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Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For example, CypA supports cancer proliferation and mediates viral infections, such as the human immunodeficiency virus 1 (HIV-1). Here, we present the design of PROTAC (proteolysis targeting chimera) compounds against CypA to induce its intracellular proteolysis and to investigate their effect on immune cells. Interestingly, upon connecting to E3 ligase ligands, both peptide-based low-affinity binders and CsA-based high-affinity binders can degrade CypA at nM concentration in HeLa cells and fibroblast cells. As the immunosuppressive effect of CsA is not directly associated with the binding of CsA to CypA but the inhibition of phosphatase calcineurin by the CypA:CsA complex, we investigated whether a CsA-based PROTAC compound could induce CypA degradation without affecting the activation of immune cells. P3, the most efficient PROTAC compound discovered from this study, could deplete CypA in lymphocytes without affecting cell proliferation and cytokine production. This work demonstrates the feasibility of the PROTAC approach in depleting the abundant cellular protein CypA at low drug dosage without affecting immune cells, allowing us to investigate the potential therapeutic effects associated with the endogenous protein in the future. [ABSTRACT FROM AUTHOR]

Cyclophilin A (CypA), the cellular receptor of the immunosuppressant cyclosporin A (CsA), is an abundant cytosolic protein and is involved in a variety of diseases. For example, CypA supports cancer proliferation and mediates viral infections, such as the human immunodeficiency virus 1 (HIV-1). Here, we present the design of PROTAC (proteolysis targeting chimera) compounds against CypA to induce its intracellular proteolysis and to investigate their effect on immune cells. Interestingly, upon connecting to E3 ligase ligands, both peptide-based low-affinity binders and CsA-based high-affinity binders can degrade CypA at nM concentration in HeLa cells and fibroblast cells. As the immunosuppressive effect of CsA is not directly associated with the binding of CsA to CypA but the inhibition of phosphatase calcineurin by the CypA:CsA complex, we investigated whether a CsA-based PROTAC compound could induce CypA degradation without affecting the activation of immune cells. P3, the most efficient PROTAC compound discovered from this study, could deplete CypA in lymphocytes without affecting cell proliferation and cytokine production. This work demonstrates the feasibility of the PROTAC approach in depleting the abundant cellular protein CypA at low drug dosage without affecting immune cells, allowing us to investigate the potential therapeutic effects associated with the endogenous protein in the future.

The Src homology 2 (SH2) and SH3 domain-containing chicken tumor virus number 10 (CT10) regulator of kinase (Crk) adaptor proteins include three cellular members that serve as integral constituents of multiple receptor-linked signal transduction pathways. CrkI and CrkII are products of alternative RNA-splicing which is transcribed from a single gene, while Crk-like (CrkL), which is highly homologous to CrkII, is encoded by a different gene. Thanks to their modular structure, the Crk adaptor proteins can simultaneously interact with activated receptors and a wide range of effector molecules, and orchestrate the assembly of complexes containing enzymes and substrates at the receptor site. They are involved in the regulation of a large number of cellular processes which control cell growth, differentiation, transformation, and apoptosis. Cell activation-dependent tyrosine phosphorylation of CrkII and CrkL serves as a major posttranslational modification mechanism that introduces conformational changes in the proteins by promoting an intramolecular interaction between the phosphotyrosine and the self SH2 domain. The resulting conformational change induces downregulation of CrkII- and CrkL-dependent biological processes. A second type of posttranslational modification mechanism regulates the structure and function of the CrkII adaptor protein by immunophilin-mediated protein isomerization. Two of the most abundant immunophilins in T lymphocytes which function as peptidyl-prolyl cis-trans isomerases (PPIases), namely cyclophilin A (CypA) and FK506-binding proteins (FKBPs), can associate with CrkII and catalyze its reciprocal cis-trans isomerization. This mechanism is of special importance for the regulation of T lymphocyte functions and for T cell-mediated immune responses, since immunophilin inhibitors, such as cyclosporin A (CsA) and FK506, function as immunosuppressive drugs that can prevent allotransplanted graft rejection. The present manuscript focuses on selected functions of Crk adaptor proteins, predominantly in T lymphocytes, and reviews in more detail the current knowledge on the immunophilin-dependent regulation of the structure and function of the CrkII adaptor protein.

Purpose: This study aims to investigate the potential of Oregon grape root extracts to modulate the activity of P-glycoprotein.Methods: We performed 3H-CsA or 3H-digoxin transport experiments in the absence or presence of two sources of Oregon grape root extracts (E1 and E2), berberine or berbamine in Caco-2 and MDCKII-MDR1 cells. In addition, real time quantitative polymerase chain reaction (RT-PCR) was performed in Caco-2 and LS-180 cells to investigate the mechanism of modulating P-glycoprotein.Results: Our results showed that in Caco-2 cells, Oregon grape root extracts (E1 and E2) (0.1–1 mg/mL) inhibited the efflux of CsA and digoxin in a dose-dependent manner. However, 0.05 mg/mL E1 significantly increased the absorption of digoxin. Ten µM berberine and 30 µM berbamine significantly reduced the efflux of CsA, while no measurable effect of berberine was observed with digoxin. In the MDCKII-MDR1 cells, 10 µM berberine and 30 µM berbamine inhibited the efflux of CsA and digoxin. Lastly, in real time RT-PCR study, Oregon grape root extract (0.1 mg/mL) up-regulated mRNA levels of human MDR1 in Caco-2 and LS-180 cells at 24 h.Conclusion: Our study showed that Oregon grape root extracts modulated P-glycoprotein, thereby may affect the bioavailability of drugs that are substrates of P-glycoprotein.

IntroductionSevere aplastic anemia(SAA)is a severe disease characterized by immune-mediated bone marrow failure and pancytopenia. Immunosuppressive therapy (ATG plus CsA, IST) is the standard treatment for patients who are not suitable for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Some patients have a delayed response after 6 months of ATG, and unnecessary to be given secondary ATG or allo-HSCT. We attempted to distinguish patients who may get potential delayed response from those who were really not responsive to IST.MethodsWe collected data from 45 SAA patients who were assessed no-response to IST at 6 months after rATG and failed to receive secondary ATG or allo-HSCT.ResultsCsA plus eltrombopag (EPAG) group has an extra 75% response rate while CsA maintenance group has an extra 44% response rate at 12 months. ATG was applied within 30 days after diagnosis, ATG dosage was suffificient (ATG/lymphocyte ≥2), and absolute reticulocyte count (ARC) was ≥30×109 /L at 6 months, indicated patients could get delayed response and benefifit from CsA maintenance. Addition of EPAG could give an even better response. Otherwise, secondary ATG or allo-HSCT treatment were recommended to be given immediately.Clinical Trial Registrationhttps://www.chictr.org.cn/searchproj.aspx, identifier ChiCTR2300067615.

Purpose: This study aims to investigate the potential of Oregon grape root extracts to modulate the activity of P-glycoprotein. Methods: We performed 3 H-CsA or 3 H-digoxin transport experiments in the absence or presence of two sources of Oregon grape root extracts (E1 and E2), berberine or berbamine in Caco-2 and MDCKII-MDR1 cells. In addition, real time quantitative polymerase chain reaction (RT-PCR) was performed in Caco-2 and LS-180 cells to investigate the mechanism of modulating P-glycoprotein. Results: Our results showed that in Caco-2 cells, Oregon grape root extracts (E1 and E2) (0.1-1 mg/mL) inhibited the efflux of CsA and digoxin in a dose-dependent manner. However, 0.05 mg/mL E1 significantly increased the absorption of digoxin. Ten µM berberine and 30 µM berbamine significantly reduced the efflux of CsA, while no measurable effect of berberine was observed with digoxin. In the MDCKII-MDR1 cells, 10 µM berberine and 30 µM berbamine inhibited the efflux of CsA and digoxin. Lastly, in real time RT-PCR study, Oregon grape root extract (0.1 mg/mL) up-regulated mRNA levels of human MDR1 in Caco-2 and LS-180 cells at 24 h. Conclusion: Our study showed that Oregon grape root extracts modulated P-glycoprotein, thereby may affect the bioavailability of drugs that are substrates of P-glycoprotein., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Fan, Zhou and Zhang.)

Introduction: Periodontitis is a set of inflammatory disorders characterized by periodontal attachment loss and alveolar bone resorption. Because of deficiency in periodontitis mechanical therapy, this study was aimed to explore the molecular influence of the erbiumdoped: yttrium aluminum garnet (Er:YAG) laser and cyclosporin A (CsA) on human gingival fibroblasts (HGFs) for improvement in periodontal diseases therapy. Methods: We focused on articles that studied the proteome profiles of HGFs after treatment with laser irradiation and application of CsA. The topological features of differentially expressed proteins were analyzed using Cytoscape Version 3.4.0 followed by module selection from the protein-protein interaction (PPI) network using Cluster ONE plugin. In addition, we performed gene ontology (GO) enrichment analysis for the densely connected region and key proteins in both PPI networks. Results: Analysis of PPI network of Er:YAG laser irradiation on HGFs lead to introducing YWHAZ, VCP, HNRNPU, YWHAE, UBA52, CLTC, FUS and IGHG1 as key proteins while similar analysis revealed that ACAT1, CTSD, ALDOA, ANXA2, PRDX1, LGALS3, ARHGDI and EEF1A1 are the crucial proteins related to the effect of drug. GO enrichment analysis of hubbottleneck proteins of the 2 networks showed the different significant biological processes and cellular components. The functional enrichments of module of Er:YAG laser network are included as fatty acid transmembrane transport, cytokinesis, regulation of RNA splicing and asymmetric protein localization. There are not any significant clusters in network of HGF treated by CsA. Conclusion: The results indicate that there are 2 separate biomarker panels for the 2 treatment methods. [ABSTRACT FROM AUTHOR]

Introduction: Severe aplastic anemia(SAA)is a severe disease characterized by immune-mediated bone marrow failure and pancytopenia. Immunosuppressive therapy (ATG plus CsA, IST) is the standard treatment for patients who are not suitable for allogeneic hematopoietic stem cell transplantation (allo-HSCT). Some patients have a delayed response after 6 months of ATG, and unnecessary to be given secondary ATG or allo-HSCT. We attempted to distinguish patients who may get potential delayed response from those who were really not responsive to IST., Methods: We collected data from 45 SAA patients who were assessed no-response to IST at 6 months after rATG and failed to receive secondary ATG or allo-HSCT., Results: CsA plus eltrombopag (EPAG) group has an extra 75% response rate while CsA maintenance group has an extra 44% response rate at 12 months. ATG was applied within 30 days after diagnosis, ATG dosage was suffificient (ATG/lymphocyte ≥2), and absolute reticulocyte count (ARC) was ≥30×109 /L at 6 months, indicated patients could get delayed response and benefifit from CsA maintenance. Addition of EPAG could give an even better response. Otherwise, secondary ATG or allo-HSCT treatment were recommended to be given immediately., Clinical Trial Registration: https://www.chictr.org.cn/searchproj.aspx, identifier ChiCTR2300067615., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wang, Wang, Liu, Shao and Fu.)

Abstract: The pancreatic adenocarcinoma remains the most aggressive human malignancy with an extremely low 5-year overall survival. Postoperative gemcitabine could significantly delay recurrence after complete resection of pancreatic cancer. However, the underlying mechanisms are not fully understood. The chemo-resistance factors against gemcitabine still need further characterizations. Here we studied the mechanism of gemcitabine-induced pancreatic cancer cell death by focusing on mammalian sterile 20-like kinase 1 (MST1) and cyclophilin D (Cyp-D). We found that MST1 and Cyp-D expressions were significantly lower in gemcitabine-resistant pancreatic cancer tissues and cell lines. In vitro, gemcitabine activated MST1 through reactive oxygen species (ROS) production, which was prevented by antioxidant n-acetyl-cysteine (NAC). We found that gemcitabine-activated MST1 translocated to mitochondria and formed a complex with the local protein Cyp-D. Gemcitabine-induced cell death was alleviated by MST1 or Cyp-D shRNA silencing, but was aggravated by MST1 or Cyp-D over-expression. Further, cyclosporin A (CsA), the Cyp-D inhibitor, prevented gemcitabine-induced MST1/Cyp-D mitochondrial complexation and cancer cell death. We suggest that gemcitabine-induced death of pancreatic cancer cells requires MST1/Cyp-D mitochondrial complexation. [Copyright &y& Elsevier]

Highlights: [•] The exposure of CS2 induced testicular germ cells apoptosis. [•] All factors of mitochondrial apoptotic pathway changed after CS2 exposure. [•] Inhibitor of MPTP could efficiently reverse the apoptosis. [•] Mitochondrial apoptotic pathway plays a role in CS2-induced testicular injury. [Copyright &y& Elsevier]

Highlights: [•] An analytical method based on LC–MS/MS was optimized for the simultaneous determination of GSH and GSSG. [•] Rat and human hepatocytes in culture were demonstrated to be suitable for the in vitro study of oxidative stress. [•] A clear GSH imbalance was observed after incubation of menadione. [•] In terms of GSH imbalance, similar results as those observed with the WST-1 cytotoxicity assay were obtained. [Copyright &y& Elsevier]

Highlights: [•] The work-loop technique provides a better understanding of cardiac contractility. [•] Doxorubicin-induced effects on the contractility of papillary muscle were assessed. [•] Doxorubicin treatment reduced in peak force, force during shortening and power output. [•] Detrimental effects of doxorubicin were abrogated when co-administered with CsA. [Copyright &y& Elsevier]

Highlights: [•] Dietary bioactive compounds protect mitochondria against oxidative stress. [•] Natural compounds modulate apoptosis signaling pathways. [•] Food plant compounds lead to mitochondria health/dysfunction in both in vitro and in vivo models. [ABSTRACT FROM AUTHOR]

Background & Aims: Many recipients of organ transplants develop chronic hepatitis, due to infection with the hepatitis E virus (HEV). Although chronic HEV infection is generally associated with immunosuppressive therapies, little is known about how different immunosuppressants affect HEV infection. Methods: A subgenomic HEV replication model, in which expression of a luciferase reporter gene is measured, and a full-length infection model were used. We studied the effects of different immunosuppressants, including steroids, calcineurin inhibitors (tacrolimus [FK506] and cyclosporin A), and mycophenolic acid (MPA, an inhibitor of inosine monophosphate dehydrogenase) on HEV replication in human hepatoma cell line Huh7. Expression of cyclophilins A and B (the targets of cyclosporin A) were knocked down using small hairpin RNAs. Results: Steroids had no significant effect on HEV replication. Cyclosporin A promoted replication of HEV in the subgenomic and infectious models. Knockdown of cyclophilin A and B increased levels of HEV genomic RNA by 4.0- ± 0.6-fold and 7.2- ± 1.9-fold, respectively (n = 6; P < .05). A high dose of FK506 promoted infection of liver cells with HEV. In contrast, MPA inhibited HEV replication. Incubation of cells with guanosine blocked the antiviral activity of MPA, indicating that the antiviral effects of this drug involve nucleotide depletion. The combination of MPA and ribavirin had a greater ability to inhibit HEV replication than MPA or ribavirin alone. Conclusions: Cyclophilins A and B inhibit replication of HEV; this might explain the ability of cyclosporin A to promote HEV infection. On the other hand, the immunosuppressant MPA inhibits HEV replication. These findings should be considered when physicians select immunosuppressive therapies for recipients of organ transplants who are infected with HEV. [Copyright &y& Elsevier]

Highlights: [•] CsA induced ER stress. [•] CHOP induced apoptosis. [•] ATF3 attenuates nephrotoxicity. [ABSTRACT FROM AUTHOR]

Abstract: Here we studied the role of mitochondrial permeability transition pore (mPTP) opening in curcumin’s cytotoxicity in melanoma cells. In cultured WM-115 melanoma cells, curcumin induced mitochondrial membrane potential (MPP) decrease, cyclophilin-D (CyPD)-adenine nucleotide translocator 1 (ANT-1) (two mPTP components) mitochondrial association and cytochrome C release, indicating mPTP opening. The mPTP blocker sanglifehrin A (SfA) and ANT-1 siRNA-depletion dramatically inhibited curcumin-induced cytochrome C release and WM-115 cell death. CyPD is required for curcumin-induced melanoma cell death. The CyPD inhibitor cyclosporin A (CsA) or CyPD siRNA-depletion inhibited curcumin-induced WM-115 cell death and apoptosis, while WM-115 cells with CyPD over-expression were hyper-sensitive to curcumin. Finally, we found that C6 ceramide enhanced curcumin-induced cytotoxicity probably through facilitating mPTP opening, while CsA and SfA as well as CyPD and ANT-1 siRNAs alleviated C6 ceramide’s effect on curcumin in WM-115 cells. Together, these results suggest that curcumin-induced melanoma cell death is associated with mPTP opening. [Copyright &y& Elsevier]

Abstract: Recently disulfide-rich head-to-tail cyclic peptides have attracted the interest of medicinal chemists owing to their exceptional thermal, chemical and enzymatic stability brought about by their constrained structures. Here we review current trends in the field of peptide-based pharmaceuticals and describe naturally occurring cyclic disulfide-rich peptide scaffolds, discussing their pharmaceutically attractive properties and benefits. We describe how we can utilise these stable frameworks to graft and/or engineer pharmaceutically interesting epitopes to increase their selectivity and bioactivity, opening up new possibilities for addressing ‘difficult’ pharmaceutical targets. [Copyright &y& Elsevier]

Background & Aims: Chronic hepatitis B and hepatitis D are global health problems caused by the human hepatitis B and hepatitis D virus. The myristoylated preS1 domain of the large envelope protein mediates specific binding to hepatocytes by sodium taurocholate co-transporting polypeptide (NTCP). NTCP is a bile salt transporter known to be inhibited by cyclosporin A. This study aimed to characterize the effect of cyclosporin A on HBV/HDV infection. Methods: HepaRG cells, primary human hepatocytes, and susceptible NTCP-expressing hepatoma cell lines were applied for infection experiments. The mode of action of cyclosporin A was studied by comparing the effect of different inhibitors, cyclophilin A/B/C-silenced cell lines as well as NTCP variants and mutants. Bile salt transporter and HBV receptor functions were investigated by taurocholate uptake and quantification of HBVpreS binding. Results: Cyclosporin A inhibited hepatitis B and D virus infections during and – less pronounced – prior to virus inoculation. Binding of HBVpreS to NTCP was blocked by cyclosporin A concentrations at 8μM. An NTCP variant deficient in HBVpreS binding but competent for bile salt transport showed resistance to cyclosporin A. Silencing of cyclophilins A/B/C did not abrogate transporter and receptor inhibition. In contrast, tacrolimus, a cyclophilin-independent calcineurin inhibitor, was inactive. Conclusions: HBV and HDV entry via sodium taurocholate co-transporting polypeptide is inhibited by cyclosporin A. The interaction between the drug and the viral receptor is direct and overlaps with a functional binding site of the preS1 domain, which mediates viral entry. [Copyright &y& Elsevier]

Abstract: Insulin resistance is associated with mitochondrial dysfunction, but the mechanism by which mitochondria inhibit insulin-stimulated glucose uptake into the cytoplasm is unclear. The mitochondrial permeability transition pore (mPTP) is a protein complex that facilitates the exchange of molecules between the mitochondrial matrix and cytoplasm, and opening of the mPTP occurs in response to physiological stressors that are associated with insulin resistance. In this study, we investigated whether mPTP opening provides a link between mitochondrial dysfunction and insulin resistance by inhibiting the mPTP gatekeeper protein cyclophilin D (CypD) in vivo and in vitro. Mice lacking CypD were protected from high fat diet-induced glucose intolerance due to increased glucose uptake in skeletal muscle. The mitochondria in CypD knockout muscle were resistant to diet-induced swelling and had improved calcium retention capacity compared to controls; however, no changes were observed in muscle oxidative damage, insulin signaling, lipotoxic lipid accumulation or mitochondrial bioenergetics. In vitro, we tested 4 models of insulin resistance that are linked to mitochondrial dysfunction in cultured skeletal muscle cells including antimycin A, C2-ceramide, ferutinin, and palmitate. In all models, we observed that pharmacological inhibition of mPTP opening with the CypD inhibitor cyclosporin A was sufficient to prevent insulin resistance at the level of insulin-stimulated GLUT4 translocation to the plasma membrane. The protective effects of mPTP inhibition on insulin sensitivity were associated with improved mitochondrial calcium retention capacity but did not involve changes in insulin signaling both in vitro and in vivo. In sum, these data place the mPTP at a critical intersection between alterations in mitochondrial function and insulin resistance in skeletal muscle. [Copyright &y& Elsevier]

Highlights: [•] Cyclosporin A (CsA) exacerbates escitalopram-induced serotonin syndrome in mice. [•] CsA pre-treatment enhances brain distribution of escitalopram in mice. [•] A combination of CsA and escitalopram reduces prefrontal cortex 5-HT turnover in mice. [•] CsA pre-treatment does not augment the activity of escitalopram in the TST in mice. [•] CsA attenuates increases in extracellular 5-HT in response to escitalopram in rats. [Copyright &y& Elsevier]

Abstract: The study aimed to determine the effect of 3′,4′-dihydroxyflavonol (DiOHF) on mitochondrial function, in particular opening of the mitochondrial permeability transition pore (mPTP), respiratory function and reactive oxygen species (ROS) production, in isolated cardiac mitochondria after coronary artery occlusion and reperfusion in vivo. Opening of the mPTP, oxygen consumption and ROS production (assessed by measurement of H2O2) was determined in mitochondria isolated from normal hearts or from the ischemic zone of rat hearts subjected to 30min coronary artery occlusion and 15min reperfusion. Treatment of sham rats with DiOHF (10mgkg−1 iv) significantly increased the concentration of Ca2+ required to stimulate mPTP opening. This was accompanied by increased state 3 oxygen consumption and decreased H2O2 release. Ischemia and reperfusion (IR) significantly decreased the concentration of Ca2+ required to stimulate mPTP opening, decreased state 3 oxygen consumption and increased H2O2 release, when pyruvate plus malate was provided as a substrate. Treatment with DiOHF prevented IR-induced changes in mPTP opening, state 3 oxygen consumption and H2O2 release so that there was no difference compared to sham. In isolated cardiac mitochondria from normal rats DiOHF had no effect on mPTP opening or on state 3 respiration but caused a small increase in state 4 respiration and decreased the respiratory control ratio. DiOHF, administered during ischemia just before reperfusion, inhibits mPTP opening and preserves mitochondrial function through a mechanism likely to be independent of its antioxidant activity or any direct effect on the mPTP. [Copyright &y& Elsevier]

Highlights: [•] In cardiomyocytes, ET-1 treatment resulted in significant upregulation of COX-2 expression and activity. [•] Both COX-2 inhibitors and COX-2 RNA interference suppressed cardiomyocyte hypertrophy induced by ET-1. [•] Calcineurin/NFATc3 signaling pathway participated in the transcriptional regulation of COX-2 by ET-1. [ABSTRACT FROM AUTHOR]

Abstract: Transient opening of the mitochondrial permeability transition pore plays a crucial role in hypoxic preconditioning-induced protection. Recently, the cyclophilin-D component of the mitochondrial permeability transition pore has been shown to interact with and regulate the F1F0-ATP synthase. However, the precise role of the F1F0-ATP synthase and the interaction between cyclophilin-D and F1F0-ATP synthase in the mitochondrial permeability transition pore and hypoxic preconditioning remain uncertain. Here we found that a 1-h hypoxic preconditioning delayed apoptosis and improved cell survival after stimulation with various apoptotic inducers including H2O2, ionomycin, and arachidonic acid in mitochondrial DNA T8993G mutation (NARP) osteosarcoma 143B cybrids, an F1F0-ATP synthase defect cell model. This hypoxic preconditioning protected NARP cybrid cells against focal laser irradiation-induced oxidative stress by suppressing reactive oxygen species formation and preventing the depletion of cardiolipin. Furthermore, the protective functions of transient opening of the mitochondrial permeability transition pore in both NARP cybrids and wild-type 143B cells can be augmented by hypoxic preconditioning. Disruption of the interaction between cyclophilin-D and F1F0-ATP synthase by cyclosporin A attenuated the mitochondrial protection induced by hypoxic preconditioning in both NARP cybrids and wild-type 143B cells. Our results demonstrate that the interaction between cyclophilin-D and F1F0-ATP synthase is important in the hypoxic preconditioning-induced cell protection. This finding improves our understanding of the mechanism of mitochondrial permeability transition pore opening in cells in response to hypoxic preconditioning, and will be helpful in further developing new pharmacological agents targeting hypoxia–reoxygenation injury and mitochondria-mediated cell death [Copyright &y& Elsevier]

Abstract: We previously demonstrated that a cellular factor, cyclosporin A (CsA) associated helicase-like protein (CAHL) that is identical to YTH domain containing 2 (YTHDC2), forms trimer complex with cyclophilin B and NS5B of hepatitis C virus (HCV) and facilitates HCV genome replication. Gene expression of YTHDC2 was shown in tumor cell lines and tumor necrosis factor (TNF)-α-treated hepatocytes, but not in untreated. However, the function of YTHDC2 in the tumor cells and the mechanism by which the YTHDC2 gene is transcribed in these cells is largely unknown. We first evaluated that the role of YTHDC2 in the proliferation of hepatocellular carcinoma (HCC) cell line Huh7 using RNA interference and found that YTHDC2-downregulated Huh7 were significantly decreased cell growth as compared to control. We next demonstrated that the cAMP response element (CRE) site in the promoter region of the YTHDC2 gene is critical for YTHDC2 transcription. To further investigate the transcription factors bound to the CRE site, we performed chromatin immunoprecipitation assays. Our findings demonstrate that c-Jun and ATF-2 bind to the CRE site in Huh7, and that TNF-α induces the biological activity of these transcription factors in hepatocytes as well as Huh7. Moreover, treatment with the HDAC inhibitor, trichostatin A (TSA), reduces YTHDC2 expression in Huh7 and in TNF-α-stimulated hepatocytes. Collectively, these data show that YTHDC2 plays an important role in tumor cells growth and activation/recruitment of c-Jun and ATF-2 to the YTHDC2 promoter is necessary for the transcription of YTHDC2, and that HDAC activity is required for the efficient expression of YTHDC2 in both of hepatocyte and HCC cells. [Copyright &y& Elsevier]

Abstract: Phosphorylated and non-phosphorylated forms of the F0F1-ATPase subunit c from rat liver mitochondria (RLM) were purified and their effect on the opening of the permeability transition pore (mPTP) was investigated. Addition of dephosphorylated subunit c to RLM induced mitochondrial swelling, decreased the membrane potential and reduced the Ca2+ uptake capacity, which was prevented by cyclosporin A. The same effect was observed in the presence of storage subunit c purified from livers of sheep affected with ceroid lipofuscinosis. In black-lipid bilayer membranes subunit c increased the conductance due to formation of single channels with fast and slow kinetics. The dephosphorylated subunit c formed channels with slow kinetics, i.e. the open state being of significantly longer duration than in the case of channels formed by the phosphorylated form that had short life spans and fast kinetics. The channels formed were cation-selective more so with the phosphorylated form. Subunit c of rat liver mitochondria was able to bind Ca2+. Collectively, the data allowed us to suppose that subunit c F0F1-ATPase might be a structural/regulatory component of mPTP exerting its role in dependence on phosphorylation status. [Copyright &y& Elsevier]

Background: Many patients with moderate-to-severe atopic dermatitis (AD) require systemic immunomodulating treatment to achieve adequate disease control. Objective: We sought to systematically evaluate the efficacy and safety of systemic treatments for moderate-to-severe AD. Methods: A systematic literature search was performed in MEDLINE, EMBASE, and CENTRAL (until June 2012). Randomized controlled trials (RCTs) evaluating systemic immunomodulating treatments for moderate-to-severe AD were included. Selection, data extraction, quality assessment, and generation of treatment recommendations using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach were performed independently by 2 reviewers. Efficacy outcomes were clinical signs, symptoms, quality of life, and the course of AD. Safety data were compared by calculating the weekly incidence rates (as percentages) for adverse events. Results: Thirty-four RCTs with 12 different systemic treatments and totaling 1653 patients were included. Fourteen trials consistently indicate that cyclosporin A efficaciously improves clinical signs of AD. Cyclosporin A is recommended as first-line treatment for short-term use. A second-line treatment option is azathioprine, but efficacy is lower, and evidence is weaker. Methotrexate can be considered a third-line treatment option. Recommendations are impossible for mycophenolate, montelukast, intravenous immunoglobulins, and systemic glucocorticosteroids because of limited evidence. A meta-analysis was not performed because of a lack of standardization in outcome measures. Conclusion: Although 12 different interventions for moderate-to-severe AD have been studied in 34 RCTs, strong recommendations are only possible for the short-term use of cyclosporin A. Methodological limitations in the majority of trials prevent evidence-based conclusions. Large head-to-head trials evaluating long-term treatments are required. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Triterpenoids are a large class of naturally occurring compounds, and some potentially interesting as anticancer agents have been found to target mitochondria. The objective of the present work was to investigate the mechanisms of mitochondrial toxicity induced by novel dimethylaminopyridine (DMAP) derivatives of pentacyclic triterpenes, which were previously shown to inhibit the growth of melanoma cells in vitro. MCF-7, Hs 578T and BJ cell lines, as well as isolated hepatic mitochondria, were used to investigate direct mitochondrial effects. On isolated mitochondrial hepatic fractions, respiratory parameters, mitochondrial transmembrane electric potential, induction of the mitochondrial permeability transition (MPT) pore and ion transport-dependent osmotic swelling were measured. Our results indicate that the DMAP triterpenoid derivatives lead to fragmentation and depolarization of the mitochondrial network in situ, and to inhibition of uncoupled respiration, induction of the permeability transition pore and depolarization of isolated hepatic mitochondria. The results show that mitochondrial toxicity is an important component of the biological interaction of DMAP derivatives, which can explain the effects observed in cancer cells. [Copyright &y& Elsevier]

Abstract: Ethnopharmacological relevance: Lotus plumule, the dried young cotyledon and radicle of the Nelumbo nucifera Gaertn. (Fam. Nymphaeaceae) ripe seed, is a famous Traditional Chinese Medicine to remove heat from the heart, anchor the mind, improve seminal emission, and arrest bleeding for centuries in China. Liensinine and its analogs neferine and isoliensinine are the major active components in lotus plumule. Aim of the study is to investigate the association of liensinine, neferine, and isoliensinine with efflux transporters. Materials and methods: Caco-2, MDCK, MDCK-MDR1, and MDCK-MRP2 were used as cell models for the transcellular transport and accumulation studies. Results: The results obtained in Caco-2 cells suggested that P-glycoprotein (P-gp) might be involved in transcellular transport. Cellular accumulation and transport experiments were further performed in MDCK-MDR1 cells. GF120918 and cyclosporine A were found to completely inhibit the efflux, and the net efflux ratios of these alkaloids exhibited saturation over the concentration range. No significant differences in liensinine accumulation and transport were observed between MDCK and MDCK-MRP2 cells. Conclusions: These results demonstrated that liensinine, neferine, and isoliensinine are substrates of P-gp, whereas MRP2 is not involved in the transport process, suggesting that P-gp might be responsible for the absorption and distribution of the 3 alkaloids. [Copyright &y& Elsevier]

Abstract: Minocycline, a tetracycline-derived compound, mitigates damage caused by ischemia/reperfusion (I/R) injury. Here, 19 tetracycline-derived compounds were screened in comparison to minocycline for their ability to protect hepatocytes against damage from chemical hypoxia and I/R injury. Cultured rat hepatocytes were incubated with 50μM of each tetracycline-derived compound 20min prior to exposure to 500μM iodoacetic acid plus 1mM KCN (chemical hypoxia). In other experiments, hepatocytes were incubated in anoxic Krebs–Ringer–HEPES buffer at pH6.2 for 4h prior to reoxygenation at pH7.4 (simulated I/R). Tetracycline-derived compounds were added 20min prior to reperfusion. Ca2+ uptake was measured in isolated rat liver mitochondria incubated with Fluo-5N. Cell killing after 120min of chemical hypoxia measured by propidium iodide (PI) fluorometry was 87%, which decreased to 28% and 42% with minocycline and doxycycline, respectively. After I/R, cell killing at 120min decreased from 79% with vehicle to 43% and 49% with minocycline and doxycycline. No other tested compound decreased killing. Minocycline and doxycycline also inhibited mitochondrial Ca2+ uptake and suppressed the Ca2+-induced mitochondrial permeability transition (MPT), the penultimate cause of cell death in reperfusion injury. Ru360, a specific inhibitor of the mitochondrial calcium uniporter (MCU), also decreased cell killing after hypoxia and I/R and blocked mitochondrial Ca2+ uptake and the MPT. Other proposed mechanisms, including mitochondrial depolarization and matrix metalloprotease inhibition, could not account for cytoprotection. Taken together, these results indicate that minocycline and doxycycline are cytoprotective by way of inhibition of MCU. [Copyright &y& Elsevier]

Abstract: Fanconi anemia is a rare genetic disease characterized by bone marrow failure, multiple congenital malformations, and an increased susceptibility to malignancy. At least 15 genes have been identified that are involved in the pathogenesis of Fanconi anemia. However, it is still a challenge to assign the complementation group and to characterize the molecular defects in patients with Fanconi anemia. In the current study, whole exome sequencing was used to identify the affected gene(s) in a boy with Fanconi anemia. A recurring, non-synonymous mutation was found (c.3971C>T, p.P1324L) as well as a novel frameshift mutation (c.989_995del, p.H330LfsX2) in FANCA gene. Our results indicate that whole exome sequencing may be useful in clinical settings for rapid identification of disease-causing mutations in rare genetic disorders such as Fanconi anemia. [Copyright &y& Elsevier]

Abstract: Contractile dysfunction and subsequent development of cardiomyopathies are well known limiting factors in the treatment of cancer with doxorubicin and have been linked to mitochondrial dysfunction. Here, using adult isolated paced cardiomyocytes, we have demonstrated that ligands of translocator protein (TSPO) 4′-chlorodiazepam and TRO40303 prevented the doxorubicin-induced alterations in contractility and improved cardiomyocyte viability. This cardioprotective effect was closely associated with both a potent reduction in reactive oxygen species production and inhibition of mitochondrial permeability transition pore opening. Thus, preventive administration of TSPO ligands may represent a novel pharmacological strategy to protect the heart during doxorubicin treatment. [Copyright &y& Elsevier]

Background & Aims: Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. The biological and therapeutic importance of host cellular cofactors for viral replication has been recently appreciated. Here we examined the roles of SNF1/AMP kinase-related kinase (SNARK) in HCV replication and pathogenesis. Methods: The JFH1 infection system and the full-length HCV replicon OR6 cell line were used. Gene expression was knocked down by siRNAs. SNARK mutants were created by site-directed mutagenesis. Intracellular mRNA levels were measured by qRT-PCR. Endogenous and overexpressed proteins were detected by Western blot analysis and immunofluorescence. Transforming growth factor (TGF)-β signaling was monitored by a luciferase reporter construct. Liver biopsy samples from HCV-infected patients were analyzed for SNARK expression. Results: Knockdown of SNARK impaired viral replication, which was rescued by wild type SNARK but not by unphosphorylated or kinase-deficient mutants. Knockdown and overexpression studies demonstrated that SNARK promoted TGF-β signaling in a manner dependent on both its phosphorylation and kinase activity. In turn, chronic HCV replication upregulated the expression of SNARK in patients. Further, the SNARK kinase inhibitor metformin suppressed both HCV replication and SNARK-mediated enhancement of TGF-β signaling. Conclusions: Thus reciprocal regulation between HCV and SNARK promotes TGF-β signaling, a major driver of hepatic fibrogenesis. These findings suggest that SNARK will be an attractive target for the design of novel host-directed antiviral and antifibrotic drugs. [Copyright &y& Elsevier]

Highlights: [•] We establish a novel genotype 3a replicon that encodes a luciferase reporter. [•] Unique adaptive mutations and Huh7-derived cell clones are identified. [•] The replicon replicates robustly in both stably-selected and transiently-transfected cells. [•] Antiviral EC50 values are reproducibly determined in 384-well assays. [•] This new replicon is aiding the development of pan-genotypic HCV therapies. [Copyright &y& Elsevier]

Highlights: [•] Chemical screening identified sulochrin as a natural product reducing HCV infection. [•] Sulochrin inhibited HCV entry. [•] The anti-HCV activity of sulochrin was conserved in different HCV genotypes. [•] Cotreatment of sulochrin with IFNα or telaprevir showed a synergistic anti-HCV effect. [•] Derivative analysis identified the structure important for anti-HCV activity. [ABSTRACT FROM AUTHOR]

Abstract: Cyclosporine A and FK506 produce immunosuppression by blocking calcineurin phosphatase activity and consequently activation of cytosolic Nuclear Factor of Activated T-cell (NFATc) transcription factor. Due to the chronic toxicity associated with their administration, the development of more specific immunosuppressants is currently an important unmet medical need. In this context, an immunosuppressant peptide derived from the CIC motif of the human Regulators of Calcineurin (RCAN) proteins has been shown to inhibit NFATc signaling without affecting general phosphatase activity of calcineurin. Here we show that protein kinase CK2 phosphorylates a conserved serine residue within the CIC motif of vertebrate RCANs, which increases its affinity for calcineurin and consequently its inhibition of NFATc-dependent gene expression in activated T-cells. Molecular modeling studies have led us to identify a positively charged interaction site on the surface of calcineurin where the phosphorylated serine residue of the CIC motif would normally locate. Finally, we have also identified RCAN3 as a new phosphoprotein with multiple phosphorylation sites. Therefore, our findings reveal for the first time a novel molecular mechanism underlying the regulation of calcineurin–NFATc signaling by means of phosphorylation of the CIC motif of RCAN proteins. The knowledge of how RCAN proteins modulate the calcineurin–NFATc pathway paves the way for the development of potent novel selective immunosuppressant drugs. [Copyright &y& Elsevier]

Abstract: P-glycoprotein (Pgp) and XIAP co-expression has been discussed in the process of the acquisition of multidrug resistance (MDR) in cancer. Here, we evaluated XIAP and Pgp expression in chronic myeloid leukemia (CML) samples, showing a positive correlation between them. Furthermore, we evaluated the effects of imatinib in XIAP and Pgp expression using CML cell lines K562 (Pgp−) and K562-Lucena (Pgp+). Imatinib increased XIAP and Pgp expression in K562-Lucena cells, while in K562 cells a downregulation of these proteins was observed, suggesting that imatinib induces an increment of MDR phenotype of CML cells that previously exhibit high levels of Pgp/XIAP co-expression. [Copyright &y& Elsevier]

Abstract: Background: Reactivation of latent human cytomegalovirus (HCMV) is a frequent complication following allogeneic haematopoietic stem cell transplantation (HSCT). Evaluation of the quantity and function of HCMV-specific CD8+ T-cell responses after HSCT may play a crucial role in the prevention of HCMV reactivation. Objectives: To investigate the mechanism of HCMV-specific T-cell immune responses after HSCT in HCMV-specific CD8+ T cells. Study design: HCMV-specific CD8+ T cells were quantified using human leucocyte antigen (HLA) pentamer staining and functionally analysed by interferon-γ-enzyme-linked immunospot (IFN-γ-ELISPOT) assay with a pp65495–503 peptide in recipients four years after HSCT. Results: The absolute number of pp65495–503-specific CD8+ T cells did not differ significantly (p >0.05) between samples with antigenaemia and those without antigenaemia given a mean of 54.5/μl and 40.5/μl, respectively, in 21 HLA-A* 0201 patients after HSCT. The level of pp65495–503-specific CD8+ T cells>20/μl of peripheral blood was maintained 90 days after transplantation. There was a significant difference in the spot count of IFN-γ-secreting T cells between samples with antigenaemia (mean, 507/2.5×105 PBMCs) and those without antigenaemia (mean, 216/2.5×105 PBMCs; p <0.05). Conclusion: pp65495–503-specific CD8+ T cells may not be sufficient to control HCMV reactivation in recipients after HSCT. However, the combination of pentamer and IFN-γ-ELISPOT assays may be valuable for evaluating HCMV-specific CD8+ T cells. Further studies on HCMV-specific T-cell immune responses continue to be performed for the prevention of persistent HCMV reactivation. [Copyright &y& Elsevier]

Abstract: The large number of drug candidates with poor dissolution characteristics seen in the past decade, has fostered interest in so-called “enabling formulations”, i.e., formulations which shall make such drugs bio-available. Development of enabling formulations is currently being guided by the following (simplified) hypothesis: If a poorly soluble drug (BCS class II drug) can be transferred into a solubilized state, one can achieve an absorption profile close to that of a soluble drug (BCS class I drug). Thus, formulation development typically endeavors to achieve the most robust solubility enhancement. Here we critically review both common in vitro approaches and experimental data available in literature pertaining to the solubility and permeability of poorly soluble drugs from enabling formulations, and discuss their interplay. Recent in vitro data indicate, that commonly employed surfactants as well as endogenous surfactants present in the intestine, although enhancing drug solubility, mostly hamper drug permeation. Mechanistic studies demonstrate a direct correlation between passive transcellular diffusion and the concentration of molecularly dissolved drug. The latter may be reduced due to partitioning into micelles or other solubilizing carriers, but enhanced in supersaturating formulations. We conclude thus that biopharmaceutical assessment approaches that rely on the amount of molecularly dissolved drug should guide us towards successful enabling formulations. [Copyright &y& Elsevier]

Abstract: Calcineurin inhibitors such as cyclosporin A (CsA) and FK506 have been used in solid organ and hematopoietic stem cell transplantations to suppress immune function. However, these immunosuppresants are associated with severe endothelial dysfunction. We investigated whether CsA and FK506 induce endothelial dysfunction using a three-dimensional culture blood vessel model, in which human umbilical vein endothelial cells form and maintain capillary-like tube and lumen structures. We found that FK506, but not CsA, induced breakdown of the tube structures and endothelial cell death. FK506 inhibited calcineurin activity, but FK506-induced tube breakdown and cell death was not suppressed by RNA interference targeting calcineurin Aα. FK506 also induced caspase activation, but caspase inhibition by zVAD(OMe)-fmk failed to suppress FK506-induced tube breakdown and cell death. FK506 induced attenuation of Akt and extracellular-regulated kinase 1/2 (ERK1/2). Furthermore, Akt inhibition by LY294002 or ERK1/2 inhibition by PD98059 induced tube breakdown and cell death. Present results suggest that FK506 induces endothelial dysfunction through attenuation of Akt and ERK1/2 independently of calcineurin inhibition and the caspase pathway. [Copyright &y& Elsevier]

Abstract: Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca2+ overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca2+ levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca2+ influx, mitochondrial network fragmentation and loss of the mitochondrial Ca2+ buffer capacity. These biochemical events increase cytosolic Ca2+ levels and trigger cardiomyocyte death via the activation of calpains. [Copyright &y& Elsevier]

Abstract: Nuclear factors of activated T-cells (NFATs) are important regulators of the cytokine gene expression in activated T-cells. In the last decade, NFATs have been shown to regulate cell cycle, differentiation and apoptosis in cells of various origins revealing their importance for cell homeostasis. In this study, we investigated the effects of NFAT1 on proliferation and differentiation of v-myb-transformed BM2 monoblasts. In contrast to many other leukemic cell lines, BM2 cells do not respond to retinoic acid. However, once overexpressing NFAT1, they became sensitive to all-trans retinoic acid (ATRA). The ATRA-treated BM2NFAT1 cells differentiated along monocyte/macrophage pathway as evidenced by changes in cell morphology, adherence, phagocytic and non-specific esterase activities, reactive oxygen species production, and vimentin expression. Furthermore, overexpressed NFAT1 either alone or in combination with the ATRA-driven signalling pathway deregulated cyclin A and retinoic acid receptor proteins in BM2 cells. Data presented in this study indicate that the NFAT1 and ATRA signalling pathways synergize in control of proliferation and differentiation of BM2 monoblasts. [Copyright &y& Elsevier]

Background: Therapeutic drug monitoring of immunosuppressive drugs is important in transplant patients. We developed and validated liquid chromatography–mass spectrometry (LC–MS/MS) assay for simultaneous quantitation of tacrolimus (TaC), sirolimus (SrL), and cyclosporin A (CsA) in dried blood spots (DBSs) to offer patients home sample collection, avoiding travel for blood draws. Methods: After extraction, samples were analyzed by LC–MS/MS in multiple reaction monitoring mode. Results: The assay was linear between 1.2–40ng/ml for TaC and SrL, and 30–1000ng/ml for CsA. Inter- and intra-assay CVs were ≤14.8% for all 3 drugs. This method correlated well with the existing clinical whole blood assay, with coefficients of determination >0.95 for all 3 drugs. DBS quality control samples were stable for at least 30days at −20, 4, and 25°C. Stability of patient DBS samples was at least 5days at temperatures up to 60°C, except for SrL where degradation was observed at 60°C within 24h. No effect of hematocrit level, blood spot volume or punch location was observed. Conclusion: Immunosuppressant levels measured in DBS correlate with whole blood LC–MS/MS assay and may contribute to successful outcome of organ transplant and patient satisfaction. [ABSTRACT FROM AUTHOR]

Abstract: Background: Mitochondrial dysfunction contributes to degenerative neurological disorders, consequently there is a need for mitochondria-targeted therapies that are effective within the brain. One approach to deliver pharmacophores is by conjugation to the lipophilic triphenylphosphonium (TPP) cation that accumulates in mitochondria driven by the membrane potential. While this approach has delivered TPP-conjugated compounds to the brain, the amounts taken up are lower than by other organs. Methods: To discover why uptake of hydrophobic TPP compounds by the brain is relatively poor, we assessed the role of the P-glycoprotein (Mdr1a/b) and breast cancer resistance protein (Bcrp) ATP binding cassette (ABC) transporters, which drive the efflux of lipophilic compounds from the brain thereby restricting the uptake of lipophilic drugs. We used a triple transgenic mouse model lacking two isoforms of P-glycoprotein (Mdr1a/1b) and the Bcrp. Results: There was a significant increase in the uptake into the brain of two hydrophobic TPP compounds, MitoQ and MitoF, in the triple transgenics following intra venous (IV) administration compared to control mice. Greater amounts of the hydrophobic TPP compounds were also retained in the liver of transgenic mice compared to controls. The uptake into the heart, white fat, muscle and kidneys was comparable between the transgenic mice and controls. Conclusion: Efflux of hydrophobic TPP compounds by ABC transporters contributes to their lowered uptake into the brain and liver. General significance: These findings suggest that strategies to bypass ABC transporters in the BBB will enhance delivery of mitochondria-targeted antioxidants, probes and pharmacophores to the brain. [Copyright &y& Elsevier]

Abstract: The steroidogenic cytochrome P450 17 (CYP17) enzyme produces dehydroepiandrosterone (DHEA), which is the most abundant circulating endogenous sex steroid precursor. DHEA plays a key role in e.g. sexual functioning and development. To date, no rapid screening assay for effects on CYP17 is available. In this study, a novel assay using porcine adrenal cortex microsomes (PACMs) was described. Effects of twenty-eight suggested endocrine disrupting compounds (EDCs) on CYP17 activity were compared with effects in the US EPA validated H295R (human adrenocorticocarcinoma cell line) steroidogenesis assay. In the PACM assay DHEA production was higher compared with the H295R assay (4.4 versus 2.2nmol/h/mg protein). To determine the additional value of a CYP17 assay, all compounds were also tested for interaction with CYP19 (aromatase) using human placental microsomes (HPMs) and H295R cells. 62.5% of the compounds showed enzyme inhibition in at least one of the microsomal assays. Only the cAMP inducer forskolin induced CYP17 activity, while CYP19 was induced by four test compounds in the H295R assay. These effects remained unnoticed in the PACM and HPM assays. Diethylstilbestrol and tetrabromobisphenol A inhibited CYP17 but not CYP19 activity, indicating different mechanisms for the inhibition of these enzymes. From our results it becomes apparent that CYP17 can be a target for EDCs and that this interaction differs from interactions with CYP19. Our data strongly suggest that research attention should focus on validating a specific assay for CYP17 activity, such as the PACM assay, that can be included in the EDC screening battery. [Copyright &y& Elsevier]

Abstract: Cell-therapy was proposed to be a promising tool in case of death or impairment of specific cell types. Correct identification of implanted cells became crucial when evaluating the success of transplantation therapy. Various methods of cell labeling have been employed in previously published studies. The use of intrinsic signaling of green fluorescent protein (GFP) has led to a well known controversy in the field of cardiovascular research. We encountered similar methodological pitfalls after transplantation of GFP-transfected embryonic stem cells into rat brains following traumatic brain injury (TBI). As the identification of implanted graft by intrinsic autofluorescence failed, anti-GFP labeling coupled to fluorescent and conventional antibodies was needed to visualize the implanted cells. Furthermore, different cell types with strong intrinsic autofluorescence were found at the sites of injury and transplantation, thus mimicking the implanted stem cells. GFP-positive stem cells were correctly localized, using advanced histological techniques. The activation of microglia/macrophages, accompanying the transplantation post TBI, was shown to be a significant source of artefacts, interfering with correct identification of implanted stem cells. Dependent on the strategy of stem cell tracking, the phagocytosis of implanted cells as observed in this study, might also impede the interpretation of results. Critical appraisal of previously published data as well as a review of different histological techniques provide tools for a more accurate identification of transplanted stem cells. [Copyright &y& Elsevier]

Abstract: Despite remarkable advances in human genetics and other genetic model systems, the fruit fly, Drosophila melanogaster, remains a powerful experimental tool to probe with ease the inner workings of a myriad of biological and pathological processes, even when evolutionary forces impart apparent divergences to some of such processes. The understanding of such evolutionary differences provides mechanistic insights into genotype-phenotype correlations underpinning biological processes across metazoans. The pioneering work developed by the William Pak laboratory for the past four decades, and the work of others, epitomize the notion of how the Drosophila system breaks new fertile ground or complements research fields of high scientific and medical relevance. Among the three major genetic complementation groups produced by the Pak's laboratory and impairing distinct facets of photoreceptor neuronal function, the nina group ( ninaA, ...., ninaJ) selectively affects the biogenesis of G protein-coupled receptors (GPCRs), mediating the photoconversion and transduction of light stimuli. Among the nina genes identified, ninaA arguably assumes heightened significance for several reasons. First, it presents unique physiological selectivity toward the biogenesis of a subset of GPCRs, a standalone biological manifestation yet to be discerned for most mammalian homologues of NinaA. Second, NinaA belongs to a family of proteins, immunophilins, which are the primary targets for immunosuppressive drugs at the therapeutic forefront of a multitude of medical conditions. Third, NinaA closest homologue, cyclophilin B (CyPB/PPIB), is an immunophilin whose loss-of-function was found recently to cause osteogenesis imperfecta in the human. This report highlights advances made by studies on some members of immunophilins, the cyclophilins. Finally, it reexamines critically data and dogmas derived from past and recent genetic, structural, biological, and pathological studies on NinaA and few other cyclophilins that support some of such paradigms to be less than definite and advance our understanding of the roles of cyclophilins in cell function, disease, and therapeutic interventions. [ABSTRACT FROM AUTHOR]

Cyclosporin A (CsA) is known to possess antiviral activity against several viruses in vitro, but the effect of CsA on BK polyoma virus (BKV) replication has not been examined. We investigated the impact of CsA on primary, chronic, and high-level BKV infection using a cell system of kidney cell origin (Vero E6 cells). During the first 2 h post infection, cells treated with CsA up to 3200 μg/L showed a near-identical BK viral load to untreated cells, with only a very minor reduction in the CsA-treated cells observed at 4 h. In chronic culture, CsA completely suppressed the primary BKV infection peak in a non-dose-dependent manner within the dose range of 200–12,800 μg/L ( P<0.05). BKV reactivation was also inhibited in the presence of CsA at doses of 200–3200 μg/L: the mean number of BKV DNA copies/mL remained stable or even decreased slightly compared with a 7-log increase in the non-CsA group ( P<0.01). CsA did not influence BKV DNA copies/mL in Vero E6 cells with high-level infection (>109 copies/mL). Cellular protein measurements indicated that the antiviral effect of CsA was not a result of cytotoxicity. These findings from a relevant in vitro kidney cell system indicate that CsA suppresses the primary BKV infection peak and inhibits escape to BKV reactivation; these effects are dose independent and not related to cytotoxicity. The intracellular antiviral mode of action of CsA against BKV does not appear to be via inhibition of viral cell entry pathways. [ABSTRACT FROM AUTHOR]

Abstract: This study compared the status of brain mitochondria isolated from 12-week streptozotocin (STZ)-diabetic rats versus STZ-diabetic animals treated with insulin during a period of 4 weeks. Brain mitochondria isolated from 12-week citrate (vehicle)-treated rats were used as control. For that purpose, several mitochondrial parameters were evaluated: respiratory indexes (respiratory control ratio (RCR) and ADP/O ratio), transmembrane potential (ΔΨm), repolarization lag phase, repolarization level, ATP, glutathione and coenzyme Q (CoQ) contents, production of H2O2, ATPase activity, and the capacity of mitochondria to accumulate Ca2+. Furthermore, the effect of Aβ1–40 was also analyzed. We observed that STZ-induced diabetes promoted a significant decrease in mitochondrial CoQ9, ATPase activity, and a lower capacity of mitochondria to accumulate Ca2+ when compared with control and insulin-treated diabetic rats. The presence of 4 μM Aβ1–40 induced a significant decrease in RCR in the three groups of rats. However, this peptide induced a significant increase in the repolarization lag phase and a significant decrease in the repolarization level in control and diabetic animals without insulin treatment. Furthermore, this peptide exacerbated significantly the production of H2O2 in STZ-diabetic rats, this effect being avoided by insulin treatment. Our data show that although diabetes induces some alterations in brain mitochondrial activity, those alterations do not interfere significantly with mitochondria functional efficiency. Similarly, insulin does not affect basal mitochondria function. However, in the presence of amyloid β-peptide, insulin seems to prevent the decline in mitochondrial oxidative phosphorylation efficiency and avoids an increase in oxidative stress, improving or preserving the function of neurons under adverse conditions, such as Alzheimer''s disease. [Copyright &y& Elsevier]