Not only calmodulin (CAM) with Ca2+ regulates the activity of many enzymes and proteins, but also free-CaM (no Ca2+bound) and Ca2+-independent CaM-binding proteins play roles in plant and animal cells. There is no in vivo method to identify the interaction between free-CaM and Ca2+-independent CaM-binding protein (CaMBP). Using site-directed mutagenesis by polymerase chain reaction (PCR), 5 mutant Arabidopsis calmodulin isoform 2 (AtCaM2) genes, mCaM21, mCoM212,mCaM2123, mCaM2124 and mCaM21234 were obtained. The mutant mCaM2 encoded glutamine in place of glutamate (E32Q; E68Q; E105Q; E141Q) in one or more EF-hand Ca2+-binding motifs of AtCaM2. The recombinant mCaM2 proteins were produced in Escherichia coli, and subsequently separated on SDS-PAGE in the presence of Ca2+ or EGTA, their electrophoresis mobilities were related with that of mutant EF-hand motifs. 45Ca2+ overlay analysis indicated that the more glutamate replaced by glutamine, the lower affinity with Ca2+ in the mCaM2 proteins. The mCaM21234 mutant protein (E32Q; E68Q; E105Q;E141Q) was unable to bind Ca2+. Using yeast two-hybrid technique with mCaM21234 as bait, it was possible to see interaction in Arabidopsis of AtCaM2 with IQD26, a calcium-independent CaM-binding protein. Site-directed mutation of AtCaM2 will aid the research of Ca2+, CaM and Ca2+-independent CaMBPs in plant biological processes.

Objectives To investigate the effect of bladder function and behavior change training on urinary retention after epidural anesthesia and the method to decrease it. Methods 265 patients receiv-ing operation under epidural anesthesia were divided into two groups, the experimental group (132 cases) and the control group (133 cases). The experimental group was further divided into 3 groups: one to two days of training, 3 to 4 days of training and above 5 days of training preoperation. The experimental group carried out bladder function and behavior change training, but the control group never undertook any train-ing. After operation the data were analyzed. Results The incidence of urinary retention was significantly different between the experimental group and the control group (P<0.01);The time length of the training before operation aorrelated with incidence of urinary retention(P<0.05). Conclusions Bladder function and behavior change training contributed to decrease urinary retention after epidural anesthesia.The training time and incidence of urinary retention showed inverse proportion. This could decrease the opportunity of suffering from urethral catheterization and urinary tract infection.

Genome of Arabidopsis has a kind of genes encoding proteins with ARM repeat domains and some of these proteins are known to play important roles in plant development and responses to hormone. An Arabidopsis mutant lfr with a distinct phenotype was got in leaf and flower development. The gene is predicted to encode a protein with ARM repeat domains. In order to study its function and molecular mechanism, recombination expression plasmid pGEX-2TGST∶LFR was constructed and transformed into the host bacteria strain Rosetta. Then IPTG was used to induce the recombinant protein expression in engineering strain. The expression products were detected by 12% SDS-PAGE. The GST∶LFR fusion protein was existed in soluble form with a relative molecular mass 77 ku, which is fit with the molecular mass supposed from gene coding frame. After purification by GST-tag affinity chromatography and electroelution, the fusion protein was used as antigen to prepare polyclonal antiserum in rabbits. After the fifth injection of antigen, the antiserum was obtained and further purified by decreased nonspecific bacteria and GST-tag antibody with method of immuno-precipitation. Western blot analysis showed that the purified antiserum, raised against the recombination LFR protein in rabbits, could react to the recombinant protein expressed in Rosetta specifically. And then the nuclear proteins of Arabidopsis wild type and mutant were extracted and separated by SDS-PAGE. Western blot assays revealed that there was a protein band, with a relative molecular mass 50 ku, indicating that antiserum could react to the native protein expressed in Arabidopsis specifically.

OBJECTIVETo investigate the reactivity of intrapulmonary arterial rings to vasoactive substances as thromboxane A2 and endothelin-1 in patients with chronic obstructive pulmonary disease (COPD).

METHODSIntrapulmonary arterial rings isolated from patients with normal lung function and COPD were mounted in a Multi Myograph system to determine the reactivity of the intrapulmonary arterial rings to 60 mmol/L KCl, thromboxane A2 analogue U46619 and endothelin-1 before and after preconditioning with the COX synthase inhibitor indomethacin.

RESULTSThe reactivity of intrapulmonary arterial rings to U46619 and endothelin-1 was significantly decreased in patients with COPD. The reactivity to U46619 was dramatically decreased in patients with normal lung function after application of indomethacin.

CONCLUSIONThe reactivity of intrapulmonary arterial rings is significantly decreased in patients with COPD.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; pharmacology ; Aged ; Endothelin-1 ; metabolism ; Female ; Humans ; In Vitro Techniques ; Indomethacin ; pharmacology ; Male ; Middle Aged ; Pulmonary Artery ; metabolism ; Pulmonary Disease, Chronic Obstructive ; metabolism ; Thromboxane A2 ; metabolism

Propofol is known to cause vasorelaxation of several systemic vascular beds. However, its effect on the pulmonary vasculature remains controversial. In the present study, we investigated the effects of propofol on human pulmonary arteries obtained from patients who had undergone surgery. Arterial rings were mounted in a Multi-Myograph system for measurement of isometric forces. U46619 was used to induce sustained contraction of the intrapulmonary arteries, and propofol was then applied (in increments from 10–300 µM). Arteries denuded of endothelium, preincubated or not with indomethacin, were used to investigate the effects of propofol on isolated arteries. Propofol exhibited a bifunctional effect on isolated human pulmonary arteries contracted by U46619, evoking constriction at low concentrations (10–100 µM) followed by secondary relaxation (at 100–300 µM). The extent of constriction induced by propofol was higher in an endothelium-denuded group than in an endothelium-intact group. Preincubation with indomethacin abolished constriction and potentiated relaxation. The maximal relaxation was greater in the endothelium-intact than the endothelium-denuded group. Propofol also suppressed CaCl₂-induced constriction in the 60 mM K⁺-containing Ca²⁺-free solution in a dose-dependent manner. Fluorescent imaging of Ca²⁺ using fluo-4 showed that a 10 min incubation with propofol (10–300 µM) inhibited the Ca²⁺ influx into human pulmonary arterial smooth muscle cells induced by a 60 mM K⁺-containing Ca²⁺-free solution. In conclusion, propofol-induced arterial constriction appears to involve prostaglandin production by cyclooxygenase in pulmonary artery smooth muscle cells and the relaxation depends in part on endothelial function, principally on the inhibition of calcium influx through L-type voltage-operated calcium channels.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid ; Arteries ; Calcium ; Calcium Channels ; Constriction ; Endothelium ; Humans* ; Indomethacin ; Myocytes, Smooth Muscle ; Propofol* ; Prostaglandin-Endoperoxide Synthases ; Pulmonary Artery* ; Relaxation ; Vasoconstriction* ; Vasodilation

OBJECTIVETo investigate the effect of dexmedetomidine on 5-HT-induced constrictions of isolated human intrapulmonary arteries and explore the mechanisms.

METHODSLung tissue was obtained from patients undergoing surgery for lung carcinoma. Intrapulmonary arteries were dissected and cut into rings, which were mounted in a Multi Myograph system to determine the effect of dexmedetomidine (0.3-3 nmol/L) on 5-HT-induced vasoconstrictions. The influences of the endothelium removal and various drugs including L-NAME, yohimbine and indomethacin were tested on the effects of dexmedetomidine.

RESULTSDexmedetomidine (0.1-100 nmol/L) did not obviously affect the resting tension of endothelium-intact human intrapulmonary arteries. 5-HT induced concentration-dependent contraction in endothelium-intact intrapulmonary arteries [pD2: 6.11∓0.05, Emax: (102.10∓1.96)%]. In the rings with intact endothelium, dexmedetomidine (0.3-3 nmol/L) significantly attenuated the Emax and pD2 of 5-HT-induced vasoconstriction [pD2: 5.94∓0.03, Emax: (79.96∓1.31)%]. 5-HT also induced concentration-dependent contraction in endothelium-denuded intrapulmonary arteries [pD2: 6.10∓0.07, Emax: (107.40∓3.20)%]. Dexmedetomidine produced no significant effects on the rings with denuded endothelium. The effects of dexmedetomidine on 5-HT-induced vasoconstriction was suppressed by L-NAME and yohimbine, but not by indomethacin.

CONCLUSIONDexmedetomidine can inhibit 5-HT-induced vasoconstriction of isolated human intrapulmonary arteries probably through α2-adrenergic acceptor and NO released from the endothelium.

Adult ; Aged ; Dexmedetomidine ; pharmacology ; Female ; Humans ; In Vitro Techniques ; Male ; Middle Aged ; Pulmonary Artery ; drug effects ; Serotonin ; pharmacology ; Vasoconstriction ; drug effects

Objective: To explore the effects of shear stress on morphology, adhesion and proliferation of human umbilical cord blood mesenchymal stem cells(hUC-MSCs). Methods: The hUC-MSCs were cultured in vitro until confluence and then placed in a flow system.The cells were subjected to different shear stress (1, 2, 3, 4 dye/cm²) for 2 hours and 6 hours, and no shear stress treatment cells served as a static control.The morphological changes of hUC-MSCs in different groups were analyzed by phase contrast microscopy and immunofluorescence.The mRNA expression levels of intercellular adhesion molecule-1 (ICAM-1) and Ki67 were detected by real-time PCR. Results: Compared with the static control group, the hUC-MSCs cells were arranged in the direction of fluid after treated with shear stress.The immunofluorescence results showed that the cytoskeletal protein F-actin filaments was prolonged after shear stress.The cytoskeleton was further elongated in the 2 dye/cm² for 6 hours group when compared with 2 dye/cm² for 2 hours group, and the cytoskeleton was loosened when time extended to 6 hours.Real-time PCR results showed that the relative expressions of ICAM-1 mRNA and Ki67 mRNA in the static control group and different gradient shear stress groups were significantly different, with significant differences among them (F=17.141, P=0.000; F=11.336, P=0.001). The relative expression of ICAM-1 mRNA in the 1, 2, 3, 4 dye/cm² shear stress group was 2.74±0.32, 9.77±1.19, 6.70±0.92 and 5.69±0.72, respectively, which was significantly higher than 1.00±0.28 in the static control group, with significant differences between them (all at P<0.05). The relative expression of Ki67 mRNA in the 3 dye/cm² and 4 dye/cm² shear stress group was 0.39±0.09 and 0.04±0.02, respectively, which was significantly lower than 1.00±0.24 in the static control group, with statistically significant differences between them (both at P<0.05). Conclusions After treated with fluid shear stress, hUC-MSCs are arranged in the direction of fluid.Shear stress can promote the adhesion of hUC-MSCs.As the increase of shear stress intensity, cell proliferation is inhibited.