The effect of the lipophilic cation lucigenin on mitochondria depends on the site of its reduction

The role of NAD(P)H-dependent oxidoreductases of the outer mitochondrial membrane (OMM) in the activation of lipophilic cationic dyes is poorly understood. In the present study we compared the rates of production of reactive oxygen species (ROS) and mitochondriotoxic effects of the redox-cycling lipophilic cationic dye lucigenin upon its activation by the respiratory chain and NAD(P)H-dependent oxidoreductases of the OMM. We found that, only in the presence of external NADH and NADPH, which are unable to penetrate the inner membrane, lucigenin stimulated a massive superoxide production and a fast permeabilization of mitochondrial membranes. The permeabilization was biphasic. The first, cyclosporin A-insensitive and Ca(2+)-independent phase was characterized by increased permeability of the inner mitochondrial membrane to solutes with molecular masses of 相似文献   

Effect of DSG on xenogeneic immune reactivity with special emphasis on human anti-pig cellular reactions in vitro

Abstract: The effect of the immunosuppressive drug 15-deoxyspergualin (DSG) on xenogeneic human anti-porcine cellular reactivity in vitro, including MLR induced proliferation, interleukin-2 (II-2) production, generation of cytotoxic cells, and the effect on antibody-dependent cellular cytotoxicity (ADCC), were compared with the effects of cyclosporin A (CsA) and/or FK506. The cytotoxic response was evaluated for both direct and indirect pathways for antigen presentation. In addition, the effects of DSG and CsA on antibody production to pig peripheral blood lymphocytes (PBL) in mice was studied. The degree of immunosuppression of xenogeneic and allogeneic cellular responses was compared. CsA and FK506 effectively inhibited proliferation and II-2 production induced by allogeneic human PBL or xenogeneic porcine PBL, whereas DSG did not have any effect on these responses. However, DSG suppressed both the allogeneic and xenogeneic in vitro induced cytotoxic responses, to the same level whether induced via the direct or indirect pathways of immune activation. In contrast, CsA inhibited cytotoxicity induced by xenogeneic cells via the direct but not via the indirect pathway. No effect of FK506 and DSG on ADCC was demonstrated.
A 5-day treatment with DSG or CsA of mice immunized with pig PBL partly suppressed antibody production. In DSG treated mice anti-pig PBL antibodies were produced, but titers were lower than in nontreated or CsA treated mice. The results indicate that DSG may be more effective than CsA/FK506 in inhibiting cytotoxic responses and antibody production induced by xenogeneic pig cells. A possible explanation could be that cytotoxicity induced via the indirect activation pathway of xenoreactivity is mediated to a high degree by CD3^- CD16⁺ (natural killer) NK-like cells, and that stimulation of these cells may be more sensitive to DSG than to CsA/FK506.     

环孢素A通过活化粘着斑激酶信号通路促进人滋养细胞的体外生长

目的:探讨粘着斑激酶(focal adhesion kinase,FAK)信号通路在环孢素A(cyclosporin A,CsA)调节人滋养细胞体外生长中的作用。方法:收集因非意愿妊娠6~9周要求行人工流产术的正常妊娠妇女的绒毛组织,体外分离培养获得滋养细胞,经CsA和/或FAK抑制剂Y15处理后,免疫荧光检测滋养细胞FAK的磷酸化水平;BrdU检测滋养细胞的增殖;Annexin V/PI检测滋养细胞的凋亡。结果:CsA可明显提高滋养细胞FAK的活化水平,FAK抑制剂Y15可阻断CsA诱导的FAK活化,阻抑CsA对细胞增殖的促进作用(P0.05)以及对滋养细胞凋亡的抑制作用(P0.05)。结论:CsA可通过活化FAK信号通路促进人早孕期滋养细胞的体外生长,从而有利于正常妊娠的维持。     

New insights in the role of nucleoporins: A bridge leading to concerted steps from HIV-1 nuclear entry until integration

Human Immunodeficiency virus type 1 (HIV-1), as well as many other viruses that depend on nuclear entry for replication, has developed an evolutionary strategy to dock and translocate through the nuclear pore complex (NPC). In particular, the nuclear pore is not a static window but it is a dynamic structure involved in many vital cellular functions, as nuclear import/export, gene regulation, chromatin organization and genome stability. This review aims to shed light on viral mechanisms developed by HIV-1 to usurp cellular machinery to favor viral gene expression and their replication. In particular, it will be reviewed both what is known and what is speculated about the link between HIV translocation through the nuclear pore and the proviral integration in the host chromatin.     

Relapse of aplastic anemia responsive to sirolimus combined with cyclosporine

Aplastic anemia (AA) is an immune-mediated disease. Although most patients are responsive to immunosuppressive therapy (IST) with a combination of anti-thymocyte globulin (ATG) and cyclosporine (CsA), some patients relapse or are refractory to IST. Sirolimus (rapamysin) inhibits the serine-threonine kinase mammalian target of rapamysin (mTOR), and blocks CsA-resistant and calcium-independent pathways late in the progression of the T-cell cycle. We report two cases of AA which relapsed after CsA and ATG plus CsA, respectively. They achieved transfusion independence after retreatment with sirolimus in combination with a CsA.     

In vitro metabolism of cyclosporine A by human kidney CYP3A5

The objectives of this study were to characterize and compare the metabolic profile of cyclosporine A (CsA) catalyzed by CYP3A4, CYP3A5 and human kidney and liver microsomes, and to evaluate the impact of the CYP3A5 polymorphism on product formation from parent drug and its primary metabolites. Three primary CsA metabolites (AM1, AM9 and AM4N) were produced by heterologously expressed CYP3A4. In contrast, only AM9 was formed by CYP3A5. Substrate inhibition was observed for the formation of AM1 and AM9 by CYP3A4, and for the formation of AM9 by CYP3A5. Microsomes isolated from human kidney produced only AM9 and the rate of product formation (2 and 20 microM CsA) was positively associated with the detection of CYP3A5 protein and presence of the CYP3A5*1 allele in 4 of the 20 kidneys tested. A kinetic experiment with the most active CYP3A5*1-positive renal microsomal preparation yielded an apparent Km (15.5 microM) similar to that of CYP3A5 (11.3 microM). Ketoconazole (200 nM) inhibited renal AM9 formation by 22-55% over a CsA concentration range of 2-45 microM. Using liver microsomes paired with similar CYP3A4 content and different CYP3A5 genotypes, the formation of AM9 was two-fold higher in CYP3A5*1/*3 livers, compared to CYP3A5*3/*3 livers. AM19 and AM1c9, two of the major secondary metabolites of CsA, were produced by CsA, AM1 and AM1c when incubated with CYP3A4, CYP3A5, kidney microsomes from CYP3A5*1/*3 donors and all liver microsomes. Also, the formation of AM19 and AM1c9 was higher from incubations with liver and kidney microsomes with a CYP3A5*1/*3 genotype, compared to those with a CYP3A5*3/*3 genotype. Together, the data demonstrate that CYP3A5 may contribute to the formation of primary and secondary metabolites of CsA, particularly in kidneys carrying the wild-type CYP3A5*1 allele.     

盐酸小檗碱与环孢素A合用对小鼠肝药物代谢酶的影响

目的：阐明盐酸小檗碱与环孢素A合用对药物代谢酶的影响，方法：采用分光光度法测定盐酸小檗碱，环孢素A及二者合用时小鼠肝微粒体CYP450，ERD，ADM和GST的含量或活性。结果：ig给药3和6d，盐酸小檗碱（200mg/kg)对CYP450，ERD没有明显的抑制作用，但对ADM和GSP有抑制作用，环孢素A（45mg/kg)对ERD，AMD和GST均有抑制作用；盐酸小檗碱与环孢素A合用对CYP450，ERD，ADM和GST均有明显抑制作用。结论：盐酸小檗碱与环孢素A的组合是药物代谢酶CYP450，ERD（CYP3A），ADM（CYP1A1，2B1，2C11）和GST的强抑制剂，其总体抑制水平至少与已知的抑制剂酮康唑和肖苯地平相当甚至更强。     

Influence of the MDR1 haplotype and CYP3A5 genotypes on cyclosporine blood level in Chinese renal transplant recipients

1. The purpose of the study was to elucidate the influence of multidrug resistance gene (MDR1) haplotype and CYP3A5 genotype on cyclosporine (CsA) blood level in Chinese renal transplant recipients.

2. CsA trough level (C₀) and peak level (C₂) of 115 patients 1 week and 1 month after renal transplantation were determined. MDR1 C1236T, G2677T/A, C3435T and CYP3A5*3 genotypes were determined by polymerase chain reaction (PCR) assays based on amplification refractory mutation.

3. Dose-adjusted C₀ (C₀/D), C₂ (C₂/D) were 50.5?±?22.5, 267.8?±?110.1 ng·kg·(ml·mg)^?1 after 1 week of therapy, and 79.3?±?29.4, 406.0?±?135.3 ng·kg·(ml·mg)^?1 after 1 month of therapy. Frequencies of MDR1 haplotype TTT, CGC, and TGC were 27.0%, 25.2% and 20.0%, respectively. After 1 month of therapy, C₂/D of TTT/TTT patients were 30% (p = 0.057) and 53% (p = 0.003) higher than CGC/TTT and CGC/CGC patients. C₀/D of CYP3A5 *1/*1, *1/*3 and *3/*3 patients after 1 month of therapy were 51.8?±?25.0, 71.5?±?27.6, and 86.7?±?28.6 ng·kg·(ml·mg)^?1 (p < 0.05).

4. MDR1 haplotypes and CYP3A5*3 genotypes can be related to C₂ and C₀ of CsA, respectively.

     

Investigation into the cardiotoxic effects of doxorubicin on contractile function and the protection afforded by cyclosporin A using the work-loop assay

Doxorubicin is known to cause cardiotoxicity through multiple routes including the build-up of reactive oxygen species and disruption of the calcium homeostasis in cardiac myocytes, but the effect of drug treatment on the associated biomechanics of cardiac injury remains unclear. Detecting and understanding the adverse effects of drugs on cardiac contractility is becoming a priority in non-clinical safety pharmacology assessment. The work-loop technique enables the assessment of force–length work-loop contractions, which mimic those of the pressure–volume work-loops experienced by the heart in vivo.During this study we evaluated whether the work-loop technique could potentially provide improved insight into the biomechanics associated with drug-induced cardiac dysfunction. In order to do this we investigated the cardiotoxic effects of doxorubicin and characterised the protection afforded by the co-administration of cyclosporin A (CsA).This study provides detailed biomechanical in vitro insight into the cardiac dysfunction associated with Doxorubicin treatment, including reduction in peak force, force during shortening and power output. These effects were significantly abrogated in doxorubicin-CsA co-treatment studies.Closely mimicking the in vivo pressure–volume muscle mechanics, this assay provides a quick and easy technique to gain a better understanding of the detailed biomechanics of drug-induced cardiac dysfunction.     

环孢素A在实验性大鼠胰性脑病中的保护作用

目的:探讨环孢素(cyclosporine A,CsA)对大鼠胰性脑病脑组织损害的保护作用,为胰性脑病的治疗提供实验依据。方法:将60只Wister大鼠随机分为对照组和CsA治疗组,两组大鼠均经颈内动脉注射精制胰磷脂酶A2(PLA2),建立大鼠胰性脑病模型,CsA治疗组在造模前5min腹腔注射CsA(30mg/kg),之后每天同时间腹腔注射CsA(30 mg/kg),直至处死,对照组用等容积生理盐水代替。对照组和CsA治疗组分别于造模后1、3及7d各处死动物10只,测定大鼠脑组织匀浆TNF-α、IL-10水平,并检测脑组织含水量、脑组织的组织学改变、中枢神经系统脱髓鞘改变。结果:经CsA处理后,大鼠脑组织匀浆TNF-α水平、脑组织含水量较对照组显著降低(P<0.05),而IL-10则较对照组升高(P<0.05),CsA治疗组脑组织神经细胞水肿较轻,脱髓鞘改变轻微。结论:CsA可以减轻胰性脑病脑损害的发生和发展。