应用PKA抑制法测定C1抑制剂功能活性

本文介绍了一简便、快速测定 Cl-INH 功能活性的方法。此法是向待检血浆中加入一定量的 PKA,在37℃培育一定时间后,测定残留的 PKA 活性,利用 Cl-INH 对 PKA 抑制作用来定量测定 Cl-INH 功能活性。结果表明该方法重复性好 Cl-INH 功能活性与其相应的抗原量完全相符(r=0.913,n=40)。用此法测定25份正常人血浆,18份 HAE 病人血浆,其活性分别为117.2±22.3%和20.08±10.6%。     

抑肽酶的提取及活性研究

以牛肺为原料,经酸沉淀,胰蛋白酶亲和层析,分离提纯抑肽酶。并建立了二种抑肽酶活性测定方法(UKIU法及PKAIU法)。提纯后抑肽酶纯度提高88.3倍。得率为96.8％。活性抑制试验表明,该抑肽酶可抑制PKA对前激肽释放酶(PK)的作用,以及尿激酶对纤溶酶原的作用,对纤溶酶原的活化及纤溶酶活性也有较强抑制作用。     

人组织激肽释放酶基因的克隆及融合蛋白的表达

目的 开发激肽释放酶基因工程产品,为开展基因治疗高血压奠定基础。方法 采用RT-PCR的方法合成人胰腺 cDNA,从中扩增出Kallikrein(KK)基因。经 XhoI和EcoR I双酶切后,连接到pET-28b(+)载体上,酶切鉴定后,进行核苷酸序列分析和融合蛋白的表达。结果 将IPTG诱导表达的菌体进行SDS-PAGE电泳,与蛋白标准品比较,在相对分子质量31800处可见明显的高表达带。免疫印迹实验表明重组蛋白具有KK的抗原性。结论 已成功克隆并表达了人胰腺组织激肽释放酶基因,为进一步开发基因工程产品及进行基因治疗高血压研究打下了基础。     

用盐析与疏水层析相偶合快速分离提纯猪胰激肽释放酶

将疏水层析技术用于从猪胰脏中分离纯化激肽释放酶,建立了一种简便、快速的分离提纯方法：将粗品溶解后经过硫酸铵沉淀处理,然后经过Butyl Sepharose FF疏水层析后得到目标蛋白,分析其纯度大于500U／mg,盐析和疏水两步纯化的收率大于85．0％,同时比较了Phenyl Sepharose FF,Octyl Sepharose FF和Butyl Sepharose FF三种疏水介质分离纯化胰K的效果,本实验工艺与传统工艺相比,具有操作简单、快速、回收率和纯化倍数高等优点,有望成为一种从动物组织中快速分离纯化药用蛋白质的有效技术平台。     

组织激肽释放酶的研究进展

组织激肽释放酶为一类性质相似的化合物,可由肝脏、胰脏、脑神经等合成。人类多种疾病与激肽释放酶基因表达相关,该基因是高血压基因治疗研究的首选靶点。本文综述了激肽释放酶基因与疾病的相关性以及该基因重组载体的构建和表达等研究进展。     

人组织激肽释放酶成熟蛋白的纯化及活性分析

目的　制备纯化人组织激肽释放酶 ,并检测其生物活性 ,为中试放大及纯化工艺奠定基础。方法　使用麦芽糖 (MBP)融合分泌载体pMBP P ,构建并筛选出 1株工程菌株 ,在 6L培养基中经IPIG诱导表达人组织激肽释放酶融合蛋白。目的蛋白经亲和层析纯化及凝血酶切割后 ,采用电位滴度法测定其活力。结果　筛选出的工程菌株表达相对分子质量约 70 0 0 0的激肽释放酶融合蛋白 ,大小与预计大小相符。纯化后共获得 2 8mg蛋白 ,并具有水解苯甲酰精胺酸乙酯 (BAEE)的能力。结论　成功进行了人组织激肽释放酶融合成熟蛋白的小量制备 ,经纯化后的产物具有生物活性 ,为中试放大、纯化工艺研究奠定了基础。     

八肽胆囊收缩素对肿瘤坏死因子-α诱导大鼠RSC-364细胞增殖及金属蛋白酶分泌的影响及作用机制

目的探讨八肽胆囊收缩素(cholecystokinin octapeptide,CCK-8)对肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)诱导大鼠RSC-364细胞增殖及金属蛋白酶(matrix metalloproteinases,MMPs)分泌的影响及作用机制。方法采用噻唑蓝(MTT)比色法检测TNF-α诱导RSC-364细胞的增殖情况,ELISA法测定细胞中MMPs(MMP-1、MMP-3、MMP-9)的分泌水平、环腺苷酸(cyclic adenosine monophosphate,c AMP)含量和蛋白激酶A(protein kinase A,PKA)活性。结果 TNF-α可促进RSC-364细胞增殖及MMP-1、MMP-3、MMP-9的分泌,CCK-8可抑制TNF-α的上述作用,Forskolin(一种c AMP诱导剂)与CCK-8的上述作用类似,而H89(PKA特异性抑制剂)可部分逆转CCK-8的上述作用。TNF-α可提高细胞中c AMP含量和PKA活性,CCK-8(10~(-10)、10~(-8)、10~(-6) mol/L)可进一步促进TNF-α的上述作用,且呈剂量依赖性,CR1409(CCK-A受体结抗剂)和CR2945(CCK-B受体结抗剂)可部分逆转CCK-8的上述作用,以CR2945为著。结论 CCK-8可通过活化c AMP-PKA信号通路,进而抑制TNF-α促细胞增殖和MMP-1、MMP-3、MMP-9的分泌,该过程由CCK受体介导。     

冻干低pH静脉注射丙种球蛋白PKA含量测定的影响因素

运用正交试验设计研究静注丙球PKA含量测定的影响因素，结果发现，制品pH值、PK活性和制品溶解液离子强度是影响试验的主要因素。用生理盐水溶解静注丙球并调高PH值至7．5左右，用较高活性的PK进行试验可获得较高的A值。低PH环境可使制品中PKA活性降低。低PH值可提高试验的灵敏度。     

95%乙醇循环系统对人血白蛋白质量的影响分析

目的分析采用低温乙醇工艺制备人血白蛋白时,95%乙醇循环系统对人血白蛋白原液的超滤收率和成品各项质量指标的影响。方法在多批次规模化生产中,分别测定对95%乙醇新增乙醇循环系统前后人血白蛋白原液的超滤收率和成品的纯度、多聚体含量、激肽释放酶原激活剂(prekallikrein activator,PKA)等指标,并将测定结果进行对比。结果新增乙醇循环系统后,人血白蛋白成品的纯度明显升高(P0.001),多聚体含量明显下降(P0.001),PKA无明显变化;同时,人血白蛋白原液超滤收率呈稳定趋势,未表现出明显变化。结论在低温乙醇工艺制备人血白蛋白的过程中,对95%乙醇使用乙醇循环系统自动控制添加过程,可降低操作难度,提高工作效率及操作精细度,更好地实现制品分离过程,进一步提升人血白蛋白产品的质量。     

室内装修中人造板的甲醛释放规律研究

本文对室内装饰用的人造板的甲醚释放规律进行研究。试验结果表明,人造板甲醛释放量随温度升高而增加;随含水率增加而增大：随人造板放置时间增长,而逐渐降低。人造板封边与否,则明显影响甲醛释放量。本论文的研究可以为室内甲醛污染的治理提供依据。     

Sphingosine Kinase Interacting Protein is an A‐Kinase Anchoring Protein Specific for Type I cAMP‐Dependent Protein Kinase

The compartmentalization of kinases and phosphatases plays an important role in the specificity of second‐messenger‐mediated signaling events. Localization of the cAMP‐dependent protein kinase is mediated by interaction of its regulatory subunit (PKA‐R) with the versatile family of A‐kinase‐anchoring proteins (AKAPs). Most AKAPs bind avidly to PKA‐RII, while some have dual specificity for both PKA‐RI and PKA‐RII; however, no mammalian PKA‐RI‐specific AKAPs have thus far been assigned. This has mainly been attributed to the observation that PKA‐RI is more cytosolic than the more heavily compartmentalized PKA‐RII. Chemical proteomics screens of the cAMP interactome in mammalian heart tissue recently identified sphingosine kinase type 1‐interacting protein (SKIP, SPHKAP) as a putative novel AKAP. Biochemical characterization now shows that SPHKAP can be considered as the first mammalian AKAP that preferentially binds to PKA‐RIα. Recombinant human SPHKAP functions as an RI‐specific AKAP that utilizes the characteristic AKAP amphipathic helix for interaction. Further chemical proteomic screening utilizing differential binding characteristics of specific cAMP resins confirms SPHKAPs endogenous specificity for PKA‐RI directly in mammalian heart and spleen tissue. Immunolocalization studies revealed that recombinant SPHKAP is expressed in the cytoplasm, where PKA‐RIα also mainly resides. Alignment of SPHKAPs' amphipathic helix with peptide models of PKA‐RI‐ or PKA‐RII‐specific anchoring domains shows that it has largely only PKA‐RIα characteristics. Being the first mammalian PKA‐RI‐specific AKAP with cytosolic localization, SPHKAP is a very promising model for studying the function of the less explored cytosolic PKA‐RI signaling nodes.     

Involvement of cAMP-Dependent Protein Kinase in the Nucleus Accumbens in Cocaine Versus Social Interaction Reward

Evidence suggests that PKA activity in the nucleus accumbens (NAc) plays an essential role in reward-related learning. In this study, we investigated whether PKA is differentially involved in the expression of learning produced by either natural reinforcers or psychostimulants. For that purpose, we inhibited PKA through a bilateral infusion of Rp-cAMPS, a specific PKA inhibitor, directly into the NAc. The effects of PKA inhibition in the NAc on the expression of concurrent conditioned place preference (CPP) for cocaine (drug) and social interaction (natural reward) in rats were evaluated. We found that PKA inhibition increased the expression of cocaine preference. This effect was not due to altered stress levels or decreased social reward. PKA inhibition did not affect the expression of natural reward as intra-NAc Rp-cAMPS infusion did not affect expression of social preference. When rats were trained to express cocaine or social interaction CPP and tested for eventual persisting preference 7 and 14 days after CPP expression, cocaine preference was persistent, but social preference was abolished after the first test. These results suggest that PKA in the NAc is involved in drug reward learning that might lead to addiction and that only drug, but not natural, reward is persistent.     

Compartmentalization Role of A-Kinase Anchoring Proteins (AKAPs) in Mediating Protein Kinase A (PKA) Signaling and Cardiomyocyte Hypertrophy

The Beta-adrenergic receptors (β-ARs) stimulation enhances contractility through protein kinase-A (PKA) substrate phosphorylation. This PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs). AKAPs coordinate multi-protein signaling networks that are targeted to specific intracellular locations, resulting in the localization of enzyme activity and transmitting intracellular actions of neurotransmitters and hormones to its target substrates. In particular, mAKAP (muscle-selective AKAP) has been shown to be present on the nuclear envelope of cardiomyocytes with various proteins including: PKA-regulatory subunit (RIIα), phosphodiesterase-4D3, protein phosphatase-2A, and ryanodine receptor (RyR2). Therefore, through the coordination of spatial-temporal signaling of proteins and enzymes, mAKAP controls cyclic-adenosine monophosphate (cAMP) levels very tightly and functions as a regulator of PKA-mediated substrate phosphorylation leading to changes in calcium availability and myofilament calcium sensitivity. The goal of this review is to elucidate the critical compartmentalization role of mAKAP in mediating PKA signaling and regulating cardiomyocyte hypertrophy by acting as a scaffolding protein. Based on our literature search and studying the structure–function relationship between AKAP scaffolding protein and its binding partners, we propose possible explanations for the mechanism by which mAKAP promotes cardiac hypertrophy.     

Serotonin/5-HT1A Signaling in the Neurovascular Unit Regulates Endothelial CLDN5 Expression

We previously reported that site-selective claudin-5 (CLDN5) breakdown and protein kinase A (PKA) activation are observed in brain microvessels of schizophrenia, but the underlying molecular basis remains unknown. The 5-HT1 receptors decline the intracellular cAMP levels and inactivate the major downstream PKA, and the 5-HT1A receptor is a promising target for schizophrenia. Therefore, we elucidated the involvement of serotonin/5-HT1A signaling in the endothelial CLDN5 expression. We demonstrate, by immunohistochemistry using post-mortem human brain tissue, that the 5-HT1A receptor is expressed in brain microvascular endothelial cells (BMVECs) and mural cells of the normal prefrontal cortex (PFC) gray matter. We also show that PKA is aberrantly activated not only in BMVECs but also in mural cells of the schizophrenic PFC. We subsequently revealed that the endothelial cell–pericyte tube-like structure was formed in a novel two-dimensional co-culture of human primary BMVECs and a human brain-derived pericyte cell line, in both of which the 5-HT1A receptor was expressed. Furthermore, we disclose that the serotonin/5-HT1A signaling enhances endothelial CLDN5 expression in BMVECs under two-dimensional co-culture conditions. Our findings provide novel insights into the physiological and pathological significance of serotonin/5-HT1A signaling in the region-specific regulation of the blood-brain barrier.     

Multi-Omics Approach Profiling Metabolic Remodeling in Early Systolic Dysfunction and in Overt Systolic Heart Failure

Metabolic remodeling plays an important role in the pathophysiology of heart failure (HF). We sought to characterize metabolic remodeling and implicated signaling pathways in two rat models of early systolic dysfunction (MOD), and overt systolic HF (SHF). Tandem mass tag-labeled shotgun proteomics, phospho-(p)-proteomics, and non-targeted metabolomics analyses were performed in left ventricular myocardium tissue from Sham, MOD, and SHF using liquid chromatography–mass spectrometry, n = 3 biological samples per group. Mitochondrial proteins were predominantly down-regulated in MOD (125) and SHF (328) vs. Sham. Of these, 82% (103/125) and 66% (218/328) were involved in metabolism and respiration. Oxidative phosphorylation, mitochondrial fatty acid β-oxidation, Krebs cycle, branched-chain amino acids, and amino acid (glutamine and tryptophan) degradation were highly enriched metabolic pathways that decreased in SHF > MOD. Glycogen and glucose degradation increased predominantly in MOD, whereas glycolysis and pyruvate metabolism decreased predominantly in SHF. PKA signaling at the endoplasmic reticulum–mt interface was attenuated in MOD, whereas overall PKA and AMPK cellular signaling were attenuated in SHF vs. Sham. In conclusion, metabolic remodeling plays an important role in myocardial remodeling. PKA and AMPK signaling crosstalk governs metabolic remodeling in progression to SHF.     

Serotonin (5-HT) 2A Receptor Involvement in Melanin Synthesis and Transfer via Activating the PKA/CREB Signaling Pathway

The 5-HT2A serotonin receptor (HTR2A) has been reported to be involved in the serotonin- or serotonin receptor 2A agonist-induced melanogenesis in human melanoma cells. However, the molecular mechanisms underlying HTR2A in regulating melanogenesis remain poorly understood. In this research, cultured mouse melanoma cell line B16F10, human skin, and zebrafish embryos were used to elucidate the downstream signaling of HTR2A in regulating melanogenesis and to verify the potential application of HTR2A in the treatment of pigment-associated cutaneous diseases. We demonstrated that HTR2A antagonists (AT1015 and ketanserin) attenuated the melanogenesis induction of serotonin in both mouse melanoma cells and zebrafish embryos. The agonists of HTR2A (DOI and TCB-2) increased melanin synthesis and transfer in B16F10 cells, human skin tissue, and zebrafish embryos. Furthermore, the HTR2A agonists increased the expression of proteins related to melanosome organization and melanocyte dendrites to facilitate the melanocyte migration and melanosome transport. HTR2A antagonists and genetic knockout of zebrafish htr2aa (the homologue of mammalian HTR2A gene) were also used to clarify that HTR2A mediates serotonin and DOI in regulating melanogenesis. Finally, through small scale screening of the candidate downstream pathway, we demonstrated that HTR2A mediates the melanogenesis induction of its ligands by activating the PKA/CREB signaling pathway. In this research, we further confirmed that HTR2A is a crucial protein to mediate melanocyte function. Meanwhile, this research supports that HTR2A could be designed as a drug target for the development of chemicals to treat cutaneous diseases with melanocytes or melanogenesis abnormality.     

Bamboo Lignin Fractions with In Vitro Tyrosinase Inhibition Activity Downregulate Melanogenesis in B16F10 Cells via PKA/CREB Signaling Pathway

Cosmetic ingredients originating from natural resources have garnered considerable attention, and the demand for whitening ingredients is increasing, particularly in Asian countries. Lignin is a natural phenolic biopolymer significantly effective as a natural sunscreen, as its ultraviolet protection efficacy ranges from 250 to 400 nm. However, using different types of lignin as cosmetic ingredients is difficult owing to the heterogeneity of lignin and the lack of in vitro and in vivo safety and efficacy data. Thus, steam-exploded lignin (SEL) was prepared from bamboo, fractionated via successive organic solvent extraction, and sequentially fractionated using ethyl acetate, methanol, and acetone to investigate its potential as a natural whitening material. Gel permeation chromatography showed that the molecular weight of acetone-soluble and acetone-insoluble SEL fractions were the lowest and the highest, respectively. Monomer structures of the four lignin fractions were elucidated using ¹H, ¹³C, and 2D heteronuclear single quantum coherence nuclear magnetic resonance and pyrolysis gas chromatography/mass spectrometry. The antioxidant and tyrosinase inhibition activities of the four fractions were compared. The methanol-soluble SEL fraction (SEL-F2) showed the highest antioxidant activity (except 2,2-diphenyl-1-picrylhydrazyl scavenging activity), and the enzyme inhibition kinetics were confirmed. In this study, the expression pattern of the anti-melanogenic-related proteins by SEL-F2 was confirmed for the first time via the protein kinase A (PKA)/cAMP-response element-binding (CREB) protein signaling pathway in B16F10 melanoma cells. Thus, SEL may serve as a valuable cosmetic whitening ingredient.     

Suppression of Lysophosphatidylcholine-Induced Human Aortic Smooth Muscle Cell Calcification by Protein Kinase A Inhibition

Lysophosphatidylcholine (lysoPtdCho) is produced mainly by the phospholipase A2-dependent hydrolysis of phosphatidylcholine (PtdCho) and can induce inflammatory activation and osteogenic gene expression in vascular smooth muscle cells. However, the mechanisms mediating these processes have not been fully elucidated. In this study, we investigated whether inhibition of protein kinase A (PKA) signaling suppressed lysoPtdCho-induced calcification of human aortic smooth muscle cells (HASMC). Calcium levels and alkaline phosphatase activity were significantly increased in HASMC treated with lysoPtdCho, but not PtdCho, compared with those in phosphate-buffered saline-treated HASMC. However, the addition of a PKA inhibitor (H-89) or PKA siRNA blocked lysoPtdCho-induced HASMC calcification. These results showed that lysoPtdCho could activate PKA-mediated HASMC calcification and that PKA may be a therapeutic target for lysoPtdCho-mediated vascular smooth muscle cell calcification.     

酿酒酵母TOR信号通路研究进展

TOR信号通路是酿酒酵母非常重要的保守信号通路,它连接着细胞所能得到的营养信号与细胞生长过程.它可以通过调节翻译、转录、核糖体生物合成、营养物质的运转以及与营养条件相关的自溶来调控细胞的生长.本文综述了酿酒酵母TOR信号通路的主要作用元件,并介绍了其与酿酒酵母时序寿命和Ras/cAMP/PKA信号通路的关系.