Characterization of protein serine/threonine phosphatase activities in human lung mast cells and basophils

1. The serine/threonine protein phosphatase (PP) inhibitors, okadaic acid and calyculin, attenuated the IgE-mediated release of histamine from human lung mast cells (HLMC) and basophils in a dose-dependent manner whereas an alternative PP inhibitor, microcystin, was ineffective. Calyculin was more potent than okadaic acid in both cell types. The concentration required to inhibit by 50% (IC₅₀) the release of histamine was 15 (HLMC) and 50 nM (basophils) for calyculin and 200 (HLMC) and 300 nM (basophils) for okadaic acid.
2. Lysates of purified HLMC and basophils dephosphorylated radiolabelled glycogen phosphorylase, a substrate for both PP1 and PP2A. The PP activity in lysates of both cell types was inhibited in a dose-dependent fashion by the PP inhibitors with the following rank order of activity, calyculin (approximate IC₅₀; 0.02–0.1 nM)⩾microcystin (0.1 nM)>okadaic acid (70 nM).
3. The PP1-selective inhibitor, inhibitor-2 (I-2), attenuated the dephosphorylation of glycogen phosphorylase in lysates of both HLMC and basophils. I-2 (20 nM) inhibited the glycogen phosphorylase PP activity by 71±3% and 49±13% in HLMC and basophil extracts, respectively. There were, approximately, 6 fold greater levels of I-2-sensitive activity in HLMC than in basophils. Qualitatively similar results were obtained with an alternative PP1-selective inhibitor, inhibitor-1 (I-1).
4. Lysates derived from HLMC and basophils dephosphorylated radiolabelled casein which is a PP2A-restricted substrate. HLMC lysates contained, approximately, 2.5 fold higher levels of casein PP activity than basophil lysates.
5. These data indicate that HLMC and basophils both contain PP1 and PP2A. The data suggest that, on a per cell basis, HLMC have higher levels of both PP1 and PP2A. Moreover, the ratio of PP1 to PP2A is higher in HLMC than in basophils.

     

Comparative study on in vitro inhibition of grass carp (Ctenopharyngodon idellus) and mouse protein phosphatases by microcystins

Microcystins isolated from toxic cyanobacteria are potent inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A). The inhibitory effects of three structural variants of microcystins (microcystin‐LR, ‐YR, and ‐RR) on protein phosphatases isolated and purified from the liver and kidney of grass carp (Ctenopharyngodon idellus) were investigated using the ³²P radiometric assay. The relationships between percentage inhibition of protein phosphatase activity and microcystin levels followed a typical dose‐dependent sigmoid curve. These results were compared to those obtained from mouse PP2A. The degree and pattern of inhibition of both fish and mouse protein phosphatases by microcystins were similar. Protein phosphatases in crude fish tissue homogenates showed similar inhibition patterns as purified fish PP2A toward microcystins. © 2000 John Wiley & Sons, Inc. Environ Toxicol 15: 71–75, 2000     

A colorimetric protein phosphatase inhibition assay for the determination of cyanobacterial peptide hepatotoxins based on the dephosphorylation of phosvitin by recombinant protein phosphatase 1

A colorimetric protein phosphatase inhibition assay based on the dephosphorylation of phosvitin by recombinant protein phosphatase 1 was developed for analysis of waters for cyanobacterial hepatotoxins. The phosphate released in the assay was determined using a malachite green reagent. Good agreement with toxin concentrations determined by HPLC was obtained. The assay was capable of determining these toxins at concentrations around 1 μg/L with high precision and without sample concentration. This is of considerable benefit as the World Health Organisation specifies a provisional guideline of 1 μg/L for microcystin‐LR. There was evidence, however, that the sample matrix might affect quantification, leading to false positive results. Thus the assay should be viewed as a screening procedure, and confirmatory analyses by an alternative procedure should be carried out for positive results. Further work is required to resolve the question of matrix interferences if phosphatase inhibition assays are used directly for measuring toxin levels in water, especially if this information is used to check compliance with water quality guidelines. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 242–252, 2001     

蛋白丝/苏氨酸磷酸酶1和2A抑制药对大鼠三叉神经元电压依赖性钠通道的影响(英文)

目的通过研究蛋白丝/苏氨酸磷酸酶1和2A特异性抑制药冈田酸对大鼠三叉神经元电压依赖性钠电流的影响,探讨磷酸酶在细胞信号转导中的作用。方法在成年大鼠三叉神经元上进行全细胞膜片钳记录。结果1μmol·L-1冈田酸显著抑制总钠电流(INa-T) ,仅轻微抑制毒素不敏感型钠电流(INa-TTX-R) ,其抑制率分别为(20±13) %(n=9,P<0 .05)和(4±3) %(n=6, P<0 .05)。冈田酸对INa-T的激活曲线产生明显的超极化位移,半激活电压从给药前的-(13±8)mV升至给药后的-(16±7)mV(P<0 .05) ,但是对INa-TTX-R的激活曲线没有影响。结论①蛋白丝/苏氨酸磷酸酶参与了大鼠三叉神经元电压依赖性钠通道的调节。②大鼠三叉神经元存在多种电压依赖性钠通道,它们对冈田酸具有不同的敏感性。     

C反应蛋白与慢性阻塞性肺疾病的临床相关性分析

目的：探讨C反应蛋白在慢性阻塞性肺疾病急性加重期（AECOPD）病情严重程度与缓解期的临床意义。方法：测定94例慢性阻塞性肺疾病患者急性加重期及缓解期（使用ELISA法检测）空腹血清中的CRP浓度和WBC总数的变化,进行肺功能第1秒用力呼气容量FEV和1s率FEV/FVC测定,患者根据肺功能测定及临床资料评估结果将COPD病情严重程度分为Ⅳ级,分析CRP水平与各级COPD之间及加重期与缓解期之间的关系。结果：随着COPD急性加重期（AECOPD）病情的加重,血清中CRP的水平明显升高（P〈0.01）,AECOPD患者CRP水平明显高于缓解期（P〈0.01）。结论：CRP可作为慢性阻塞性肺疾病急性加重期感染的敏感及重要指标,并与AECOPD病性严重程度分级呈正相关。     

榅桲果实及种子不同提取部位的蛋白酪氨酸磷酸酶1B抑制活性筛选

目的探讨榅桲果实和种子的甲醇提取物及其不同溶剂提取部位对蛋白酪氨酸磷酸酶1B(PTP1B)的抑制作用,筛选抗糖尿病活性部位。方法利用PTP1B体外筛选系统,对榅桲果实和种子不同提取部位进行细致筛选,确定其降糖活性部位。结果榅桲果实和种子各部位均有不同程度抑制PTP1B活性的作用,其中榅桲种子乙酸乙酯部位、榅桲果实二氯甲烷部位和榅桲果实水部位的体积分数为50%的乙醇溶液洗脱部位对PTP1B抑制作用强,其半数抑制质量浓度(IC50)分别为0.844,0.423和0.395mg·L-1,初步推测榅桲果实及种子提取物具有抗2型糖尿病的潜力。结论榅桲种子乙酸乙酯部位、榅桲果实二氯甲烷部位和榅桲果实水部位的体积分数为50%的乙醇溶液洗脱部位是主要的抗糖尿病活性部位。     

急性胰腺炎患者实验室指标与疾病严重程度的关系

摘要： 目的 探讨急性胰腺炎（AP）患者血钙、尿素氮（BUN）、D-二聚体、C-反应蛋白（CRP）、纤维蛋白原及淀粉酶水平与疾病严重程度的关系。 方法 114 例 AP 患者中轻度 AP（MAP 组）70 例, 中度 AP（MSAP 组）18 例, 重度 AP （SAP 组）26 例, 比较各组实验室指标水平, 并分析各指标与急性生理学与慢性健康评估系统Ⅱ（APACHE Ⅱ）评分的相关性; ROC 曲线分析 CRP、D-二聚体、纤维蛋白原诊断 SAP 的敏感性。 结果 MSAP 组、SAP 组 BUN、CRP、D-二聚体、纤维蛋白原和 APACHE Ⅱ 评分均高于 MAP 组, 血钙低于 MAP 组; SAP 组 APACHE Ⅱ 评分高于 MSAP 组（P＜ 0.05）; 各组间血淀粉酶差异无统计学意义。 APACHE Ⅱ 评分与 CRP、D-二聚体、纤维蛋白原呈正相关（r 分别是 0.407、0.404、0.245, 均 P＜ 0.05）, 与血钙呈负相关（r= -0.333, P＜ 0.05）。 ROC 曲线显示 CRP 诊断 SAP 曲线下面积最大为 0.752（95%CI 为 0.644～ 0.860）, cut-off 值是 74.45 mg/L, 灵敏度为 86.4%, 特异度为 68.2%。 结论 联合监测 BUN、凝血指标、CRP、血钙等实验室指标, 有助于综合评价 AP 患者的病情, 改善患者的预后。     

急性脑梗死患者血清高敏C反应蛋白水平与颈动脉斑块性质的关系

目的 探讨急性脑梗死患者血清高敏C反应蛋白（hs-CRP）水平与颈动脉斑块性质的关系. 方法 73例急性脑梗死患者采用彩色多普勒超声检测仪进行检测,并根据结果将其分为不稳定斑块组（A组,23例）、稳定斑块组（B组,25例）和无斑块组（C组,25例）,同时选取25例健康体检者为对照组（D组）.分别检测四组hs-CRP水平及颈动脉斑块进行统计比较. 结果 A组、B组、C组颈动脉斑块检出率及hs-CRP水平明显高于D组（均P＜0.05）,A组hs-CRP水平明显高于B组及C组（均P＜0.05）. 结论 急性脑梗死患者的颈动脉斑块检出率及hs-CRP水平明显高于健康人群,hs-CRP水平与颈动脉斑块性质有相关性.     

In vivo kinetic analysis of covalent binding between N-acetyl-L-cysteine and plasma protein through the formation of mixed disulfide in rats

Purpose. This investigation was undertaken to study the relationship between plasma drug clearance and covalent protein-binding kinetics of N-acetyl-L-cysteine (NAC). Methods. NAC was intravenously administered to rats via a bolus injection or continuous infusion. Plasma concentrations of protein-unbound and total NAC were analyzed using a compartment model, taking into consideration of the protein binding process, and the apparent first-order binding and dissociation rate constants (k_on and k_off) were obtained. Results. Plasma total NAC after a bolus injection showed biphasic elimination with an inflection point at 1 hr. After 1 hr, NAC was largely present in the covalent protein-bound form. During the steady state of the infusion, approximately 30%-40% of plasma NAC bound with protein covalently. The k_on, k_off, and the elimination rate constant of protein-unbound drug (k_e) were 0.23, 0.57, and 4.3 hr^-1. The dissociation half-life of NAC from protein estimated from k_off was in agreement with the elimination half-life of plasma total NAC. This suggests that the dissociation of NAC from protein rate-limited the drug elimination in plasma (k_off < k_e). Conclusion. We demonstrated that plasma total drug clearance is kinetically limited by covalent protein binding. The compartmental model described here is useful for analyzing its kinetics in vivo.     

超敏C反应蛋白水平与急性脑梗死病情的相关性

目的：观察急性脑梗死患者血清超敏C反应蛋白水平与病情发展及演变的关系。方法急性脑梗死患者（151例）作为试验组,依据神经功能缺损程度再分为轻、中、重型3个亚组;选取健康者作为对照组（151例）。试验组进行常规治疗（低分子右旋糖酐250 mL连续7 d、丹红注射液4 mL连续14 d、5％碳酸氢钠250 mL连续3 d;视情况予以肝素总量不超过12500 u抗凝）。2组均进行血清超敏C反应蛋白（ hs-CRP）、D-二聚体及低密度脂蛋白胆固醇（ LDL-C）水平监测。比较2组血清hs-CRP水平与急性脑梗死严重程度的关系。结果急性脑梗死患者,血清 hs -CRP 水平、D-二聚体阳性率均明显高于对照组（ P＜0.05）;其hs-CRP与神经系统损伤评分呈高度正相关（ P＜0.05）;其D-二聚体水平与神经系统损伤评分也呈正相关（ P＜0.05）。结论 hs-CRP水平可以独立反映脑梗死疾病的发生发展,与病情有相关性。     

进展性脑卒中患者血浆C-反应蛋白的研究

目的：研究血浆C-反应蛋白（CRP）与进展性脑卒中（SIP）的关系。方法：运用免疫比浊法测定40例SIP患者及40例稳定性脑卒中患者血浆CRP水平。结果：SIP患者血浆CRP水平明显高于稳定性脑卒中患者,差异具有显著性（P〈0.01）。结论：高CRP可能参与了SIP的发生和发展。     

丁苯酞对老年急性脑梗死的疗效及对血清尿酸、C反应蛋白和血液流变学的影响

目的 研究丁苯酞对老年急性脑梗死的疗效及对血清尿酸、C反应蛋白和血液流变学的影响。方法 选择2015年1月-2015年12月在西安市第一医院进行诊治的老年急性脑梗死患者86例,随机分为两组,对照组采用常规急性脑梗死对症治疗,观察组加用丁苯酞胶囊。观察两组的美国国立卫生研究院卒中量表（NIHSS）评分、Barthel指数和疗效,检测并比较治疗前后两组患者血清尿酸、C反应蛋白、血小板聚集率和血黏稠度。结果 观察组的有效率为90.70%,明显高于对照组的72.09%（P<0.05）;治疗后两组的NIHSS评分、血清尿酸和C反应蛋白水平、血小板聚集率和血黏稠度均明显降低,Barthel指数明显升高（P<0.05）,且观察组的变化较对照组更为明显（P<0.05）。结论 丁苯酞对老年急性脑梗死患者的疗效显著,能降低患者的血清尿酸、C反应蛋白水平和神经功能缺损程度,改善其血液流变学状态和生活自理活动能力,具有脑保护作用。     

Integrative Monitoring of Marine and Freshwater Harmful Algae in Washington State for Public Health Protection

The more frequent occurrence of both marine and freshwater toxic algal blooms and recent problems with new toxic events have increased the risk for illness and negatively impacted sustainable public access to safe shellfish and recreational waters in Washington State. Marine toxins that affect safe shellfish harvest in the state are the saxitoxins that cause paralytic shellfish poisoning (PSP), domoic acid that causes amnesic shellfish poisoning (ASP) and the first ever US closure in 2011 due to diarrhetic shellfish toxins that cause diarrhetic shellfish poisoning (DSP). Likewise, the freshwater toxins microcystins, anatoxin-a, cylindrospermopsins, and saxitoxins have been measured in state lakes, although cylindrospermopsins have not yet been measured above state regulatory guidance levels. This increased incidence of harmful algal blooms (HABs) has necessitated the partnering of state regulatory programs with citizen and user-fee sponsored monitoring efforts such as SoundToxins, the Olympic Region Harmful Algal Bloom (ORHAB) partnership and the state’s freshwater harmful algal bloom passive (opportunistic) surveillance program that allow citizens to share their observations with scientists. Through such integrated programs that provide an effective interface between formalized state and federal programs and observations by the general public, county staff and trained citizen volunteers, the best possible early warning systems can be instituted for surveillance of known HABs, as well as for the reporting and diagnosis of unusual events that may impact the future health of oceans, lakes, wildlife, and humans.     

依达拉奉对急性脑梗死患者血清C反应蛋白的影响及临床意义

目的研究依达拉奉对急性脑梗死患者血清C反应蛋白(CRP)含量的影响,并分析其临床意义。方法选择80例经CT或MRI确诊的脑梗死患者,随机分为2组,2组均予常规治疗,治疗组在此基础上予依达拉奉30mg静滴,一日两次,分别在确诊后24h内和治疗2周后使用免疫比浊法测定2组患者的血清CRP水平及进行中国卒中评分(CSS)。结果急性脑梗死患者血清CRP水平增高,治疗组治疗2周后血清CRP水平明显降低,CSS评分改善,与对照组相比有显著性差异。结论依达拉奉可以显著降低急性脑梗死患者血清CRP水平,对脑梗死病情变化和预后具有重要意义。     

心型脂肪酸结合蛋白与急性冠状动脉综合征关系的研究

目的探讨心型脂肪酸结合蛋白（H—FABP）与急性冠状动脉综合征（ACS）早期诊断、危险分层及预后的关系。方法分别测定90例ACS患者（ACS组）、20例稳定型心绞痛（SAP）患者（SAP组），及30例健康体检者（正常对照组）的血清H—FABP浓度，比较ACS组内H—FABP与肌钙蛋白（CTnT）的变化，比较ACS组中低、中、高危患者的H—FABP浓度的不同，并于4周内观察各组住院期间并发症情况及预后的情况。结果ACS组发病3h内的患者H—FABP阳性率为40％，CTnT阳性率为10％（P〈0．05）；发病6h内的患者H—FABP阳性率65％，CTnT阳性率30％（P〈0．05）；所有样本中H．FABP阳性诊断ACS的敏感性是95．7％，CTnT阳性诊断ACS的敏感性是81．5％（P〈0．05）。ACS组内低、中、高危及急性心肌梗死患者H．FABP浓度差异具有统计学意义，发生心脏意外事件患者血清H—FABP浓度均高于未发生心脏意外事件患者（P〈0．01）。结论H—FABP的定量测定对ACS患者的早期诊断、危险分层及预后评估具有重要的临床价值。     

重症急性胰腺炎患者高迁移率族蛋白B1的变化及其与多器官功能障碍综合征的关系

目的动态观察急性胰腺炎（SAP）患者血浆中高迁移率族蛋白B1（HMGB1）水平的变化并探讨其与多器官功能障碍综合征（MODS）的关系。方法31例SAP患者,根据是否并发MODS分组,MODS组（／7,=17）与非MODS组（n=14）,采用酶联免疫吸附实验检测患者入院后1、3、5、7、14、21d血浆HMGB1含量的变化。结果SAP患者入院后1d血浆中HMGB1含量显著升高,其中入院7、21dMODS组HMGB1含量显著高于非MODS组（P〈0．05）。MODS患者存活组入院后3、21dHMGB1含量显著低于非存活组（P〈0．05）。结论HMGB1作为重要的晚期炎性介质参与SAP后MODS的病理生理过程,动态观察其水平有助于SAP病程监测及患者预后判断。     

46例重症急性胰腺炎并发症的诊治分析

目的探讨重症急性胰腺炎严重并发症的原因和诊治方法。方法对46例重症急性胰腺炎并发症进行回顾性分析。结果46例重症急性胰腺炎中型26例,型20例。各类并发症共105次。其中以休克、成人呼吸窘迫综合征(ARDS)、多器官功能障碍综合征(MODS)、腹腔内感染、急性肾功能衰竭、胃肠瘘、胰瘘常见,病死率34.8%。结论重症急性胰腺炎主要的并发症应早期诊断,及时治疗。     

急性冠状动脉综合征患者高敏C反应蛋白与糖化血红蛋白的相关性研究

目的探讨急性冠状动脉综合征（ACS）患者血浆高敏C反应蛋白（hs-CRP）和糖化血红蛋白（HbA1c）水平与冠状动脉病变严重程度的关系及hs-CRP与HbA1c的相关性。方法选择因ACS行冠状动脉造影术患者80例（ACS组）,其中急性心肌梗死（AMI）33例,不稳定型心绞痛（UA）47例。按病变支数分为单支病变组（27例）,双支病变组（23例）,j支病变组（30例）。另选择因怀疑冠心病行冠状动脉造影术排除冠心病的患者32例作为对照组。以液相层析法检测HbA1c浓度,以乳胶免疫增强比浊法检测hs-CRP浓度。结果ACS组和其中的单支、双支、3支病变组以及AMI组、UA组的hs-CRP水平均明显高于对照组[（7．85±5．89）、（4．49±2．68）、（8．79±5．09）、（9．37±5．49）、（10．93±4．56）、（4．89±2．09）mg／L比（1．56±1．03）mg／L,均P〈0．01],HbA1c水平与对照组比较差异均无统计学意义[（6．48±1．32）％、（6．33±1．60）％、（6．54±1．68）％、（6．79±1．32）％、（6．49±1．46）％、（6．52±1．32）％比（6．32±1．20）％,均P〉0．05]。hs-CRP与病变支数、病变类型呈正相关（r=0．63,r=0．74,均P〈0．01）。HbA1c与病变支数和病变类型无相关性（r=0．09,r=0．06,均P〉0．05）？结论ACS患者中血浆hs-CRP水平升高,hs-CRP与冠状动脉病变不稳定程度具相关性。HbAIC不能反映冠状动脉病变的不稳定程度。ACS急性期hs-CRP与HbA1c无相关性,HbA1c与动脉粥样硬化斑块慢性进展、累及范围相关,hs-CRP更多反映动脉粥样斑块的不稳定状态。     

血清胱抑素C表达及尿液蛋白标记物水平在急性心力衰竭患者预后评估中的价值

目的：探讨血清胱抑素C（ Cys-C）表达及尿液蛋白标记物水平在急性心力衰竭患者预后评估中的价值。方法选取因急性心力衰竭治疗的90例患者作为观察组,并且取健康体检的90例患者作为对照组,分别检测其Cys-C、脑钠肽（BNP）以及血肌酐和尿素氮、左心室射血分数（LVEF）,分析对比不同心功能其分级和对照组的差异以及ACEI的干预性的影响效果,并且检测观察组在刚入院时,心力衰竭后24 h后其血Cys-C、肌酐（ Scr）以及尿β2-微球蛋白（β2-MG）和白蛋白（ ALb）的变化情况,并进行对比分析。结果观察组Cys-C的浓度明显高于对照组（ P ＜0构．05）,而且随着心力衰竭患者的心功能的级别的增高而明显升高（ P ＜0．05）;观察组住院时血Scr和尿β2-MG、Alb水平与心力衰竭24 h比较,差异无统计学意义（ P ＞0．05）,而血Cys-C比较差异有统计学意义（ P ＜0．01）;高Cys-C患者其在6个月内的全因病死率、心血管事件的发生率以及再次入院概率均显著高于低Cys-C患者,差异有统计学意义（ P ＜0．05）;且心功能Ⅳ级患者肌酐水平明显高于对照组,差异有统计学意义（ P ＜0．05）。结论 Cys-C的水平和心功能历程密切相关,其可反映出心力衰竭患者的早期肾脏功能的有关损伤情况,Cys-C水平对全因死亡以及心血管事件的预测意义重大,其明显优于其他的标志物。其被认为是评价心力衰竭患者早期肾损伤的一项快速、可靠的指标。