Parthenogenetic activation of mouse oocytes by strontium chloride: a search for the best conditions

Strontium has been successfully used to induce activation of mouse oocytes in nuclear transfer and other experiments, but the optimum treatment conditions have not been studied systematically. When cumulus-free oocytes were treated with 10mM SrCl(2) for 0.5-5h, activation rates (88.4+/-4.1 to 91.2+/-2.7%) did not differ (mean+/-S.E.; P>0.2), but rate of blastulation (57.3+/-3.5%) and cell number per blastocyst (45.0+/-2.4) were the highest after treatment for 2.5h. When treated with 1-20mM SrCl(2) for 2.5h, the activation rate and cell number per blastocyst were higher (P<0.02) after 10mM SrCl(2) treatment than other treatments. The best activation and development were obtained with Ca(2+)-free Sr(2+) medium, but the activation rate was low (37.7+/-1.6%) in Ca(2+)-containing medium. Activation rates were the same, regardless of the presence or absence of cytochalasin B (CB) in the activating medium, but the blastulation rate was higher (P<0.001) in the presence of CB. Only 70% of the cumulus-enclosed oocytes were activated and 10% blastulated after a 10 min exposure to 1.6mM SrCl(2), and many lysed, with increased intensity of Sr(2+) treatment. The presence of CB in SrCl(2) medium markedly reduced lysis of cumulus-enclosed oocytes. Media M16 and CZB did not differ when used as activating media. Only 10.5% of the oocytes collected 13 h post hCG were activated by Sr(2+) treatment alone, with 34% blastulating, but rates of activation and blastulation increased (P<0.001) to 94 and 60%, respectively, when they were further treated with 6-dimethylaminopurine (6-DMAP). The total and ICM cell numbers were less (P<0.001) in parthenotes than in the in vivo fertilized embryos. In conclusion, the concentration and duration of SrCl(2) treatment and the presence or absence of CB in activating medium and cumulus cells had marked effects on mouse oocyte activation and development. To obtain the best activation and development, cumulus-free oocytes collected 18 h post hCG should be treated for 2.5h with 10mM SrCl(2) in Ca(2+)-free medium supplemented with 5 microg/mL of CB.     

Optimization of theaflavin biosynthesis from tea polyphenols using an immobilized enzyme system and response surface methodology

Theaflavins were synthesized from tea polyphenols extracted from green tea using an immobilized polyphenol oxidase system. To optimize the production of theaflavins, response surface methodology was applied to determine the effects of five critical variables and their mutual interactions on theaflavin biosynthesis at five levels. A total of 52 individual experiments were performed and a statistical model predicted that the highest theaflavin concentration was 0.766mgml^–1 at optimized conditions. Using these optimal parameters under experimental conditions in three independent replicates, the average value of the biosynthesized theaflavin concentration reached 0.75±0.017mgml^–1 and matched the value predicted by the modelRevisions requested 03 November 2004; Revisions received 7 December 2004     

Evaluation of 18F-labeled acetylcholinesterase substrates as PET radiotracers

Four 18F-labeled acetylcholinesterase (AChE) substrates, (S)-N-[18F]fluoroethyl-2-piperidinemethyl acetate (1), (R)-N-[18F]fluoroethyl-3-pyrrolidinyl acetate (2), N-[18F]fluoroethyl-4-piperidinyl acetate (3), and (R)-N-[18F]fluoroethyl-3-piperidinyl acetate (4), were evaluated for in vivo blood and brain metabolism in mice, brain pharmacokinetics in rats monkeys (M. nemistrina) using PET imaging. All 18F-labeled compounds were compared to N-[11C]methyl-4-piperidinyl propionate (PMP). Compound 1 was completely metabolized within 1 min in mouse blood and brain. This compound had relatively fast regional brain pharmacokinetics and poor discrimination between brain regions with different AChE concentration. Compound 4 showed relatively slower blood metabolism and slower pharmacokinetics than compound 1 but again poor discrimination between brain regions. Both compounds 1 and 4 showed different kinetic profiles than PMP in PET studies. Compound 3 had the slowest blood metabolism and slower pharmacokinetics than PMP. Compound 2 showed highly encouraging characteristics with an in vivo metabolism rate, primate brain uptake, and regional brain pharmacokinetics similar to [11C]PMP. The apparent hydrolysis rate constant k3 in primate cortex was very close to that of [11C]PMP. This compound has potential to be a good PET radiotracer for measuring brain AChE activity. The longer lifetime of 18F would permit longer imaging times and allows preparation of radiotracer batches for multiple patients and delivery of the tracer to other facilities, making the technique more widely available to clinical investigators.     

内源性儿茶酚胺参与白细胞介素-2对大鼠离体心脏的作用

目的: 研究内源性儿茶酚胺是否参与白细胞介素-2(IL-2)的心脏作用.方法: 采用Langendorff离体心脏灌流模型,观察室性早搏次数、左室发展压(LVDP)、左室舒张末压(LVEDP)、心率(HR)和冠脉流量(CF)的变化.结果: ①50 U/ml IL-2增加离体心脏的室性早搏次数,增加LVDP、LVEDP、HR和CF.②利舍平(reserpine)或普萘洛尔(propranolol)预处理后,50 U/ml IL-2对离体心脏的作用消失;phentolamine预处理后,不改变50U/ml IL-2的离体心脏作用.③200 U/ml IL-2增加离体心脏的室性早搏次数,对LVDP、LVEDP、HR和CF无显著增加作用.④reserpine或propranolol预处理后,200 U/ml IL-2增加离体心脏的室性早搏次数作用不明显,LVDP、HR和CF降低、LVEDP增高.结论: 内源性儿茶酚胺介导了IL-2的致离体心脏心律失常、正性变时和变力作用,较高浓度的IL-2抑制离体心脏的功能.     

超声破裂载基因微泡增强心肌细胞报告基因的转染与表达

目的:通过超声破裂载基因微泡介导报告基因心肌细胞转染,探讨其能否增强心肌细胞外源基因转染与表达.方法:以β-galactosidase质粒为报告基因,将其与自制氟碳气体微泡粘附,制备载基因微泡.利用诊断性超声破裂微泡进行体外心肌细胞基因转染;以磷酸钙共沉淀转染为阳性对照并将其以不同方式与超声破裂微泡技术联合应用,以期进一步增强基因转染效果.分别采用原位染色及酶学定量检测β-galactosidase表达水平,同时进行细胞活性检测.结果:超声破裂载基因氟碳气体微泡(PESDA)转染组心肌细胞β-galactosidase表达水平可达单纯质粒转染组60倍(P＜0.01).磷酸钙共沉淀转染3.67倍(P＜0.01)超声强度、微泡浓度对超声破裂介导基因转染效果有明显影响.超声破裂微泡技术与磷酸钙共沉淀联合应用可进一步提高报告基因的表达(P＜0.05),即使在磷酸钙转染后6 h,超声破裂微泡仍能明显增强报告的基因的表达(P＜0 05).结论:超声破裂微泡技术是一种高效基因转染方法,其不但能增加DNA转染,而且增强入胞后基因的表达.超声破裂微泡与其它基因转染技术联合应用能进一步增加基因转染效率.     

The mitochondrial permeability transition pore and the Ca2+-activated K+ channel contribute to the cardioprotection conferred by tumor necrosis factor-alpha

Pretreatment with tumor necrosis factor-alpha (TNF-alpha) is known to trigger cardioprotection and it can activate multiple downstream signaling cascades. However, it is not known whether the mitochondrial permeability transition pore and the Ca(2+)-activated K(+) channel (K(Ca) channel) are involved in the TNF-alpha-induced cardioprotection. In the present study, we examined whether TNF-alpha inhibits pore opening and activates the K(Ca) channel in the cardioprotection. In isolated rat hearts subjected to 30 min of regional ischemia and 120 min of reperfusion, pretreatment with 10 U/ml TNF-alpha for 7 min followed by 10 min washout improved the recovery of rate-pressure product (RPP=left ventricular developed pressure x heart rate) and coronary flow (CF) during reperfusion, and reduced the infarct size and release of lactate dehydrogenase (LDH). Administration of 20 micromol/L atractyloside, a pore opener, for the last 5 min of ischemia and first 15 min of reperfusion, and pretreatment with 1 micromol/L paxilline, an inhibitor of the K(Ca) channel, for 5 min before ischemia, attenuated the recovery of RPP and CF, and the reductions of infarct size and release of LDH induced by TNF-alpha. On the other hand, administration of 10 micromol/L NS 1619, an opener of the K(Ca) channel, for 10 min before ischemia, decreased the infarct size and LDH release, and improved contractile functions and CF; these effects were attenuated by atractyloside. Pretreatment with 0.2 micromol/L cyclosporin A for the last 5 min of ischemia and first 15 min of reperfusion showed similar effects to those of TNF-alpha, and they were not attenuated by paxilline. In mitochondria isolated from hearts pretreated with 10 U/ml TNF-alpha for 7 min, a significant inhibition of Ca(2+)-induced swelling was observed. Furthermore, paxilline attenuated the inhibition of Ca(2+)-induced mitochondrial swelling by TNF-alpha. These findings indicate that TNF-alpha protects the myocardium against ischemia and reperfusion injury by inhibiting mitochondrial permeability transition pore opening as well as activating K(Ca) channels, probably the mitochondrial K(Ca) channel, which is upstream from the pore.     

New amperometric and potentiometric immunosensors based on gold nanoparticles/tris(2,2'-bipyridyl)cobalt(III) multilayer films for hepatitis B surface antigen determinations

Two generic, fast, sensitive and novel electrochemical immunosensors have been developed. Initially, a layer of plasma-polymerized Nafion film (PPF) was deposited on the platinum electrode surface, then positively charged tris(2,2'-bipyridyl)cobalt(III) (Co(bpy)(3)(3+)) and negatively charged gold nanoparticles were assembled on the PPF-modified Pt electrode by layer-by-layer technique. Finally, hepatitis B surface antibody (HBsAb) was electrostatically adsorbed on the gold nanoparticles surface. Electrochemical behavior of the {Au/Co(bpy)(3)(3+)}(n) multilayer film-modified electrodes was studied. Cyclic voltammetry, electrochemical impedance spectroscopy (EIS) were adopted to monitor the regular growth of the multilayer films. The performance and factors influencing the performance of the resulting immunosensors were studied in detail. The multilayer film-modified immunosensor was used for hepatitis B surface antigen (HBsAg) determination via the amperometric and potentiometric immunosensor systems, and both systems provided the same linear ranges from 0.05 to 4.5 microg/mL with different detection limits for the amperometric system 0.005 microg/mL and for the potentiometric system 0.015 microg/mL. The immunosensors were used to analyse HBsAg in human serum samples. Analytical results of clinical samples show that the developed immunoassay is comparable with the enzyme-linked immunosorbent assays (ELISAs) method, implying a promising alternative approach for detecting HBsAg in the clinical diagnosis. In addition, the multilayer films also showed better stability for 1 month at least.     

An electrochemical stripping metalloimmunoassay based on silver-enhanced gold nanoparticle label

A novel, sensitive electrochemical immunoassay has been developed based on the precipitation of silver on colloidal gold labels which, after silver metal dissolution in an acidic solution, was indirectly determined by anodic stripping voltammetry (ASV) at a glassy-carbon electrode. The method was evaluated for a noncompetitive heterogeneous immunoassay of an immunoglobulin G (IgG) as a model. The influence of relevant experimental variables, including the reaction time of antigen with antibody, the dilution ratio of the colloidal gold-labeled antibody and the parameters of the anodic stripping operation, upon the peak current was examined and optimized. The anodic stripping peak current depended linearly on the IgG concentration over the range of 1.66 ng ml(-1) to 27.25 microg ml(-1) in a logarithmic plot. A detection limit as low as 1 ng ml(-1) (i.e., 6 x 10(-12) M) human IgG was achieved, which is competitive with colorimetric enzyme linked immuno-sorbent assay (ELISA) or with immunoassays based on fluorescent europium chelate labels. The high performance of the method is attributed to the sensitive ASV determination of silver (I) at a glassy-carbon electrode (detection limit of 5 x 10(-9) M) and to the catalytic precipitation of a large number of silver on the colloidal gold-labeled antibody.     

The ligand-dependent interaction of mineralocorticoid receptor with coactivator and corepressor peptides suggests multiple activation mechanisms

We investigated the coregulator (coactivator and corepressor) interactions with the mineralocorticoid receptor (MR) that lead to activation and inhibition of the receptor in the presence of agonist and/or antagonist. Our results indicate that MR ligand binding domain (LBD) interacts strongly with only a few specific coactivator peptides in the presence of the agonist aldosterone and that these interactions are blocked by the antagonist eplerenone. We also discovered that cortisol, the preferred physiological ligand for the glucocorticoid receptor in humans, is a partial MR agonist/antagonist, providing a possible molecular explanation of the tissue-selective effects of glucocorticoids on MR. However, when we examined the coactivator and corepressor peptide interactions in the presence of cortisol, we found that MR bound with cortisol or aldosterone interacted with the same set of peptides. Thus, the partial agonism shown by cortisol is unlikely to be the result of differential interaction with known coactivators and corepressors. On the other hand, we have identified coactivator binding groove mutations that are critical for cortisol activation but not for aldosterone activation, suggesting that the two steroids induce different MR LBD conformations. In addition, we also show that cortisol becomes full agonist when S810L mutation is introduced in the LBD of MR. Interestingly, MR antagonists, such as eplerenone and progesterone, become partial agonist/antagonist of S810L but are still able to recruit LXXLL peptides to the mutant receptor. Together, these findings suggest a model to explain the MR activation by various ligands.