Objective To explore the roles and possible mechanism of calcium-sensing receptor(CaSR) in cell cardiac hypertrophy model using angiotensin Ⅱ (Ang Ⅱ ).Methods The cultured neonatal rat ventricular myocytes were treated with Ang Ⅱ as cell cardiac hypertrophy model.Hypertrophic neonatal rat cardiomyocytes were treated with GdCl3(a specific agonist of CaSR) and/or with Ro318220(a specific inhibitor of PKC pathway).To evaluate the status of cardiac hypertrophy,cell diameter was observed by HE dyeing,and protein content was determined through coomassie brilliant blue protein kit.The intracellular calcium concentration( [ Ca2+]i) was determined by laser scanning confocal microscope.The protein expression of CaSR and PKC pathway were analyzed using Western blotting.Results ①Compared to the control group(0.1263 ± 0.0443),the protein expression of CaSR was increased in Ang Ⅱ group and in GdCl3 group(0.1963 ± 0.0375,0.2778 ± 0.0564,all P＜ 0.05).Moreover,compared with Ang Ⅱ alone,the increase was significant in GdCl3 group(P ＜ 0.05).②Compared to control group(222.70 ± 22.09),AngⅡ group(392.16 ± 36.85) remarkably increased [Ca2+]i(P＜ 0.05),and this increase of [Ca2+]i was further enhanced in GdCl3 group (502.60 ± 44.21) versus Ang Ⅱ group (P ＜ 0.05).③Compared to control group,Ang Ⅱ could induce cardiomyocyte hypertrophy,and GdCl3 enhanced the effect.Moreover,this enhancement was attenuated by Ro318220.④Compared to control group(0.27 ± 0.07,0.69 ± 0.06,0.87 ± 0.04),the protein expression of PKCα,PKCε and PKCδ was increased in Ang Ⅱ group(0.60 ± 0.16,1.02 ± 0.13,1.20 ± 0.18,all P＜ 0.05) and the protein expression of PKCα,PKCε was increased in GdCl3 group(0.82 ± 0.16,1.34 ± 0.12,all P ＜ 0.05).Moreover,compared with Ang Ⅱ group,the protein expression of PKCα,PKCε was obviously increased in GdCl3 group (all P ＜ 0.05);compared with GdCl3 group,the protein expression of PKCα,PKCε(0.41 ± 0.10,0.85 ± 0.14) was obviously decreased in Ro318220 group(all P ＜ 0.05).Conclusions CaSR is involved in cardiac hypertrophy induced by Ang Ⅱ through PKC pathway in cultured neonatal rat cardiomyocytes.

This study is designed to obtain recombinant human acetylcholinesterase (rhAChE) and apply it in screening acetylcholinesterase inhibitors. The rhAChE was overexpressed in HEK293 cells transfected by plasmid of pCMV-AChE with the cationic liposome and rhAChE was found to be secreted into cell culture medium. AChE activity was assayed according to modified Ellman method to obtain kinetic parameters. IC so50 values for donepezil compounds of rhAChE were calculated to determine their activities of inhibition. The results showed that Km value was 151.9 micromol.L-1 donepezil inhibited rhAChE in a mixed competitive-noncompetitive way (Ki= 16.03 nmol.L-1, Ki = 18.36 nmol.L-1) and that most new compounds tested exhibited high activities of inhibition on rhAChE. The study suggests that rhAChE is available to be applied in screening AChE inhibitors in vitro.
Acetylcholinesterase ; genetics ; metabolism ; Cholinesterase Inhibitors ; analysis ; pharmacology ; HEK293 Cells ; Humans ; Indans ; analysis ; pharmacology ; Inhibitory Concentration 50 ; Kinetics ; Piperidines ; analysis ; pharmacology ; Plasmids ; Recombinant Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo observe the effects of electroacupuncture (EA) at "Sanyinjiao" (SP 6) on urodynamics indices in rats with overactive bladder (OAB) after cystostomy, and to explore its regulation mechanism on bladder function.

METHODSForty-eight Sprague-Dawley female rats which received cystostomy were randomly divided into a blank group (group A), a blank Sanyinjiao group (group B), a blank non-acupoint group (group C), a model group (group D), a model Sanyinjiao group (group E) and a model non-acupoint group (group F), 8 rats in each one. The model of OAB was established with 1% acetic acid solution perfused into the bladder in the group D, group E and group F. No treatment was given to the group A and group D. Acupuncture was applied at bilateral "Sanyinjiao" (SP 6) in the group B and group E, followed by EA after the arrival of qi. Acupuncture was applied at bilateral non-acupoint in the group C and group F, followed by EA with continuous wave, 2 Hz of frequency for 30 min. The treatment was given for continuous 5 urination cycles. The BL-420 E+ biological function experiment system was used to measure and record the changes of indices of bladder pressure and urodynamics.

RESULTSCompared with the group A, the bladder capacity and urine output in the group B were significantly increased (both P<0.05), and the urination rate was increased in the group C (P<0.05); the differences of each index between group C and group B were not statistically significant (all P>0.05). Compared with the group D, the capacity pressure, bladder capacity, detrusor pressure, urinary output and urination rate in the group E were all increased (all P<0.05). Compared with the group F, the capacity pressure and detrusor pressure in the group E were increased (both P<0.05).

CONCLUSIONThe EA at "Sanyinjiao" (SP 6) could significantly improve urine function in rats with OAB after cystostomy, but its regulation effect on urination is not obvious in rats with non-OAB.

Acupuncture Points ; Animals ; Cystostomy ; Disease Models, Animal ; Electroacupuncture ; Female ; Humans ; Rats ; Rats, Sprague-Dawley ; Urinary Bladder ; physiopathology ; surgery ; Urinary Bladder, Overactive ; physiopathology ; surgery ; therapy

BACKGROUNDRecent studies have demonstrated that dexamethasone (DEX) interferes with immune responses by targeting key functions of dendritic cells (DCs) at the earliest stage. However, the cellular and molecular mechanisms are still incompletely understood. This study aimed to explore the possible mechanisms by investigating the roles of DEX on differentiation, maturation & function of murine DCs and the effects of DEX on DCs via Toll-like receptor 4 (TLR4)-nuclear factor (NF)-kappaB mediated signal pathway.

METHODSImmature DCs (imDCs) were cultured from murine bone marrow (BM) cells. We added DEX into culture medium at different time. The expression of CD11c, CD86 and I-A(b) (mouse MHC class II molecule) was determined by flow cytometry. We determined the expression of NF-kappaB and its inhibitory protein I-kappaBalpha by electrophoretic mobility shift assay (EMSA) and Western blotting, respectively. The productions of interleukin (IL)-12p70 and IL-10 in cell culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA).

RESULTSDEX impaired differentiation of DCs from murine bone marrow progenitors, and inhibited lipopolysaccharide (LPS) induced maturation of DCs. DEX significantly inhibited NF-kappaB expression of normal DCs, the higher the DEX concentration or the longer the DEX treatment time, the more obvious the effect. However, DEX had little effect on LPS-induced NF-kappaB activation, and partially impaired LPS-induced I-kappaBalpha degradation. DEX significantly decreased LPS induced IL-12p70 production by DCs. Interestingly, our results showed a synergistic effect between DEX and LPS on the production of IL-10 by DCs.

CONCLUSIONSDEX inhibits the differentiation and maturation of murine DCs involved in TLR4-I-kappaB-NF-kappaB pathway, and also indirectly impairs Th1 development and interferes with the Th1-Th2 balance through IL-12 and/or IL-10 secretion by DCs.

Animals ; Blotting, Western ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; metabolism ; Dexamethasone ; pharmacology ; Electrophoretic Mobility Shift Assay ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Male ; Mice ; NF-kappa B ; metabolism ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; metabolism

Eleven compounds were isolated from the culture of Streptomyces sp. CPCC 202950 by a combination of various chromatographic techniques including column chromatography over macroporous resin HP-20, MCI, and reversed-phase HPLC. Their structures were identified as 1H-pyrrole-2-carboxamide(1),5'-deoxy-5'-methylthioinosine(2), vanillamide(3), trans-3-methylthioacrylamide(4), 1,2,3,4-Tetraydro-1H-pyrido[3,4-b]indole-3-carboxylic acid(5), cyclo(L-pro-L-tyr) (6), N-[2-(4-hydroxyphenyl)]ethylacetamide(7), benzamide (8), cyclo ('L-leucyl-trans-4-hydroxy-L-proline)(9), cyclo-(Phe-Gly) (10), and tryptophan (11). Among them, compounds 1 and 2 were new natural products. In the preliminary assays, none of the compounds exhibited obvious inhibition of HIV-1 protease activity (IC50 > 10 micromol x L(-1)).
Culture Media ; chemistry ; metabolism ; HIV Protease ; analysis ; HIV Protease Inhibitors ; chemistry ; isolation & purification ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; Streptomyces ; chemistry ; metabolism

OBJECTIVETo explore effects of nordy on biological behaviors of human malignant glioblastoma cell line U87MG in vitro and transplanted tumor in vivo, and to identify the differential proteome upon Nordy induced differentiation.

METHODSGlioblastoma U87MG cells were induced to differentiate by synthetic lipoxygenase inhibitor, Nordy. The drug was also given via peritoneal injection to nude mice (27 mg/kg body weight) bearing orthotopic transplanted tumors of U87MG cells in the brain. The tumor volumes and GFAP expression were measured. Total proteins of U87MG cells after Nordy treatment were analysed by two-dimensional gel electrophoresis. PDQuest 7.1 computer software was used to compare protein profiles of the treated cells with that of untreated control. Differentially expressed proteins were then selected and characterized by matrix assisted laser desorption ionization-time of flight-mass spectrometry. The functional aspects of these proteins were analyzed by bioinformatics.

RESULTSNordy suppressed both the proliferation of U87MG cells in vitro and the tumor growth of orthotopic transplanted tumors in vivo (P < 0.01). The differentially expressed proteins induced by Nordy included proliferation-associated gene A, alternative splicing factor ASF-3, eukaryotic translation initiation factor 5A, coffilin 1 (non-muscle), beta galactoside binding lectin, glyceraldehyde-3-phosphate dehydrogenase, enolase 1 and an unknown protein.

CONCLUSIONSNordy promotes the differentiation of glioblastoma cells, by which it may serve as a therapeutic agent. Various proteins identified during Nordy-induced differentiation are involved in the cell proliferation, metabolism, differentiation, apoptosis and gene transcription.

Animals ; Antineoplastic Agents ; pharmacology ; Brain Neoplasms ; metabolism ; pathology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Expression Regulation, Neoplastic ; Glial Fibrillary Acidic Protein ; metabolism ; Glioblastoma ; metabolism ; pathology ; Humans ; Lipoxygenase Inhibitors ; pharmacology ; Male ; Masoprocol ; analogs & derivatives ; pharmacology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Protein Array Analysis ; Proteome ; genetics ; metabolism ; Proteomics ; methods ; Random Allocation ; Tumor Burden

OBJECTIVETo study the content of phytoestrogen in dissimilarity herbs.

METHODThe activity of phytoestrogen in heat-clearing drugs, drugs for relieving exterior syndrome, diuretic, anastaltics, tonics and astringents were detected based on the recombinant yeast cell (W303-1A/hER-ERE-Lac Z). The estrogenic activity in traditional Chinese materia medica were assayed quantitatively by determining the expression of beta-galactosidase.

RESULTThe phytoestrogen concentration (6.35 x 10(-3) nmol x g(-1) E2 equivalent) in heat-clearing drugs was the highest while that in anastaltic and tonic drugs was the lowest, which was less than the detected limit.

CONCLUSIONCompared with the other traditional Chinese materia medica, the content of phytoestrogen, which can bind to estrogen receptor, in giant knotweed rhizome, forsythia suspense, ash bark, baical skullcap root and ophiopogonis tuber were higher.

Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Phytoestrogens ; analysis ; metabolism ; Plants, Medicinal ; chemistry ; Receptors, Estrogen ; metabolism ; Recombination, Genetic ; Saccharomyces cerevisiae ; chemistry ; cytology ; drug effects ; genetics ; beta-Galactosidase ; analysis

Hairy root clones of Saussurea involucrata transformed with Agrobacterium rhizogenes strains R1601, R1000, and LBA9402 were established to investigate the flavonoid production. Opine synthesis and PCR analysis confirmed the integration of the T-DNA fragment of Ri plasmid from A. rhizogenes strain R1601 into the transformed root genome. The frequency of hairy root formation from root segments, which were pre-cultured 2 days in N6 solid medium without plant growth regulators, amounted to 100% following infection with R1601 strain of A. rhizogenes. The transformed roots were kept in hormone-free N6 liquid medium in the dark at 25 degrees C, 110r/min and routinely subcultured every 20 - 24 days. One hairy root clone, which grew vigorously with lateral branches, was periodically examined for the ability to produce flavonoid. The maximum of biomass and flavonoid yield achieved 66.7 g/L (fresh weight) and 102.3mg/g dry weight after incubation 20 days. The calli were induced from the hairy root culture in the presence of 0.5mg/L IBA and intact plantlets were regenerated from these calli. The regeneration plantlets from hairy roots, in which the flavonoid content were 53% in that of untransformed plants, weren't different in growth and morphology of the untransformed plantlets. Therefore plant regeneration from hairy roots may be also a means for producing transformed S. involucrata plants. Hairy root cultures of S. involucrata clearly showed higher flavonoid contents compared to the wild plant or the regeneration seedlings. As the wild S. involucrata grows only in special regions with peculiar climate, and cultivation of this species in a normal climate has been unsuccessful so far. The success in obtaining a method for high production of flavonoid might very well be one of the solutions for this problem in the future.
Culture Techniques ; Flavonoids ; biosynthesis ; Plant Roots ; growth & development ; Rhizobium ; physiology ; Saussurea ; growth & development

Two strategies, direct ligation after enzyme digestion and over-lap PCR technology, were adopted to construct a fusion gene which was composed of the antimelanoma single chain antibody gene and the staphylococcal enterotoxin A gene without N-terminal signal sequence. The fusion gene was subcloned into pET28-a vector and transformed into E. coli BL21(DE3). Ni-NTA system was selected to separate and purify the expresstd products. The inhibition ratio of the fusion protein was tested by MTT method. It is shown that the 6His-ScFv-SEA fusion protein can be expressed stably in E. coli BL21 (DE3). The quantity of the fusion protein was shown up to 30% of the total protein of the bacteria and mainly in inclusion body. By activation the effective cells, the fution protein can inhibit the melanoma cell whith expressed corresponding antigen.
Cell Line, Tumor ; Cell Survival ; drug effects ; Cells, Cultured ; Electrophoresis, Polyacrylamide Gel ; Enterotoxins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Humans ; Inclusion Bodies ; genetics ; metabolism ; Melanoma ; drug therapy ; immunology ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology ; therapeutic use ; Single-Chain Antibodies ; genetics ; metabolism

OBJECTIVETo explore the effect of low-energy semiconductor laser intranasal irradiation of the blood on blood coagulation status in healthy pregnant women at term.

METHODSLow-energy semiconductor laser was introduced into the nasal cavity in 126 healthy pregnant women at term and 123 healthy young unmarried women as the control group. The plasma prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen levels were examined using transmissive turbidimetry after the therapy.

RESULTSPT, APTT and TT levels were significantly lowered, whereas fibrinogen level significantly increased in the healthy pregnant women before the laser therapy as compared with those in the control group (P<0.01). After intranasal laser therapy, these parameters were significantly improved in the healthy pregnant women (P<0.05) although there were differences from those of the control group.

CONCLUSIONLow-energy semiconductor laser intranasal irradiation of the blood can effectively improve high blood coagulation status in healthy pregnant women at term.

Adult ; Blood Coagulation ; radiation effects ; Blood Coagulation Tests ; Female ; Humans ; Labor Onset ; blood ; Low-Level Light Therapy ; Nasal Cavity ; radiation effects ; Partial Thromboplastin Time ; Pregnancy ; Prothrombin Time ; Semiconductors ; Thrombin Time ; Young Adult