The Bbeta-sheet in the PAI-1 molecule plays an important role for its stability

We have investigated the B beta-sheet in PAI-1 regarding its role for the stability of the molecule. The residues from His(219) to Tyr(241) (except for Gly(230) and Pro(240)), covering the s2B and s3B strands, and in addition His(185) and His(190)) were substituted by amino acids with opposite properties. The 23 generated single-site changed mutants and also wild type PAI-1 (wtPAI-1) were expressed in E. coli. Subsequently they were purified by heparin-Sepharose and anhydrotrypsin agarose affinity chromatographies. The stability of the purified PAI-1 variants was analyzed at 37 degrees C and at different pHs (5.5, 6.5 or 7.5). At pH 7.5 and 37 degrees C, single substitutions of the residues in the central portions of both strands 2 and 3 in the B beta-sheet (Ile(223) to Leu(226) on s2B and Met(235) to Ile(237) on s3B), caused a significant decrease in stability, yielding half-lives of about 10-25% as compared to wtPAI-1. On the other hand, mutations at both sides of the central portion of the B beta-sheet (Tyr(221), Asp(222), Tyr(228) and Thr(232)) frequently resulted in an increased PAI-1 stability (up to 7-fold). While wtPAI-1 exhibited prolonged half-lives at pH 6.5 and 5.5, the PAI-1 variant Y228S was more stable at neutral pH (half-life of 9.6 h at pH 7.5) as compared to its half-life at pH 5.5 (1.1 h). One of the 4 modified histidine residues (His(229)) resulted in a variant with a clearly affected stability as a function of pH, suggesting that it may, at least in part, be of importance for the pH dependence of the PAI-1 stability. Thus, our data demonstrate that the B beta-sheet is of great importance for the stability of the molecule. Modifications in this part causes decreased or increased stability in a certain pattern, suggesting effects on the insertion rate of the reactive center loop into the A beta-sheet of the molecule.     

Plasma levels of t-PA free PAI-1 and a complex of t-PA with PAI-1 in human males and females at various ages

The concentration of tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1) and the complex of t-PA and PAI-1 (t-PA-PAI-1 complex) were measured using an enzyme immunoassay, where the first antibody was monoclonal antibody against PAI-1 and the second one was polyclonal anti-t-PA Fab' antibody. Plasma levels of t-PA and t-PA-PAI-1 complex increased with increase in age in the total population of males and females, but there was no age related change in free and total PAI-1. Plasma levels of t-PA antigen were higher in males than in females, and those in females gradually increased with increase in age, reaching the same levels as those of males at their 60s. Plasma levels of t-PA-PAI-1 complex were also higher in males than in females, but the difference was low at their 50s and no difference was observed at their 60s. Plasma levels of free PAI-1 were higher in males than in females, but those of males declined at age group higher than their 50s. At their 60s plasma levels of free PAI-1 were higher in females than in males. These results indicate that fibrinolytic parameters such as t-PA and PAI-1 were balanced at higher levels in plasma in males than in females.     

Heme oxygenase-1 plays an important protective role in experimental autoimmune encephalomyelitis.

Increasing evidence shows that oxidative stress plays an important role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of the human disease, multiple sclerosis (MS). Heme oxygenase-1 (HO-1) is a heat shock protein induced by oxidative stress. HO-1 metabolizes heme to the antioxidant bilirubin and carbon monoxide, and represents a powerful endogenous defensive mechanism against free radicals in many diseases. However, the role of this important enzyme in EAE remains unknown. In this study, we showed high expression of HO-1 in lesions of EAE, and demonstrated that hemin, an inducer of HO-1, inhibited EAE effectively. In contrast, tin mesoporphyrin, an inhibitor of HO-1, markedly exacerbated EAE. Our results suggest that endogenous HO-1 plays an important protective role in EAE, and that targeted induction of HO-1 overexpression may represent a new therapy for the treatment of multiple sclerosis.     

Relationships between euglobulin clot lysis time and the plasma levels of tissue plasminogen activator and plasminogen activator inhibitor 1

The relationships between tissue plasminogen activator (tPA), its fast acting inhibitor (PAI-1) and euglobulin clot lysis time (ELT) were investigated with healthy volunteers' plasma. Turbidimetric clot lysis assay by the microtiter plate reader was utilized for ELT with a slight modification. Both tPA and PAI-1 showed the significant correlation with ELT. tPA had a significantly positive, not negative, correlation with ELT (R = 0.387, p less than 0.001). Higher correlation coefficients (R = 0.580, p less than 0.001 and R = 0.599, p less than 0.001) were obtained between ELT and total PAI-1 or free PAI-1 than tPA or tPA-PAI-1 complex (R = 0.427, p less than 0.001). The positive correlation was also obtained between tPA and PAI-1. These data suggest that PAI-1 is a highly important factor for ELT, especially, the amounts of free PAI-1 being the key factor to determine the ELT, which can represent the potential activity of the fibrinolytic system.     

Transferrin receptor 1 plays an important role in muscle development and denervation-induced muscular atrophy

Previous studies demonstrate an accumulation of transferrin and transferrin receptor 1(TfR1) in regenerating peripheral nerves.However, the expression and function of transferrin and TfR1 in the denervated skeletal muscle remain poorly understood.In this study, a mouse model of denervation was produced by complete tear of the left brachial plexus nerve.RNA-sequencing revealed that transferrin expression in the denervated skeletal muscle was upregulated, while TfR1 expression was downregulated.We also investigated the function of TfR1 during development and in adult skeletal muscles in mice with inducible deletion or loss of TfR1.The ablation of TfR1 in skeletal muscle in early development caused severe muscular atrophy and early death.In comparison, deletion of TfR1 in adult skeletal muscles did not affect survival or glucose metabolism, but caused skeletal muscle atrophy and motor functional impairment, similar to the muscular atrophy phenotype observed after denervation.These findings suggest that TfR1 plays an important role in muscle development and denervation-induced muscular atrophy.This study was approved by the Institutional Animal Care and Use Committee of Beijing Institute of Basic Medical Sciences, China(approval No.SYXK 2017-C023) on June 1, 2018.     

Fibrin clot lysis by thrombolytic agents is impaired in newborns due to a low plasminogen concentration.

Although thrombolytic drugs have been extensively used in adults, there is sparse information on their effectiveness in newborns whose fibrinolytic system differs significantly from adults. The purpose of this study was to determine if low plasma levels of plasminogen in cord plasma limited the therapeutic effectiveness of thrombolytic agents. Urokinase (UK), streptokinase (SK) and tissue plasminogen activator (TPA) were compared for their ability to lyse washed 125I-labelled adult or cord fibrin clots suspended in cord or adult plasma. 125I-labelled fibrin clots were prepared by recalcifying cord or adult plasma spiked with labelled fibrinogen and then placed into cord or adult plasma which contained either saline or differing amounts of a specific thrombolytic agent. After a 60 min incubation, the remaining 125I-fibrin in clots released 125I-fibrin fragments, and concentrations of fibrinogen, alpha 2-antiplasmin, and plasminogen in the bathing plasma were measured and compared to starting values. Cord fibrin clots were more resistant than adult fibrin clots to all thrombolytic drugs tested (p less than 0.001). On average, the cord system retained 27% more 125I-fibrin in clots, and released 32% less 125I-fibrin fragments into plasma. Fibrinogenolysis was also decreased in cord plasmas compared to adult plasmas. The degree of fibrinolysis and fibrinogenolysis in cord plasma increased to adult values when plasminogen concentrations were increased in the bathing plasma. Thus, cord fibrin clots have an impaired response to thrombolytic agents secondary to low levels of plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

兴奋毒性在肌萎缩侧索硬化的发病机制中具有重要作用

目的:分析比较肌萎缩侧索硬化病人和神经系统正常的受试者脑脊液中的谷氨酸水平,以明确由谷氨酸介导的“兴奋毒性”是否在ALS的发病机制中具有作用。方法:肌萎缩侧索硬化病人15例,神经系统正常的外科手术腰麻病人20例,采集脑脊液后用氨基酸自动分析仪进行检测。结果:肌萎缩侧索硬化病人脑脊液中谷氨酸水平与对照比较增高有极显著差异(两组分别为48.81±31.67μmol/L和15.85±6.70μmol/L)(P(0.01)。结论:由谷氨酸介导的“兴奋毒性”在肌萎缩侧索硬化的发病过程具有重要作用。     

The hardness of food plays an important role in food selection behavior in rats

To examine the importance of the hardness of foods, we conducted behavioral and electromyographical experiments in Wistar male rats using three kinds of pellets, a hard commercially made pellet (MF), a hard privately produced pellet (H) and a soft privately produced pellet (S). MF and H had the same hardness but contained different ingredients, and S and H had the same ingredients but different degrees of hardness (S相似文献   

Evidence that the lateral tegmental field plays an important role in the central sympathoinhibitory action of 8-OH-DPAT.

We examined the effects of 8-OH-DPAT and 5-HT on three types of sympathetic-related neurons identified in the lateral tegmental field of anesthetized cats using spike-triggered averaging techniques. Based on their response to baroreceptor activation, these neurons could be classified as sympathoexcitatory, sympathoinhibitory, or baroreceptor activation unresponsive. 8-OH-DPAT administered intravenously was found to inhibit sympathoexcitatory neurons with a high degree of correlation to inhibition of sympathetic activity, and to excite sympathoinhibitory neurons in a dose-dependent manner. Iontophoretic application of 8-OH-DPAT and 5-HT to the majority of sympathoexcitatory neurons caused inhibition of spontaneous activity while iontophoretic application of 8-OH-DPAT to sympathoinhibitory neurons had variable effects although 5-HT consistently caused excitation. Baroreceptor unresponsive neurons were insensitive to iontophoretic 8-OH-DPAT and showed only limited response to 5-HT. It is concluded that the lateral tegmental field plays an important role in the sympathoinhibitory action of 8-OH-DPAT.     

The gender-specific role of polymorphisms from the fibrinolytic, renin-angiotensin, and bradykinin systems in determining plasma t-PA and PAI-1 levels

Tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) directly influence thrombus formation and degradation and thus risk for arterial thrombosis. We report here results from a genetic analysis of plasma t-PA and PAI-1 levels in a large population-based sample from the PREVEND study in Groningen, the Netherlands (n = 2,527). We measured polymorphisms from genes of the fibrinolytic system, the renin-angiotensin system (RAS), and the bradykinin system. We found that males had higher levels of natural-log transformed t-PA, and PAI-1 (P < 0.01) compared to females. When stratifying females by menopausal status, PAI-1 levels were only significantly different between pre-menopausal females and males (p < 0.001). Furthermore, we found that age, body mass index, and waist-to-hip ratio were significant predictors of t-PA and PAI-1 in both females and males, and that the regression relationships between these factors and plasma t-PA and PAI-1 were dependent on gender. In addition, we found that the PAI-1 4G/5G polymorphism was a significant predictor of PAI-1 levels in both females and males, that the angiotensin II type I receptor A1166C was a significant predictor of t-PA and PAI-1 levels in females, and that the bradykinin receptor B2 58CT polymorphism was a significant predictor of t-PA levels in females. In conclusion, this large population-based study showed that t-PA and PAI-1 levels are determined by several demographic and genetic factors involved in the fibrinolytic, RAS and bradykinin system. In addition, the results support the idea that the biology of t-PA and PAI-1 is different between females and males.     

Regulation of PAI-1 expression by genetic polymorphisms. Impact on atherogenesis

Increased expression of plasminogen activator inhibitor type 1 (PAI-1) is now considered as an independent risk factor for cardiovascular disease. Numerous biochemical factors have been found to regulate the expression of PAI-1 gene and the synthesis of PAI-1 protein. In the recent past, polymorphisms in the PAI-1 gene have been identified and their impact on PAI-1 expression has been characterized. This article will review the current knowledge of these PAI-1 gene polymorphisms (in cell culture epidemiological, and clinical studies) and their role in the development of cardiovascular disease.     

Regulation of PAI-1 concentration in platelets by systemic administration of antisense oligonucleotides to rats.

In this report we tested the effect of oligodeoxyribonucleotides antisense to PAI-1 mRNA administered into rats on PAI-1 concentration in platelets. Low doses of the antisense oligonucleotide (MPO-16R) reduced PAI-1 activity, both in rat blood plasma and platelet lysates by 20.5% and 28.7%, respectively. There was no change in platelet count after treatment with MPO-16R but treated platelets showed lower aggregability as compared with controls (37 +/- 13% and 54 +/- 12%, respectively). In an experimental model of rat arterial thrombosis, low doses of MPO-16R caused a significant delay in the occlusion time (31.8%). These data further support for the role of PAI-1 as a major determinant of arterial thrombolysis resistance and for the first time demonstrate the possibility of reduction of platelet PAI-1 concentration by antisense approach.     

Diurnal variation in PAI-1 activity predominantly confined to the 4G-allele of the PAI-1 gene

We examined the diurnal pattern in Plasminogen Activator Inhibitor-type 1 (PAI-1) activity and Plasminogen activator (t-PA) in relation to the 4G/5G-polymorphism in the promoter of the PAI-1 gene. The analyses were performed in the Arnhem Elderly Study, a population-based study of 598 elderly. A single blood sample was drawn and the time of blood sampling was recorded (between 8 a.m. and 5.30 p.m.). Plasma PAI-1 activity was strongly associated with time of blood sampling, showing the highest values in the early morning. The diurnal pattern was clearly present in the 4G/4G (n = 184) and 4G/5G (n = 275) genotypes, but not in the 5G/5G-genotype (n = 139). T-PA antigen showed a weak diurnal variation, which did not differ across the variants of the 4G/5G-polymorphism. Our findings raise the hypothesis that 5G-homozygotic persons may be relatively protected from diurnal variation in the occurrence of coronary events.     

NOD2 plays an important role in the inflammatory responses of microglia and astrocytes to bacterial CNS pathogens

While glial cells are recognized for their roles in maintaining neuronal function, there is growing appreciation that resident central nervous system (CNS) cells initiate and/or augment inflammation following trauma or infection. We have recently demonstrated that microglia and astrocytes constitutively express nucleotide-binding oligomerization domain-2 (NOD2), a member of the novel nucleotide-binding domain leucine-rich repeat region containing a family of proteins (NLR) that functions as an intracellular receptor for a minimal motif present in all bacterial peptidoglycans. In this study, we have confirmed the functional nature of NOD2 expression in astrocytes and microglia and begun to determine the relative contribution that this NLR makes in inflammatory CNS responses to clinically relevant bacterial pathogens. We demonstrate the increased association of NOD2 with its downstream effector molecule, Rip2 kinase, in primary cultures of murine microglia and astrocytes following exposure to bacterial antigens. We show that this cytosolic receptor underlies the ability of muramyl dipeptide to augment the production of inflammatory cytokines by glia following exposure to specific ligands for disparate Toll-like receptor homologues. In addition, we demonstrate that NOD2 is an important component in the in vitro inflammatory responses of resident glia to N. meningitidis and B. burgdorferi antigens. Finally, we have established that NOD2 is required, at least in part, for the astrogliosis, demyelination, behavioral changes, and elevated inflammatory cytokine levels observed following in vivo infection with these pathogens. As such, we have identified NOD2 as an important component in the generation of damaging CNS inflammation following bacterial infection.     

GCN2 kinase plays an important role triggering the remission phase of experimental autoimmune encephalomyelitis (EAE) in mice

Experimental autoimmune encephalomyelitis (EAE) has been widely employed as a model to study multiple sclerosis (MS) and indeed has allowed some important advances in our comprehension of MS pathogenesis. Several pieces of evidence suggest that infiltrating Th1 and Th17 lymphocytes are important players leading to CNS demyelination and lesion during the peak of murine EAE. Subsequently, effector T cell responses rapidly decline and the recovery phase of the disease strongly correlates with the expression of anti-inflammatory cytokines and the enrichment of Foxp3+ regulatory T (Treg) cells within the target organ. However, the mechanisms leading to the increased presence of Treg cells and to the remission phase of the disease are still poorly understood. Recent researches demonstrated that chemically induced amino-acid starvation response might suppress CNS immune activity. Here we verified an important participation of the general control nonrepressible 2 (GCN2), a key regulator kinase of the amino-acid starvation response, in the development of the remission phase of EAE in C57BL/6 mice. By immunizing wild type C57BL/6 (WT) and GCN2 knock-out mice (GCN2 KO) with myelin oligodendrocyte glycoprotein peptide (MOG_35–55), it was noticed that GCN2 KO mice did not develop the remission phase of the disease and this was associated with higher levels of CNS inflammation and increased presence of effector T cells (Th1/Th17). These animals also showed lower frequency of Treg cells within the CNS as compared to WT animals. Higher expression of indoleamine 2,3-dioxygenase (IDO) and higher frequency of plasmacytoid dendritic cells (pDCs) were found at the peak of the disease in the CNS of WT animals. Our results suggest that the GCN2 kinase-dependent sensing of IDO activity represents an important trigger to the EAE remission phase. The IDO-mediated immunoregulatory events may include the arresting of effector T cell responses and the differentiation/expansion of Treg cells within the target organ.     

急性脑血管病患者血浆t-PA、PAI-1活性测定及意义

目的 研究急性脑血管病（脑梗死和脑出血）患者的血浆组织型纤溶酶原激活物（t-PA）和纤溶酶原激活物抑制物（PAI-1）的活性变化及临床意义.方法 采用酶联免疫吸附法测定67例急性脑梗死和53例脑出血患者的t-PA和PAI-1的活性,并计算P/t值,与50例正常对照组进行比较分析.结果 急性脑血管病组患者急性期（7d内）血浆t-PA及PAI-1活性均升高,与正常对照组比较有显著性差异（P＜0.05） 脑梗死组P/t值与正常对照组比较有显著性差异（P＜0.05） 脑出血组P/t值与正常对照组比较无显著性差异（P＞0.05）.结论 脑梗死和脑出血患者急性期血浆P/t改变不同 脑梗死患者急性期P/t降低,体内处于相对纤溶亢进状态 脑出血患者P/t变化不明显,体内凝血纤溶机制相对平衡P/t值可以更好地反映血液中的纤溶活性.     

颈动脉粥样硬化斑块内纤溶酶原激活抑制物-1表达的研究

目的观察颈动脉粥样硬化(AS)斑块内纤溶酶原激活抑制物-1(PAI-1)的mRNA和抗原的表达情况.方法分别应用原位杂交和免疫组织化学测定颈AS斑块内PAI-1mRNA和抗原表达.结果相对于正常动脉内膜,颈AS病变内膜PAI-1阳性细胞数明显增多(P＜0.05),其中颈AS严重病变肩区阳性细胞数最多,且以单核-巨噬细胞为主.结论PAI-1与颈AS发生有关.     

Regular exercise, plasminogen activator inhibitor-1 (PAI-1) activity and the 4G/5G promoter polymorphism in the PAI-1 gene.

The aim of this controlled randomised clinical trial was to investigate the effects of regular low to moderate intensity physical activity on plasminogen activator inhibitor-1 (PAI-1) activity during three years while taking into account the 4G/5G polymorphism in the promoter of the PAI-1 gene. Male subjects (age 52-62 years, n = 132) were randomised into an exercise or a (non-intervention) reference group. Aerobic threshold increased by 8.8% (p = 0.025) in the exercise group, and decreased by 1.1% in the non-intervention group, while PAI-1 activity did not change significantly in either study group. However, homozygotes for the 4G allele in the exercise group showed a 36% reduction in PAI-1 (p = 0.025). In conclusion, although regular moderate physical activity did not decrease PAI-I activity in the whole group, regular exercise may be effective for controlling elevated PAI-1 level in subjects homozygous for the 4G allele.