中国汉坦病毒H82O5株G2糖蛋白基因的克隆及在真核细胞中的瞬时表达

从感染病毒乳鼠脑组织提取总RNA，采用RT－PCR和分子克隆技术将扩增到的G2糖蛋白基因插入含CMV启动子的pcDNA3.1/His质粒载体中，通过脂质体介导转染COS－7细胞，用SDS－PAGE、Western-blot及IFIA方法分别测定表达产物的相对分子量及特异性。结果证明获得正向插入的G2－pcDNA3.1/His重组表达质粒，表达产物的相对分子量为56ku，与理论预期大小一致，并且可与汉坦病毒H8205株的腹水抗体起特异反应。表明构建的G2－pcDNA3.1/His重组质粒所表达的蛋白为中国汉坦病毒株特有，能在哺乳动物细胞中表达并具有抗原性，重组质粒可应用于汉坦病毒的DNA疫苗研究。     

Stereoselectivity of actions of the calcium sensitizer [+]-EMD 60263 and its enantiomer [-]-EMD 60264

The thiadiazinone derivative [+]-EMD 60263 ((+)-5-(1-(α-ethylimino-3,4-dimethoxybenzyl)-1,2,3,4- tetrahydroquinoline-6-yl)-6-methyl-3,6-dihydro-2H-1,3,4 -thiadiazine-2-on) is a Ca²⁺-sensitizing agent with only minor phosphodiesterase inhibitory activity. Our aim was to characterize the inotropic and electrophysiological effects of [+]-EMD 60263 and its enantiomer [-]-EMD 60264 in several cardiac muscle preparations. The Ca²⁺-sensitizing activity resided in the [+]-enantiomer only. [+]-EMD 60263 (3 μM) shifted the EC₅₀ of Ca²⁺ for contractile activation of skinned fibers of pig heart from 2.41 μM to 0.73 μM, whereas [-]-EMD 60264 (30 μM) was ineffective. In Langendorff-perfused guinea pig hearts, [+]-EMD 60263 and [-]-EMD 60264 induced concentration-dependent positive and negative inotropic effects, respectively; both enantiomers reduced spontaneous heart rate but did not influence perfusion pressure. The maximum increase in force of human atrial trabeculae was 35 % of pre-drug control with [+]-EMD 60263 in comparison to 113 % with forskolin. In guinea-pig papillary muscles, [+]-EMD 60263 and [-]-EMD 60264 had opposite inotropic responses, however, both agents similarly prolonged action potential duration. Both enantiomers concentration-dependently blocked the rapidly activating component I_Kr of the delayed rectifier in guinea-pig myocytes. The block saturated at potentials positive to +30 mV, closely resembling the effects of the antiarrhythmic agent E-4031 which had been originally used to define I_Kr. It is concluded, that the positive inotropic action of [+]-EMD 60263 can be explained by prevalence of the Ca²⁺-sensitizing effect. The accompanying prolongation in action potential duration is caused by block of the I_Kr component of the delayed rectifier. While the inotropic effects are stereoselective, most of the electrophysiological actions are clearly independent of sterical configuration. The combination of Ca²⁺-sensitizing with class-III antiarrhythmic action may provide an interesting pharmacological profile of potential therapeutic use. Received: 7 January 1997 / Accepted: 25 February 1997     

老年充血性心衰患者地高辛药代动力学研究

目的 :探讨老年心衰患者地高辛维持量的合理应用。　 方法 :用荧光偏振免疫分析法测定了 14例老年心衰患者血清地高辛浓度 ,并计算出药代动力学参数。　 结果 :14例老年心衰患者地高辛 t1/ 2 β,β和 Vd分别为 72 .769± 2 9.768h、0 .0 11± 0 .0 0 6h-1和 3.190± 3.30 0 L/ kg,与非老年心衰患者 ( t1/ 2 β 36.1± 2 .0 h,56.3± 12 h,β 0 .0 19± 0 .0 0 1h-1,Vd 7.37± 1.65L / kg)相比差异显著。　 结论 :老年心衰患者地高辛维持量以小量分次给药为宜 ,且更应注意个体化     

Investigation of Role of Nitric Oxide in Protection from Bordetella pertussis Respiratory Challenge

The mechanism whereby whole-cell pertussis vaccines (WCV) confer protection against Bordetella pertussis is still not fully understood. We have previously reported that macrophage activation produced by vaccination with WCV is associated with induction of NO synthesis by macrophages in response to in vitro stimulation with B. pertussis antigens. To determine whether NO production is an effector of protection or simply a marker of activation, the susceptibility of inducible nitric oxide synthase (type II, iNOS) knockout mice to infection with B. pertussis was examined. We showed that iNOS knockout mice were more susceptible to B. pertussis respiratory challenge than wild-type mice. iNOS-deficient mice also developed a less effective protective response than wild-type mice after the same immunization with WCV. This suggests that NO plays an important role in effecting protection against B. pertussis challenge.     

Spermidine synthase of Escherichia coli: localization of the speE gene.

We have obtained Escherichia coli mutants lacking spermidine synthase (putrescine aminopropyltransferase) and have found that the mutated gene (speE) is located immediately upstream from the gene coding for S-adenosylmethionine decarboxylase (speD); these genes are located at 2.7 minutes on the E. coli chromosome. Both genes are present in a 1795-base-pair fragment of E. coli DNA that was cloned into pBR322. Deletion of 105 bases upstream of speE caused a coordinate loss of both activities, indicating that speE and speD constitute a single operon. speE and speD have also been cloned separately in a high-expression vector; strains carrying these plasmids overproduce the respective enzymes.     

Effect of gradual rise in plasma calcium concentration on the impairment of atrioventricular nodal conduction due to verapamil

The possible reversal by calcium of the inhibitory action of verapamil on the atrioventricular (AV) node was investigated in anesthetized, atropinized dogs, with cardiac pacing. The His bundle potentials were recorded by endocavitory electrode and the AV node effective refractory period measured by the extrastimulus method. Calcium infusion was effective against the impairment of AV nodal conduction induced by verapamil, provided it remained moderate: the gradual rise in the plasma calcium concentration counteracted the effects of an infusion of verapamil on conduction time and effective refractory period in the AV node, as long as it did not exceed 5 mmol/L. However, beyond this level, calcium appeared less and less capable of reversing the effects of verapamil. Thus, the protective action of calcium had a bell-shaped dose-response curve, with the optimum at 5 mmol/L. This biphasic influence is consistent with the opposite opinions previously given concerning the antagonism between calcium and calcium blockers, depending on whether hypercalcemia brought into play was mild or major. In any case, the prominent role played by calcium in the slow inward current in the AV node accounts for the antagonism, observed in vivo, between calcium and verapamil. The pacemaker activity of the sinoatrial (SA) node was less influenced by both calcium blocker and calcium.     

Factors influencing the variations of ejection fraction during exercise in chronic aortic regurgitation

The influence of left ventricular volume variations and regurgitant fraction variations upon left ventricular ejection fraction, during exercise was examined using equilibrium radionuclide angiography in patients suffering from aortic regurgitation. Ejection fraction (EF), regurgitant fraction (RF), end diastolic volume (EDV) and end systolic volume (ESV) variations from rest to peak exercise were determined in 44 patients suffering from chronic aortic regurgitation (AR) and in 8 healthy volunteers (C). In C, EF increased (+0.10±0.03, P<0.01) and ESV decreased significantly (-23%±12%, P<0.01) RF and EDV did not vary significantly. In AR patients, EF, EDV and ESV did not vary significantly because of important scattering of individual values. Changes in EF and ESV were inversely correlated (r=-0.79, P<0.01) and RF decreased significantly (-0.12±0.10, P<0.01). Volumes and EF changes during exercise occurred in three different ways. In a 1st subgroup of 7 patients, EF increased (+0.09±0.03, P<0.05) in conjunction with a reduction of ESV (-24%±12%, P<0.05) without a significant change in EDV. In a 2nd group of 22 patients. EF decreased (-0.04±0.07, P<0.01) in association with an increase in ESV (+17%±16%, P<0.01) and no change in EDV. In a 3rd subgroup of 15 patients, EF decreased (-0.02±0.06, P<0.01) despite a reduction in ESV (-7%±6%, P<0.01) because of a dramatic EDV decrease (-10%±6%, P<0.05). In this subgroup, changes in EF were inversely correlated with changes in ESV (r=-0.55, P<0.01) and positively related to EDV variations (r=0.42, P=0.02). EDV related to EDV variations (r=0.42, P=0.02). EDV changes were weakly, but significantly, correlated to RF decrease (r=0.39, P<0.05). We conclude that changes in left ventricular ejection fraction during exercise in patients with chronic aortic regurgitation are significantly related in some patients to changes in ventricular loading conditions as well as contractile state. Therefore, a correct interpretation of EF changes during exercise requires the simultaneous determination of changes in LV volumes.Abbreviations EDV end diastolic volume - EF ejection fraction - ESV end systolic volume - LV left ventricle - RV right ventricle     

Functional recognition of the neuronal tyrosine hydroxylase cAMP regulatory element in different cell types.

Transient transfection of pLB2CAT constructs bearing short synthetic oligonucleotides derived either from the tyrosine hydroxylase (TH) promoter or other sources was used to examine functional cAMP regulatory element (CRE) activity in a variety of cell lines. The region containing only the putative TH CRE was found to be as or more effective in conferring cAMP responsiveness onto pLB2CAT (which employs the TK promoter) than the immediate 272 bp region of the TH promoter. Increases in CAT activity of 10- to 20-fold were observed in JEG-3 cells with a single insert of the TH CRE region (-31 to -54) in pLB2CAT, and the presence of a second insert generated only a modest further increase. This construct also responded to cAMP in 4 other cell lines tested but the degree of increase was less dramatic. Inserts containing the consensus 8 bp CRE motif embedded in other natural or artificial contexts served generally as weak functional CREs in all cell lines tested. In vitro analysis revealed that a specific protein-DNA complex apparently containing a single protein with a MW of 45-50 kDa was formed equally well with JEG-3 cell nuclear extract and CRE-bearing-TH and other fragments which produced dramatically different cAMP effects in vivo. These results suggest specificity in the effects of cAMP on different CREs which are dictated by contextual differences.     

颈丛阻滞、硬膜外阻滞下甲状腺手术应激反应的比较

目的 :比较颈丛阻滞、硬膜外阻滞下甲状腺手术应激反应的大小。方法 :选择ASAⅠ～Ⅱ级 ,女性 ,甲状腺手术患者 30例 ,年龄 2 2～ 5 5岁 ,术前无呼吸、循环和内分泌疾病 ,随机分为颈丛阻滞组 (颈丛组 ) 15例 ,硬膜外阻滞组(硬膜外组 ) 15例 ;颈丛阻滞选用 0 .8%利多卡因和 0 .2 5 %布比卡因混合液 ,以C4一点法行双侧深浅丛阻滞 ;硬膜外阻滞选用 1.3%利多卡因和 0 .15 %丁卡因混合液 ,穿刺点选择C4～ 5或C5～ 6间隙 ,采用侧卧位直入法 ,并向头置管 3cm ;分别测定并记录麻醉前、麻醉后 2 0min、切皮、分上极、切腺体和术毕共六个时点的血糖、血压和心率的变化。结果 :两组病例各时点血糖均逐步上升 ,于分上极、切腺体和术毕血糖值与麻醉前比较有显著性差异 (P <0 .0 1) ;硬膜外组只在分上极时SBP与麻醉前比较有差异外 (P <0 .0 5 ) ,而颈丛组在分上极、切腺体时DBP与麻醉前比较有差异 (P <0 .0 5 ) ,SBP、MAP与麻醉前比较有显著性差异 (P <0 .0 1)。结论 :本研究表明颈丛阻滞、硬膜外阻滞均不能完全抑制甲状腺手术的应激反应 ,在稳定甲状腺手术循环功能方面硬膜外阻滞优于颈丛阻滞