgery. The survival of the transplanted adipose tissue is not always constant, and one of reasons is the accelerated apoptosis of the implanted preadipocytes. We have recently established highly homogeneous preadipocytes, named ccdPAs. The aim of the current study was to evaluate the regulation of the potency of platelet-rich plasma (PRP) on the apoptosis of ccdPAs in vitro. PRP stimulated the proliferation of the preadipocytes in a dose-dependent manner, and the stimulatory activity of 2% PRP was significantly higher than that of 2% FBS or 2% platelet-poor plasma (PPP). The presence of 2% PRP significantly inhibited serum starvation- or TNF-α/cycloheximide-induced apoptosis in comparison to 2% FBS or 2% PPP. DAPK1 and Bcl-2-interacting mediator of cell death (BIM) mRNAs were reduced in the preadipocytes cultured with 2% PRP in comparison to those cultured in 2% FBS. The gene expression levels were significantly higher in cells cultured without serum in comparison to cells cultured with 2% FBS, and the levels in the cells with 2% PRP were reduced to 5-10% of those in the cells without serum. These results indicated that ccdPAs exhibit anti- apoptotic activities, in addition to increased proliferation, when cultured in 2% PRP in comparison to the same concentration of FBS, and that this was accompanied with reduced levels of DAPK1 and BIM mRNA expression in in vitro culture. PRP may improve the outcome of transplantation of adipose tissue by enhancing the anti-apoptotic activities of the implanted preadipocytes.     

Activity of T Cells Stimulated by Hemagglutinin-neuraminidase of Newcastle Disease Virus in vivo

To investigate the stimulated activity of T cells and the anti-tumor properties of hemagglutinin-neuraminidase(HN) of Newcastle disease virus(NDV) strain Changchun(NDVcc), the expression of HN gene in hepatoma cells(human HepG-2 and mouse H22 cells) infected with the recombinant adenovirus(Ad-HN) was identified by Western blot analysis and flow cytometry. Sialidase activity of NDVcc HN expressed by Ad-HN was assayed by the periodate-resorcinol method. The in vivo anti-tumor effects of NDVcc HN were evaluate...     

Simultaneous separation of angiotensin and endothelin peptide families by high-performance liquid chromatography: application to the specific radioimmunoassay measurement of angiotensin II or endothelin-1 from tissue

Currently available measurements of endogenous angiotensin II (ANG II) and endothelin-1 (ET-1) concentrations by radioimmunoassay (RIA) lack specificity to ANG II or ET-1. ANG II and ET-1 antibodies cross-react with immuno-reactive angiotensin and endothelin family members, respectively. We have therefore developed an ion-pair reversed-phase high-performance liquid chromatography (HPLC) for simultaneously separating angiotensin and endothelin peptides and enhancing RIA specificity in the measurement of ANG II and ET-1. The developed HPLC separation was applied to canine myocardium extracts; ANG II or ET-1 fractions were collected and quantified by RIA. Elution times for both peptide families, ANG I, ANG II, ANG III, ANG IV, ANG II(4–8), bET-1, ET-1, ET-2 and ET-3 were within 25 min. In normal canine myocardium from the right atrium, right ventricle, left atrium and left ventricle, ANG II concentrations were 39±11, 28±21, 31±11 and 21±8 fmol/g and ET-1 concentrations were 43±16, 42±19, 55±21 and 57±34 fmol/g (mean±SD, N=7), respectively. The combination of HPLC with RIA renders the measurement of ANG II or ET-1 specific and convenient, and saves time. This HPLC separation may be applied to the specific measurement of other immuno-reactive angiotensin and endothelin peptides.     

Induction of Effective Antitumor Immune Response by Combined Administration of hIL-18 and NDV HN

To analyze the antitumor potential and mechanism of action of simultaneous Newcastle disease virus (NDV) hemagglutinin-neuraminidase(HN) and human interleukin 18(hIL-18) gene transfer in C57BL/6 mice with H22 hepatoma,the mouse model with H22 hepatoma was established in C57BL/6 mice, and the antitumor effects of the combined application of NDV HN and hIL-18 were evaluated in vivo. The results show that the growth of established tumors in mice immunized with adenovirus(Ad)-HN in conjunction with Ad-hIL-18 was significantly inhibited compared with that in mice immunized with Ad-HN, Ad-hIL-18 alone, or the empty vector(Ad-mock). Furthermore, the immunization of mice with Ad-HN in conjunction with Ad-hIL-18 elicited strong natural killer activity and H22 tumor-specific cytotoxic T lymphocyte(CTL) responses in vivo. In addition, T cells from the lymph nodes of mice immunized with Ad-hIL-18 or Ad-HN+Ad-hIL-18 secreted high levels of the Th1 cytokine IL-2 and interferon-γ (IFN-γ), indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with NDV HN together with hIL-18 may be a novel and powerful strategy for cancer immunotherapy.     

Inequivalence of substitution pairs in hydroxynaphthaldehyde: A theoretical measurement by intramolecular hydrogen bond strength,aromaticity, and excited‐state intramolecular proton transfer reaction

The inequivalence of substitution pair positions of naphthalene ring has been investigated by a theoretical measurement of hydrogen bond strength, aromaticity, and excited state intramolecular proton transfer (ESIPT) reaction as the tools in three substituted naphthalene compounds viz 1‐hydroxy‐2‐naphthaldehyde (HN12), 2‐hydroxy‐1‐naphthaldehyde (HN21), and 2‐hydroxy‐3‐naphthaldehyde (HN23). The difference in intramolecular hydrogen bond (IMHB) strength clearly reflects the inequivalence of substitution pairs where the calculated IMHB strength is found to be greater for HN12 and HN21 than HN23. The H‐bonding interactions have been explored by calculation of electron density ρ(r) and Laplacian ?²ρ(r) at the bond critical point using atoms in molecule method and by calculation of interaction between σ* of OH with lone pair of carbonyl oxygen atom using NBO analysis. The ground and excited state potential energy surfaces (PESs) for the proton transfer reaction at HF (6‐31G**) and DFT (B3LYP/6‐31G**) levels are similar for HN12, HN21 and different for HN23. The computed aromaticity of the two rings of naphthalene moiety at B3LYP/6‐31G** method also predicts similarity between HN12 and HN21, but different for HN23. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Reactions of hydrazoic acid on TiO2 nanoparticles: an experimental and computational study

This article reports the results of a computational and experimental study on the reaction of hydrazoic acid, HN3, adsorbed on 15-20 nm TiO2 particle films. Experimentally, FTIR spectra of HN3(a) have been measured by varying HN3 dosage, UV irradiation time and surface annealing temperature. Three sharp peaks, related to v(a)(NNN) of HN3(a) and N3(a) with different configurations in the 2000-2200 cm(-1) region, and a broad band absorption, related to associated and isolated HN(a) and HO(a) adsorptions in the 3000-3800 cm(-1) region, have been detected. Computationally, molecular structures, vibrational frequencies and adsorption energies of possible adsorbates including HN3 and its derivatives, N3, N2, NH, and H, have been predicted by first-principles calculations based on the density functional theory (DFT) and the pseudopotential method. On the basis of the experimental and computational results, the peak appeared at 2075 cm(-1), which increases at a faster rate with HN3 exposure time, is attributed to a stable adsorbate, N3-Ti(a), with the predicted adsorption energy, E(ads) = 13 kcal/mol. The peak at 2118 cm(-1), which survives at the highest surface temperature in the heating experiment, is attributable to the most stable adsorbate, Ti-N2N(H)-O(a) with E(ads) = 36 kcal/mol. The peak at 2170 cm(-1), which vanishes most readily in all of the aforementioned experiments, is related to less stable molecular adsorbates, end-on HN3-Ti(a) with E(ads) = 5 kcal/mol and side-on HN(N2)-Ti(a) with E(ads) = 8 kcal/mol. A potential energy diagram for the formation of various absorbates with their transition states has been established for the HN3/TiO2 system. On the basis of the predicted desorption energies, the four most stable products of the HN3 reaction on TiO2 are H-O(a), 118 kcal/mol; HN-O(a), 85 kcal/mol; Ti-N2N(H)-O(a), 36 kcal/mol; and N3-O(a), 19 kcal/mol.     

Antitumor Effects of Newcastle Disease Virus Hemagglutinin-neuraminidase Used as a Molecular Adjuvant

Gene therapy is a potentially powerful tool used in cancer therapy. The strength of immune responses induced by some strategies is usually low, therefore, the development of agents capable of enhancing these responses is highlighted. The authors investigated the potential of an approach based on the hemagglutinin-neuraminidase(HN) of Newcastle disease virus(NDV) as a potential immune adjuvant. It was found that recombinant adenovirus(Ad) infected SGC7901 cells expressing HN exhibited both hemagglutinin(HA) and neuraminidase(NA) activities. It was demonstrated that administration of HN induced higher levels of the effector cytokines TNF-α, IFN-α and IFN-γ and increased natural killer(NK) cell activity. Based on the therapeutic tumor model, the results show that the administration of HN with Apoptin led to improved survival and tumor suppression. In conclusion, this study indicates that HN stimulates innate immune responses to make the activity of NK cells increased, which highlights the potential adjuvant activity of HN in cancer gene therapy.     

On-chip characterization of cryoprotective agent mixtures using an EWOD-based digital microfluidic device

For tissue engineering and regenerative medicine, cryopreservation, a technique for preserving biomaterials in the frozen state with cryoprotective agents (CPAs), is critically important for preserving engineered tissues (ETs) as well as cells necessary to create ETs. As more diverse ETs are produced using various cell types, CPAs and corresponding freeze/thaw (F/T) protocols need to be developed cell/tissue-type specifically. This is because CPAs and F/T protocols that have been successful for one cell/tissue type have proven to be difficult to adapt to other cell/tissue types. The most critical barrier to address this challenge is the inability to screen and identify CPA or CPA mixtures efficiently. In this paper, we developed an "electro-wetting-on-dielectic" (EWOD) based digital microfluidic platform to characterize and screen CPA mixtures cell-type specifically. The feasibility of the EWOD platform was demonstrated by characterizing and optimizing a mixture of dimethlysulfoxide (DMSO) and PBS for human breast cancer cell line as model CPA mixture and cell line. The developed platform multiplexed droplets of DMSO and PBS to create an array of DMSO-PBS mixtures, and mapped the phase change diagram of the mixture. After loading cell suspensions on the platform, the mixture was further screened on-chip for toxicity and cryoprotection. The results were discussed to illustrate the capabilities and limitations of the EWOD platform for cell and tissue-type specific optimization of CPA mixtures and F/T protocols.     

Rhenium-mediated coupling of acetonitrile and pyrazoles. New molecular clefts for anion binding

The reaction of fac-[ReBr(CO)3(NCMe)2] (1) with either pyrazole (Hpz) or 3,5-dimethylpyrazole (Hdmpz) in a 1:2 Re/pyrazole ratio affords the known complexes fac-[ReBr(CO)3(Hpz)2] (2) and [ReBr(CO)3(Hdmpz)2] (3). Using a 1:1 ratio, MeCN as solvent, and longer reaction times led to a mixture in which the major components are the pyrazolylamidino complexes fac-[ReBr(CO)3(HN=C(CH3)pz-kappa2N,N)] (4) and fac-[ReBr(CO)3(HN=C(CH3)dmpz-kappa2N,N)] (5). The complexes fac-[ReBr(CO)3(Hpz)(NCMe)] (6) and fac-[ReBr(CO)3(Hdmpz)(NCMe)] (7) (along with 2 and 3) were found to be minor components of these reactions. Analogous reactions of fac-[Re(OClO3)(CO)3(NCMe)2] yielded fac-[Re(NCCH3)(CO)3(HN=C(CH3)pz-kappa2N,N)]ClO4 (8), fac-[Re(NCCH3)(CO)3(HN=C(CH3)dmpz-kappa2N,N)]ClO4 (9), fac-[Re(Hpz)(CO)3(HN=C(CH3)pz-kappa2N,N)]ClO4 (10), and fac-[Re(Hdmpz)(CO)3(HN=C(CH3)dmpz-kappa2N,N)]ClO4 (11). The X-ray structure of 11 showed the perchlorate anion to be hydrogen-bonded by the N-H groups of the pyrazole and pyrazolylamidino ligands. The behavior of the compound fac-[Re(Hdmpz)(CO)3(HN=C(CH3)dmpz-kappa2N,N)]BAr'4 (13) (synthesized by reaction of [ReBr(CO)3(Hdmpz)2] (3) with (i) AgOTf and (ii) NaBAr'(4)/MeCN) as an anion receptor has been studied in CD3CN solution. In addition, the structure of the supramolecular adduct fac-[Re(CO)3(Hdmpz)(HN=C(CH3)dmpz-kappa2N,N)].Cl (14), featuring chloride binding by the two N-H groups, was determined by X-ray diffraction.     

S-allyl Cysteine Enhances Testosterone Production in Mice and Mouse Testis-Derived I-10 Cells

Hypogonadism, associated with low levels of testosterone synthesis, has been implicated in several diseases. Recently, the quest for natural alternatives to prevent and treat hypogonadism has gained increasing research interest. To this end, the present study explored the effect of S-allyl cysteine (SAC), a characteristic organosulfur compound in aged-garlic extract, on testosterone production. SAC was administered at 50 mg/kg body weight intraperitoneally into 7-week-old BALB/c male mice in a single-dose experiment. Plasma levels of testosterone and luteinizing hormone (LH) and testis levels of proteins involved in steroidogenesis were measured by enzymatic immunoassay and Western blot, respectively. In addition, mouse testis-derived I-10 cells were also used to investigate the effect of SAC on steroidogenesis. In the animal experiment, SAC significantly elevated testosterone levels in both the plasma and the testis without changing the LH level in plasma and increased phosphorylated protein kinase A (p-PKA) levels. Similar results were also observed in I-10 cells. The findings demonstrating the increasing effect of SAC on p-PKA and mRNA levels of Cyp11a suggest that SAC increases the testosterone level by activating the PKA pathway and could be a potential target for hypogonadism therapeutics.     

Alkylation of Hydroquinone with tert-Butyl Alcohol over Bis[（perfluoroalkyl）sulfonyl]imides Supported on MCM-41

Bis[（perfluoroalkyl）sulfonyl]imides [HN（SO2Rf）2, and Rf represents the perfluorinated alkyl group] supported on MCM-41 were synthesized and characterized by XRD, FTIR, SEM, TGA and N2-adsorption techniques. The supported catalysts, HN（SO2Rf）2/MCM-41, were used as the catalysts for the tert-butylafion of hydroquinone （HQ） with tert-butyl alcohol （TBA） in the liquid phase. A high yield （52.0%） of 2-tert-butyl hydroquinone （TBHQ） could be obtained in the presence of 5 tool% HN（SO2C4F9）2/MCM-41 under the optimized reaction conditions and the heterogeneous catalyst could be recycled at least 6 times without substantial loss of activity.     

Inhibition of endothelin (ET)-1-and ET-2-induced vasoconstriction by anti-ET-1 monoclonal antibody

We produced a monoclonal antibody to endothelin (ET)-1, tested cross-reactivities with the related peptides by enzyme immunoassay, and investigated the effects of the antibody on ET-1- or ET-2-induced vasoconstriction of rat isolated thoracic aorta. The antibody recognized ET-1, ET-2 and ET-3, and the immunoreactive site proved to be the N-terminal region but not the C-terminal region of ET-1. Moreover, at an approximate molar-equivalent concentration, the antibody absorbed ET-1 and ET-2, and significantly inhibited ET-1- and ET-2-induced vasoconstriction notwithstanding the presence of the endothelin receptor.     

[HN(C2H5)3][B5O6(OH)4]的合成、结构和热稳定性

利用水热法合成了五硼酸三乙胺[HN(C2H5)3][B5O6(OH)4], 并利用单晶X射线衍射技术解析了其结构, 同时利用傅立叶红外分析、元素分析及热分析技术加以佐证. 该化合物属于单斜晶系, 其空间群为P21/c, 相应的晶胞参数为a=1.0036(2) nm, b=1.1353(2) nm, c=1.4843(3) nm和β=106.54(3)°. 它由孤立的五硼酸阴离子[B5O6(OH)4]-和三乙胺阳离子[HN(C2H5)3]+构成. 五硼酸阴离子[B5O6(OH)4]-通过氢键构成三维网状结构, [HN(C2H5)3]+位于其中的孔道中.     

C_(59)F_(2n)HN(n=1,2)异构体的结构与稳定性的理论研究

分别用MNDO和AM1两种半经验方法,对C59F2nHN (n = 1, 2) 的异构体进行几何构型全优化,结合频率分析及HF/6-31G单点能计算,确定了各异构体的基态结构及其相对稳定性。计算结果表明,C59HN的F加成物的立体选择性规律与C60的不同,最稳定异构体不是1-2加成物。C59F2HN的最稳定异构体是1-4加成的6, 18-或12, 15-异构体; C59F4HN的最稳定异构体是1-4,1-4加成的6, 18; 12, 15-异构体,其能量远小于其它各异构体的能量。N原子取代碳笼骨架C原子后,改变了碳笼F加成物的立体选择性规律。