Subcellular Localization of Small GTP-binding Protein DsRab in Dunaliella salina

With the total RNA of Dunaliella salina as a template,the cD NA sequence of D. salina small GTP-binding protein gene was amplified by RT-PCR technique,and cloned onto pM Dl8-T simple vector,the recon was subjected to PCR detection and restriction endonuclease analysis,and the total sequence of DNA was determined. The results showed that the cloned fragment was 612 bp,and shared 100% homology with reported D. salina DsRab gene（ GenB ank： JN989548）. The target gene fragment was inserted downstream of pM DCG 35 S promoter,constructing subcellular localization recombinant vector pM DCG-DsRab. The successfully constructed subcellular localization recombinant vector pM DCG-DsRab was transformed into Agrobacterium tumefaciens LBA4404,and positive single clones were screened and used for transinfection of onion epidemical cells by Agrobacterium-mediated method,and the instant expression of DsRab was observed under fluorescence microscope. The results showed that the fusion protein GFP-DsRab was successfully expressed in onion epidemical cells,and mainly distributed on cytomembrane. This study will provide reference for further illumination of the function and action mechanism of D. salina small GTP-binding protein DsRab.

Mechanisms mediating cholinergic antral circular smooth muscle contraction in rats

AIM:To investigate the pathway(s)mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent,bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. METHODS:Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer.Isometric tension was recorded.Cumulative concentration-response curves were obtained for(+)-cis- dioxolane(cD),a nonspecific muscarinic agonist,at 10^(-8)- 10^(-4)mol/L,in the presence of tetrodotoxin(TTX,10^(-7)mol/L). Results were normalized to cross sectional area.A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1(pirenzepine), M2(methoctramine)and M3(darifenadn)muscarinic receptor subtypes.The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment.The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. RESULTS:A dose-dependent contractile response observed with bethanechol,was not affected by TTx.The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol.Lack of calcium as well as the presence of the L-type calcium channel blocker,nifedipine,also inhibited the cholinergic contraction,with a reduction in response from 2.5±0.4 g/mm^2 to 1.2±0.4 g/mm^2(P<0.05).The dose- response curves were shifted to the right by muscarinic antagonists in the following order of affinity:darifenacin (M_3)>methocramine(M_2)>pirenzepine(M_1). CONCLUSION:The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s)involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels.The presence of the residual contractile response after the treatment with nifedipine,suggests that an additional pathway could mediate the cholinergic contraction.The involvement of more than one muscarinic receptor(functionally predominant type 3 over type 2)also suggests more than one pathway mediating the cholinergic contraction in rat antrum.

Study on Lymphocyte Activation and Proliferation Induced by Anti-CD3 McAb

T cell activation and proliferation via CD3-TCR complex were investigated by lymphocyte DNA synthesis in vitro.Several interfering factors were also discussed.The result indicated that lymphocyte activation and proliferation are calciumdependent.A rise of cytoplasmic free Ca2+ quickly following activation with CD3 McAb is mainly due to intracellular mobilization of Ca2+,while lymphocyte proliferation needs both intracellular mobilization of Ca2+ as well as influx of extracellular Ca2+, It was confirmed that CTX sensitive G protein plays a role in regulating T cell proliferation by pretreatment with CTX suppressing lymphocyte H-TdR incorporation obviously.PLC and PKC inhibitor neomycin and P.S.S could also decrease T cell proliferation.

Gene Expression Profiling of the Proliferative Effect of Periplocin on Mouse Cardiac Microvascular Endothelial Cells

Objective： Periplocin is an active digitalis-like component from Cortex Periplocae, which has been widely used in the treatment of heart diseases in China for many years. According to the recommendations on the cardiovascular effect of periplocin from in vivo experiments, subsequent in vitro experiments are greatly needed for the global assessment of periplocin. The objective of this study is to investigate the cell proliferation effect and the mechanism of periplocin on endothelial cells. Methods： The proliferative activity of periplocin （0.4, 2, 10, 50, 250 pmol/L; 6, 12, 24, 48, 72 h） was investigated by a comparison with the well-reported cardiac glycoside, ouabain, on mouse cardiac microvascular endothelial cells （CMEC）. 3-（4,5-dimethylthiazolyl）- 2,5-diphenyltetrazolium bromide （MTT）, lactate dehydrogenase （LDH） and 5-bromo-2-deoxyuridine （BrdU） assays were used to evaluate cell proliferation and viability. Subsequently, cDNA microarray experiments were performed on periplocin- （50 pmol/L） and ouabain- （50 pmol/L） treated cells, and data was analyzed by ArrayTrack software. Results： Periplocin could increase cell viability to a level lower than ouabain in the MIF analysis, but decrease LDH release simultaneously. The BrdU incorporation assay showed an increase in cell proliferation with 2-50 μmol/L periplocin. Genes related to protein serine/threonine kinase were the most significantly enriched in the 160 genes identified in periplocin versus the control. In the 165 genes regulated by periplocin versus ouabain, GTP-binding was the most altered term. Conclusions： The results demonstrated the proliferation action of periplocin on CMEC. Meanwhile, its lower cytotoxicity compared to ouabain provides a new insight into the treatment of heart failure.

Maxingxiongting mixture attenuates hypoxia pulmonary arterial hypertension to improve right ventricular hypertrophy by inhibiting the rho-kinase signaling pathway

OBJECTIVE:To explore the mechanism of Maxingxiongting mixture(MXXTM)on pulmonary hypertension in a rat model established by intraperitoneal injection of monocrotaline solution,smoking and forced swimming.METHODS:A total of 30 male Sprague-Dawley rats were randomly divided into five groups:control group,model group,high-dose of MXXTM group(HM),low-dose of MXXTM group(LM),and fasudil group.The mean pulmonary artery pressure(m PAP)was measured by using a miniature catheter.Lung tissue and right ventricular tissue sections were stained with hematoxylin-eosin.The right ventricle(RV)and left ventricle+septum(LV+S)were weighted.RV/(LV+S)was calculated to reflect the degree of right ventricular hypertrophy.Rho/Rho-kinase signaling pathway key proteins(Rho A,ROCKⅠand ROCKⅡ)in rat right ventricular tissue were measured by Western blot analysis.The levels of serum hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF)and the levels of plasma renin activity(PRA),angiotensinⅡ(ANG-Ⅱ),aldosterone(ALD)in rat anticoagulated plasma were all measured by enzyme-linked immunosorbent assay.RESULTS:Compared with the control group,the m PAP and RV/(LV+S)in the model group were significantly increased.Administration of fasudil resulted in a significant decrease of m PAP and RV/(LV+S).In the HM group and LM group,m PAP and RV/(LV+S)were significantly lower than the model group.Compared with the control group,the contents of HIF-1α,VEGF,PRA,ANG-Ⅱand ALD in the model group were significantly increased.The administration of fasudil and high-dose MXXTM significantly reduced the contents of HIF-1α,VEGF,PRA,ANG-II and ALD.Compared with the control group,the expression of Rho A,ROCKⅠand ROCKⅡin the right ventricle of the model group were significantly increased.The administration of fasudil and high-dose MXXTM significantly reduced the expression of Rho A and RockⅡproteins.Our results indicated that high-dose of MXXTM had similar effects on reducing pulmonary artery pressure and improving right ventricular remodeling to fasudil.However,MXXTM was unable to restore parameters above to control levels.CONCLUSIONS:MXXTM attenuates hypoxia pulmonary arterial hypertension to improve right ventricular hypertrophy by inhibiting the Rho-kinase signaling pathway.

Cloning of the Full-length cDNA of the Wheat Involved in Salt Stress：Root Hair Defective 3 Gene （RHD3）

: The full-length cDNA of the wheat (Triticum aestivum L.) root hair defective 3 gene (RHD3) has been cloned from the salt-tolerant hybrid wheat variety Shanrong No. 3 (Za3) using the mRNA differential display and 5’rapid amplification of cDNA ends (RACE) methods. Analysis of the amino acid sequence deduced from the wheat RHD3, gene shows that two conservative GTP-binding motifs, namely GXXXXGKS and DXXG, in eukaryotes also exist at the N-terminal of wheat RHD3. In addition, an 18 amino acid residue transmembrane domain, namely FYLAVMFVVFLVGKAIWV, exists at positions 701—718 of the C-terminal of the deduced protein of wheat RHD3 obtained, but this domain is absent in another three proteins aligned, including rice RHD3, Arabidopsis RHD3, and yeast homologue SEY1. Northern blot revealed that transcription of the wheat RHD3, gene is down-regulated in both the salt-tolerant line and in JN177 under saline stress. A possible stress-responsive mechanism for this gene is discussed.

Reconstitution of Gs and Adenylate Cyclase From Bovine Brain Cortices on Asolectin Liposomes

Signal transduction across cell membranes is an important subject of the current studies on biomembranes. Hormonally regulated adenylate cyclase signalling system is composed of three distinct types of plasma-membrane associated proteins: the receptor, adenylate cyclase (AC) and stimulatory GTP-binding protein (Gs) which me-

Purification of β-adrenergic receptors from Beijing duck erythrocyte plasma membranes and their reconstitution with Gs and adenylate cyclase on asolectin liposomes

Catecholamines such as adrenaline, norepinephrine and isoproterenol regulate a wide variety of physiological responses via their specific binding to adrenergic receptors located in the plasma membrane. Adrenergic receptors have been divided into two major types, α and β. Binding of ligands to β-adrenergic receptors (β-AR) first triggers the activation of stimulatory GTP-binding protein (Gs). The activated Gs then interacts