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Biochemical Pharmacology (v.69, #1)
Lisofylline: a potential lead for the treatment of diabetes by Zandong Yang; Meng Chen; Jerry L. Nadler (pp. 1-5).
Lisofylline (LSF), a synthetic modified methylxanthine, was originally designed and tested as an agent to reduce mortality during serious infections associated with cancer chemotherapy. Experimental studies and several clinical trials showed that LSF inhibited the generation of phosphatidic acid and free fatty acids. LSF also blocked the release of pro-inflammatory cytokines in oxidative tissue injury, in response to cancer chemotherapy and in experimental sepsis. Recent research has revealed a new potential to extend the therapeutic application of LSF especially for diabetes mellitus. These new studies demonstrate multiple actions of LSF in the regulation of immune cell function and autoimmune response by inhibition of IL-12 signalling and cytokine production. Supporting the new potential for LSF is the discovery of beneficial effects in protecting pancreatic β cells and in preventing autoimmunity. In this article, these new observations about LSF are reviewed and a strategy proposed for using this compound in new clinical applications. LSF may, thus, have therapeutic value in the prevention of autoimmune disorders, including Type 1 diabetes, and autoimmune recurrence following islet transplantation, and in preservation of β cell functional mass during islet isolation.
Keywords: Abbreviations; FFA; free fatty acids; LSF; lisofylline; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NOD mouse; nonobese diabetic mouse; PA; phosphatidic acid; scid; severe combined immune deficiency; STAT; signal transducers and activators of transcription; STZ; streptozotocinAnti-inflammation; Cytokine; Autoimmunity; Type 1 diabetes; Pancreatic β cell; Islet transplantation
Sequence specificity of formaldehyde-mediated covalent binding of anthracycline derivatives to DNA by Agata Szulawska; Marek Gniazdowski; Malgorzata Czyz (pp. 7-17).
Daunorubicin (DRB) and doxorubicin (DOX) in the presence of formaldehyde (CH2O) form covalent adducts with DNA. A G-specific adduct is formed by producing an aminal bridge between the C-3′ of daunosamine and the C-2 of guanine. New derivatives of DRB, DOX and epidoxorubicin (EDOX) with an amidine group bonded to the C-3′ of the daunosamine moiety, with either a morpholine or hexamethyleneimine ring attached to the amidine group, were studied in this paper. DNase I footprinting and analyses with restriction endonucleases were applied to compare the specificity of adduct formed by the amidine derivatives and their parent compounds. These approaches provide consistent results, proving that a GC pair is required for covalent binding of anthracycline derivatives to DNA and that different flanking sequences are able to modify the sequence preference of the drugs. The 5′-GC-3′, 5′-CG-3′ and 5′-TC-3′ sequences were protected most efficiently by the parent compounds and their morpholine derivatives and some increased protection of 5′-TC-3′ sequence was observed for morpholine analogues. Hexamethyleneimine derivatives bind to DNA with much lower efficiency. Finally, the sequence specificity of anthracycline derivatives was correlated with their ability to inhibit binding of transcription factors Sp1 and AP-1 to their DNA recognition sequences. The anthracycline derivatives were more potent in inhibiting Sp1 binding to its cognate GC box than in preventing AP-1 from binding to its mixed A·T and G·C site. Overall, the results indicate that the amidine derivatives of anthracyclines show similar, but not identical sequence specificity as parent compounds, though they exert their effect at a higher concentration.
Keywords: Abbreviations; DRB; daunorubicin; DOX; doxorubicin; EDOX; epidoxorubicin; DRBM; morpholine derivative of daunorubicin; DOXM; morpholine derivative of doxorubicin; EDOXM; morpholine derivative of epidoxorubicin; DRBH; hexamethyleneimine derivative of daunorubicin; DOXH; hexamethyleneimine derivative of doxorubicin; HUVEC; human umbilical vein endothelial cells; TNF-α; tumor necrosis factor alpha; EMSA; Electrophoretic Mobility Shift Assay; DTT; dithiothreitol; oc; open circular plasmid DNA; ccc; covalent closed circular plasmid DNA; ds-oligonucleotide; double-stranded oligonucleotideAnthracycline antibiotics; Covalent binding; Footprinting; Transcription factors; Drug specificity
Synthesis and biological activities of bisnaphthalimido polyamines derivatives: cytotoxicity, DNA binding, DNA damage and drug localization in breast cancer MCF 7 cells by Anne-Marie Dance; Lynda Ralton; Zoe Fuller; Lesley Milne; Susan Duthie; Charles S. Bestwick; Paul Kong Thoo Lin (pp. 19-27).
New bisoxynaphthalimidopolyamines (BNIPOPut, BNIPOSpd and BNIPOSpm) were synthesized. Their cytotoxic properties were evaluated against breast cancer MCF 7 cells and compared with bisnaphthalimidopolyamines BNIPSpd and BNIPSpm. Among the bisoxynaphthalimido polyamines, BNIPOSpm and BNIPOSpd exhibited cytotoxic activity with an IC50 of 29.55 and 27.22μM, respectively, while BNIPOPut failed to exert significant cytotoxicity after 48-h drug exposure. DNA binding was determined by midpoint of thermal denaturation ( Tm) measurement, ethidium bromide displacement and DNA gel mobility. Both BNIPOSpm and BNIPOSpd exhibited strong binding affinities with DNA. BNIPOPut had the least effect. The results were compared with other cytotoxic bisnaphthalimido compounds (BNIPSpm and BNIPSpd) previously reported by us. Using the single cell gel electrophoresis assay, it was found that BNIPSpm and BNIPSpd caused substantial DNA damage to MCF 7 treated cells while BNIPOSpm showed no significant effect over a range of drug concentrations after 4-h drug exposure. However, after 12-h drug exposure, BNIPOSpm had induced significant DNA damage similar to that of BNIPSpm (after 4-h drug exposure). Fluorescence microscopic analysis revealed that at 1μM drug concentration and after 6-h drug exposure, both BNIPSpm and BNIPSpd were located within the cell while the presence of BNIPOSpm, was not observed. Therefore, we conclude that BNIPSpd, BNIPSpm and BNIPOSpm induce DNA damage consistent with their rate of uptake into the cells.
Keywords: Abbreviations; BNIPPut; bisnaphthalimidopropylputrescine; BNIPSpd; bisnaphthalimidopropylspermidine; BNIPSpm; bisnaphthalimidopropylspermine; BNIPOPut; bisnaphthalimidooxypropylputrescine; BNIPOSpd; bisnaphthalimidooxypropylspermidine; BNIPOSpm; bisnaphthalimidooxypropylspermine; DMF; dimethylformamide; THF; tetrahydrofuran; MTT; 3-(4,5-diemthylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; T; m; midpoint of thermal denaturationBisnaphthalimides; Synthesis; Cytotoxicity; DNA binding and damage; Single cell gel electrophoresis assay; Fluorescence microscopy
Reversal by aminoguanidine of the age-related increase in glycoxidation and lipoxidation in the cardiovascular system of Fischer 344 rats by Régis Moreau; Binh T. Nguyen; Catalin E. Doneanu; Tory M. Hagen (pp. 29-40).
Non-enzymatic glycoxydation and lipoxidation of proteins continues to stimulate great interest in gerontology as both markers and promoters of aging. The first aim of the study was to determine the age-related changes in levels of Nɛ-(carboxymethyl)lysine (CML) and 4-hydroxy-2-nonenal (HNE) present on proteins of the cardiovascular system of Fischer 344 rats and identify the particular polypeptides being modified. The second objective was to evaluate whether pharmacological administration of aminoguanidine (1g/L in the drinking water) could reverse protein glycoxidation and lipoxidation. CML content in serum, aorta, and heart proteins from 28-month-old rats was double of that found in 4-month-old animals. AG administration to old rats for 3months from the age of 25months lowered CML content by 15 ( P=.2275), 44 ( P<.0001), and 28% ( P=.0072) in serum, aorta, and heart, respectively. Serum albumin, transferrin and immunoglobulins were most prominently adducted by both CML and HNE. While the extent of albumin and transferrin modification was comparable between age groups, CML and HNE bound to immunoglobulins increased in the sera of old rats as a result of the accumulation of immunoglobulin heavy and light chains. AG treatment prevented immunoglobulin accumulation in serum, suggesting a beneficial action on renal filtration. Lipoxidation of heart mitochondrial proteins was prevalent over glycoxidation, either as CML or pentosidine. Although AG prevented HNE-induced inactivation of the α-ketoglutarate dehydrogenase complex in vitro, it had no effect in rat hearts, suggesting AG could not reach the mitochondrial matrix.
Keywords: Abbreviations; CML; N; ɛ; -(carboxymethyl)lysine; HNE; 4-hydroxy-2-nonenal; AG; aminoguanidine; AGE; advanced glycation end-products; ALE; advanced lipoxidation end-products; KGDC; α-ketoglutarate dehydrogenase complex; iNOS; inducible nitric oxidase synthase; NO; nitric oxide N; ɛ; -(Carboxymethyl)lysine; 4-Hydroxy-2-nonenal; Amyloid; Heart; Mitochondria; Aging
Mechanism of concentration-dependent induction of heme oxygenase-1 by resveratrol in human aortic smooth muscle cells by Shu-Hui Juan; Tzu-Hurng Cheng; Hui-Chen Lin; Yen-Ling Chu; Wen-Sen Lee (pp. 41-48).
Resveratrol-mediated heme oxygenase-1 (HO-1) induction has been shown to occur in primary neuronal cultures and is thought to have potential neuroprotective action. Further, antioxidant properties of resveratrol have been reported to protect against coronary heart disease. We attempted to examine resveratrol's HO-1 inducing potency and its induction regulation in human aortic smooth muscle cells (HASMC). We showed that resveratrol-mediated HO-1 induction occurred in concentration- and time-dependent manners, but only at low concentrations (1–10μM), and that it was modulated at both the transcription and translation levels. Additionally, the results of our study showed that nuclear factor-kappa B (NF-κB) inhibitors eliminated resveratrol-mediated HO-1 induction and promoter activity, and that deletion of NF-κB binding sites in the HO-1 promoter region strongly reduced promoter activity, suggesting involvement of the NF-κB pathway in HO-1 induction by resveratrol. Suppression of NF-κB activity by resveratrol at high concentrations (≥20μM) has been reported to be attributed to its anti-inflammatory and anti-oxidative properties. Likewise, we showed that resveratrol at concentrations of ≥20μM blocked the activity of NF-κB through suppression of I kappa-B alpha (IκBα) phosphorylation, which caused inhibition of HO-1 induction. Conversely, resveratrol in a range of 1–10μM enhanced the phosphorylation and degradation of IκBα, a key step in NF-κB activation, resulting in HO-1 induction. Collectively, we suggest that resveratrol-mediated HO-1 expression occurs, at least in part, through the NF-κB pathway, which might contribute to resveratrol's vascular-protective effect at physiological concentrations after moderate red wine consumption.
Keywords: Abbreviations; HASMC; human aortic smooth muscle cell; HO-1; heme oxygenase-1; NF-κB; nuclear factor-kappa B; IκBα; I kappa-B alpha; MAPKs; mitogen-activated protein kinases; ERK1/2; extracellular signal-regulated kinase 1/2; JNK; c-Jun N-terminal kinase; NBT; 4-nitroblue tetrazolium; BCIP; 5-bromo-4-chloro-3-indolyl-phosphate; Act D; actinomycin D; Cyclo; cycloheximideHuman aortic smooth muscle cell; Heme oxygenase-1; Resveratrol; Nuclear factor-κB; I kappa-B alpha; Mitogen-activated protein kinases; Extracellular signal-regulated kinase 1/2; c-Jun N-terminal kinase
Effects of Y-27632, a selective Rho-kinase inhibitor, on myocardial preconditioning in anesthetized rats by Şeniz Demiryürek; Ali F. Kara; Ahmet Çelik; Mehmet Tarakçıoğlu; Cahit Bağcı; Abdullah T. Demiryürek (pp. 49-58).
The objective of this study was to examine the effects of Y-27632, a selective Rho-kinase inhibitor, on ischemic preconditioning (IP) and carbachol preconditioning (CP) in anesthetized rats. Administration of Y-27632 (0.1mg/kg) produced slight, but not significant, reduction in mean arterial blood pressure and suppressed the total number of ventricular ectopic beats (VEBs). IP, induced by 5min coronary artery occlusion and 5min reperfusion, decreased the incidence of ventricular tachycardia (VT) from 100 ( n=30) to 25% ( n=24) and abolished the occurrence of ventricular fibrillation (VF) (40% in control group) during 30min of ischemia. The incidences of VT and VF in Y-27632+IP group were found to be similar to IP group. Carbachol (4μg/kg/min for 5min) induced marked depressions in mean arterial blood pressure, heart rate and attenuated the total number of VEBs, but significant reductions in VT and VF incidences were noted in Y-27632+CP group. Y-27632 infusion for 5min abolished VF occurrence. Marked reductions in plasma lactate levels were observed in all treatment and preconditioning groups. IP led to marked decrease in malondialdehyde levels. Decreases in infarct size were also observed with all groups when compared to control. These results suggest that infusion of Y-27632 was able to produce cardioprotective effects on myocardium against arrhythmias, infarct size or biochemical parameters and mimic the effects of ischemic preconditioning in anesthetized rats. Therefore, it is likely that inhibition of Rho-kinase is involved in the signaling cascade of myocardial preconditioning.
Keywords: Abbreviations; CK-MB; creatine kinase-MB; CP; carbachol preconditioning; IP; ischemic preconditioning; LAD; left anterior descending; MDA; malondialdehyde; PKC; protein kinase C; PMA; phorbol-12-myristate-13 acetate; TBARS; thiobarbituric acid-reacting substances; VEBs; ventricular ectopic beats; VF; ventricular fibrillation; VT; ventricular tachycardiaArrhythmias; Cardioprotection; Infarct size; Preconditioning; Rho-kinase; Y-27632
Alterations of insulin secretion following long-term manipulation of ATP-sensitive potassium channels by diazoxide and nateglinide by Andrew J. Ball; Peter R. Flatt; Neville H. McClenaghan (pp. 59-63).
Previous studies have shown that prolonged exposure to drugs, which act via blocking KATP channels, can desensitize the insulinotropic effects of drugs and nutrients acting via KATP channels. In this study, effects of prolonged exposure to diazoxide, a KATP channel opener, on beta cell function were examined using clonal BRIN-BD11 cells. The findings were compared to the long-term effects of KATP channel blockers nateglinide and tolbutamide. Following 18h exposure to 200μM diazoxide, the amounts of insulin secreted in response to glucose, amino acids and insulinotropic drugs were increased. Secretory responsiveness to a variety of agents acting via KATP channels was retained following prolonged diazoxide exposure. In contrast, 18h exposure to 100μM nateglinide significantly attenuated the insulin secretory responses to tolbutamide, nateglinide and BTS 67 582. Glucose- andl-alanine-stimulated insulin release were unaffected by prolonged nateglinide exposure, however responsiveness tol-leucine andl-arginine was diminished. Prolonged exposure to nateglinide had no effect on forskolin- and PMA-stimulated insulin release, and the overall pattern of desensitization was similar to that induced by 100μM tolbutamide. We conclude that in contrast to chronic long-term KATP channel blockade, long-term diazoxide treatment is not harmful to KATP channel mediated insulin secretion and may have beneficial protective effects on beta cell function.
Keywords: Abbreviations; K; ATP; channels; adenosine triphosphate-sensitive potassium channels; PKA; protein kinase A; PKC; protein kinase C; PMA; phorbol 12-myristate 13-acetateClonal pancreatic β-cells; Insulin release; K; ATP; channels; Diazoxide; Nateglinide; Tolbutamide
Suppression of respiratory burst in human neutrophils by new synthetic pyrrolo-benzylisoquinolines by Tsong-Long Hwang; Yang-Chang Wu; Shang-Hsin Yeh; Reen-Yen Kuo (pp. 65-71).
Reactive oxygen species produced by neutrophils contribute to the pathogenesis of inflammatory diseases. In this study, the inhibition of superoxide anion (O2−) generation in human neutrophils by new synthetic pyrrolo-benzylisoquinoline derivatives was determined. We found that KW-2, KW-5, and KW-7 (8,9-dimethoxyl-1-( R-phenyl)-5,6-dihydro-pyrrolo[2,1- a]isoquinoline-2,3-dione; where R is 3-chloro, 3-bromo, and 4-methoxy, respectively) were the most effective inhibitors of formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP)-induced O2− release in human neutrophils. KW-2, KW-5, and KW-7 displayed no antioxidant or O2−-scavenging ability. The inhibition of O2− generation was reversed by the protein kinase (PK)A inhibitor, N-(2-(( p-bromocinnamyl)amino)ethyl)-5-isoquinolinesulfonamide (H89), but not by the PKG inhibitor (8 R,9 S,11 S)-(−)-2-methyl-9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta(cde)trinen-1-one (KT5823), or the soluble guanylate cyclase (sGC) inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). KW derivatives increased cellular cyclic AMP concentrations through the inhibition of phosphodiesterase (PDE) activity but not the elevation of adenylate cyclase (AC) activity. These results indicate that inhibition of FMLP-induced respiratory burst in human neutrophils by KW derivatives are cyclic AMP/PKA-dependent and are due to inhibition of PDE. The new chemical skeleton of PDE inhibitors may protect against the progression of inflammation.
Keywords: Abbreviations; AC; adenylate cyclase; CB; cytochalasin B; cyclic AMP; cyclic adenosine 3′,5′-monophosphate; cyclic GMP; cyclic guanosine 3′,5′-monophosphate; DMSO; dimethyl sulfoxide; FMLP; formyl-; l; -methionyl-; l; -leucyl-; l; -phenylalanine; H89; N; -(2-((; p; -bromocinnamyl)amino)ethyl)-5-isoquinolinesulfonamide; KT5823; (8; R; ,9; S; ,11; S; )-(−)-2-methyl-9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cyclocta(cde)trinen-1-one; O; 2; −; superoxide anion; PDE; phosphodiesterase; PKA; protein kinase A; PKC; protein kinase C; PKG; protein kinase G; LDH; lactate dehydrogenase; PMA; phorbol myristate acetate; sGC; soluble guanylate cyclase; SOD; superoxide dismutase; Ro318220; 3-(1-(3-(amidinothio)propyl-1H-indol-3-yl))-3-(1-methyl-1H-indol-3-yl)maleimide; WST-1; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium saltAdenylate cyclase; Cyclic AMP; Neutrophil; Phosphodiesterase; Protein kinase A; Pyrrolo-benzylisoquinoline
Changes in spectrin organisation in leukaemic and lymphoid cells upon chemotherapy by Patrycja M. Dubielecka; Bożena Jaźwiec; Stanisław Potoczek; Tomasz Wróbel; Joanna Miłoszewska; Olga Haus; Kazimierz Kuliczkowski; Aleksander F. Sikorski (pp. 73-85).
The aim of the present study was to investigate changes in spectrin and protein kinase C θ (PKC θ) organisation in human lymphoid and leukaemic cells undergoing chemotherapeutically induced apoptosis. An analysis of spectrin arrangement in human peripheral lymphoid (non-Hodgkin lymphoma) and leukaemic (acute lymphoblasic leukaemia) cells before and after chemotherapy revealed radical differences in the distribution of this protein. By using immunofluorescent technique, in lymphocytes isolated before chemotherapy, we found spectrin evenly distributed in the cytoplasm and the plasma membrane, while after the therapy changes in spectrin organisation occurred. Moreover, in lymphocytes after chemotherapy, extraction with buffer containing non-ionic detergent (Triton X-100) revealed presence of an insoluble fraction of spectrin. In normal or malignant cells before chemotherapy spectrin was totally soluble, however it should be mentioned that in total cell extracts and supernatants (but not in pellets) apoptotic fragments of spectrin (in addition to intact α and β chains) were also found. In malignant cells after chemotherapy changes in PKC θ organisation, similar to this observed in the case of spectrin, were shown by the immunofluorescence technique. In contrast, no differences in the distribution of other isoforms of protein kinase C: βI and βII, before and after chemotherapy, were found. Apoptotic phosphatidyloserine (PS) externalisation, as well as cell shrinkage, membrane protrusions and blebbing were observed in lymphocytes after chemotherapy and treatment with cytostatics in vitro. The overall results may suggest that spectrin redistribution/aggregation is the phenomenon involved in programmed cell death (PCD) of normal and neoplastic lymphocytes and lymphoblasts, however molecular basis of this phenomenon should be further investigated.
Keywords: Abbreviations; PKC; protein kinase C; SMAC; supramolecular activation complex; NF-κB; nuclear factor κB; IS; immunological synapse; TCR; T-cell receptor; TGF β; transforming growth factor β; ELF; embryonic liver fodrin; PBMCs; peripheral blood mononuclear cellsNonerythroid spectrin (fodrin); PKC θ; Chemotherapy; Apoptosis; ALL; nHL
Inhibitory effects of mevastatin and a geranylgeranyl transferase I inhibitor (GGTI-2166) on mononuclear osteoclast formation induced by receptor activator of NFκB ligand (RANKL) or tumor necrosis factor-α (TNF-α) by Je-Tae Woo; Hiroshi Nakagawa; Annette M. Krecic; Kazuo Nagai; Andrew D. Hamilton; Said M. Sebti; Paula H. Stern (pp. 87-95).
We have previously reported that the statin mevastatin (compactin) reversibly inhibits the fusion of TRAP-positive mononuclear preosteoclasts (pOCs) into multinucleated osteoclasts and disrupts the actin ring in mature osteoclasts through the inhibition of protein prenylation. Protein geranylgeranylation, specifically, is known to be required for pOC fusion and for the function and survival of mature osteoclasts. However, it has not been determined whether protein geranylgeranylation is involved in early differentiation of osteoclasts (pOC formation). The current study shows that statins and the geranylgeranyl transferase I inhibitor GGTI-2166 inhibit the pOC formation induced by RANKL or TNF-α in cultures of both mouse marrow-derived macrophage-colony-stimulating factor (M-CSF) dependent monocytes (MD cells) and the mouse monocyte cell line RAW 264.7 (RAW cells). Mevastatin, 0.1–0.6μM, inhibited the formation of pOCs induced by receptor activator of nuclear factor-κB ligand (RANKL) or tumor necrosis factor (TNF-α) in both cell cultures. The inhibitory effects of mevastatin were overcome by the addition of mevalonate, farnesyl pyrophosphate or geranylgeranyl pyrophosphate. GGTI-2166 inhibited TRAP activity induced by RANKL or TNF-α in both cell cultures and prevented the incorporation of [3H]all- trans geranylgeraniol into prenylated proteins in RAW cells. However, the farnesyl transferase inhibitor FTI-2153 did not inhibit TRAP activity although FTI prevented the incorporation of [14C]mevalonate into farnesylated proteins in RAW cells. Clostridium difficile cytotoxin B (toxin B) inhibited pOC formation induced by RANKL or TNF-α in both cell cultures. The inhibitory effects of statins and GGTI-2166 on pOC formation may result from the inhibition of the geranylgeranylation of G-proteins, such as Rho or Rac, suggesting that the geranylgeranylation of these proteins is involved in the early differentiation of progenitor cells into pOCs.
Keywords: Abbreviations; pOC; preosteoclast; RANKL; receptor activator of NFκB ligand; M-CSF; macrophage colony-stimulating factor; GGTI; geranylgeranyl transferase inhibitor; GGOH; geranylgeraniol; FTI; farnesyl transferase inhibitor; TRAP; tartrate-resistant acid phosphatase; TNF; tumor necrosis factor; GTP; guanosine triphosphate; HMG-CoA; hydroxymethylglutaryl coenzyme A; MEM; minimal essential medium; MD; marrow derived M-CSF dependent monocytes; MTT; methyl thiazole tetrazoliumOsteoclast differentiation; Statin; GGTI; FTI; Geranylgeranylation; Small G-protein
Accessibility of endothelial and inducible nitric oxide synthase to the intracellular citrulline–arginine regeneration pathway by Li-Jiuan Shen; Karin Beloussow; Wei-Chiang Shen (pp. 97-104).
This study investigates our hypothesis that argininosuccinate synthase (AS), the rate-limiting enzyme for arginine (l-arg) regeneration from citrulline (l-cit), plays a pivotal role in supplyingl-arg to endothelial (eNOS), but not inducible (iNOS) nitric oxide synthase, for nitric oxide (NO) production. Transgenic rat blood–brain barrier (TR-BBB) endothelial cells were used as a model to elucidate the accessibility of thel-arg compartments for NOS isozymes. NO production via eNOS or iNOS, with or without α-methyl-dl-aspartic acid (MDLA), an AS inhibitor, was measured by a fluorometric method. NO production via eNOS was activated by the calcium ionophore A23187, while via iNOS was induced by cytokines. AS activity was assayed by the amount of argininosuccinate regenerated from radioactive aspartic acid from cell extracts. Upon increased AS activity (5.9-fold) in cells grown inl-arg-free/l-cit-supplemented medium, A23187-activated NO production also significantly increased, however cytokine-induced NO production was not detected. A23187-activated NO production was observed not only inl-arg containing medium, but alsol-arg-free andl-arg-free/l-cit-supplemented medium, and was abolished by MDLA regardless of medium type. Cytokine-induced NO production was only observed inl-arg containing medium, not inl-arg-free orl-arg-free/l-cit-supplemented medium, and it was not inhibited by MDLA in thel-arg containing medium. Our results indicate that extracellularl-arg was the onlyl-arg pool for cytokine-induced NO production and intracellularl-arg regenerated froml-cit via AS pathway was the majorl-arg pool for A23187-activated NO production in TR-BBB endothelial cells. Therefore, modulation of AS activity could be a promising strategy to selectively alter NO production via eNOS, but not iNOS.
Keywords: Abbreviations; CAT; cationic amino acid transporter; eNOS; endothelial nitric oxide synthase; iNOS; inducible nitric oxide synthase; HPLC; high performance liquid chromatography; NO; nitric oxide; ADI; arginine deiminase; TR-BBB; transgenic rat-blood–brain barrier; AS; argininosuccinate synthase; l; -arg; l; -arginine; l; -cit; l; -citrulline; MDLA; α-methyl-; dl; -aspartic acidArgininosuccinate synthase; Nitric oxide synthases; Endothelial; Arginine; Nitric oxide; MDLA
4-Hydroxynonenal inhibits cell proliferation and alters differentiation pathways in human fetal liver hematopoietic stem cells by Craig G. Moneypenny; Evan P. Gallagher (pp. 105-112).
During fetal development, the liver serves as the primary hematopoietic organ in which hematopoietic stem cells (HSC) comprise a large proportion of hepatic cell populations. Because HSC are capable of initiating long-term hematopoiesis, injury to these cells may have ramifications with regard to the etiology of blood-borne diseases. In the current study, we examined the effects of 4-hydroxynonenal (4-HNE), a mutagenic α,β-unsaturated aldehyde that can be produced in utero, on HSC proliferation, differentiation, viability and apoptosis. Exposure of HSC to acute single doses of 4-HNE as low as 1nM inhibited HSC proliferation. Because 4-HNE rapidly disappears from culture media, a multiple dosing régime was also employed to approximate short-term steady state 4-HNE concentrations relevant to physiological oxidative stress. 4-Hydroxynonenal steady state concentrations as low as 1μM altered HSC differentiation pathways, but did not affect apoptosis or cause cell death. In contrast, exposure to steady state 5μM 4-HNE elicited a loss in viability, and increased the rate of apoptosis in total HSC populations. Collectively, our data indicate that cellular levels of 4-HNE associated with a low level of oxidative stress cause a loss of proliferation and viability and alter differentiation pathways in human fetal HSC.
Keywords: 4-Hydroxynonenal; Human fetal hematopoietic stem cells; Differentiation; Apoptosis
Thiocolchicine dimers: a novel class of topoisomerase-I inhibitors by Giuseppina Raspaglio; Cristiano Ferlini; Simona Mozzetti; Silvia Prislei; Daniela Gallo; Nandita Das; Giovanni Scambia (pp. 113-121).
During a cellular screening of thiocolchicine analogs, thiocolchicine dimers resulted particularly active in cisplatin-resistant A2780-CIS cells. In order to discover by which mechanism(s) thiocolchicine dimers overcame cisplatin resistance, p53, p21waf1 and MLH1 were assessed by Western blot. Results pointed out that, when combined with cisplatin, dimers increased the amount of all the three proteins with respect to the levels obtained by single drug exposure, thereby suggesting an interference in the process of repair of the cisplatin-induced DNA lesions. Moreover, in isolated nuclei drugs were able to produce DNA breaks, as demonstrated by Comet assay, thereby proving that the compounds were able to target cell nucleus independently from microtubules. Since Topo-I (topoisomerase I) is directly involved in the DNA repair and such activity is overexpressed in cisplatin-resistant cells, Topo-I was investigated as a potential target. Using DNA relaxation assay, thiocolchicine dimers inhibited Topo-I, a property not shared by thiocolchicine. At variance with camptothecin, dimers did not produce cleavable complexes, thereby indicating that Topo-I inhibition occurs upstream of the religation step. To assess the mechanism of inhibition, an electrophoretic mobility shift assay between DNA and Topo-I was performed and revealed that thiocolchicine dimers specifically interfere with binding of Topo-I to DNA. The interference is specific since the same compounds did not modulate DNase activity and did not act as intercalating agents in the DNA unwinding assay. Finally, behaviour of dimers as spindle poisons was investigated and no relevant changes with respect to thiocolchicine in terms of interaction with microtubules were found.
Keywords: Abbreviations; EMSA; electrophretic mobility shift assay; Topo-I; topoisomerase-I; SSB; single strand break; NER; nucleotide excision repair; CPT; camptothecinThiocolchicine; Topoisomerase-I; Drug-resistance; Spindle poisons; DNA repair; Cisplatin
Lack of an effect of breast cancer resistance protein (BCRP/ABCG2) overexpression on methotrexate polyglutamate export and folate accumulation in a human breast cancer cell line by Myung S. Rhee; Erasmus Schneider (pp. 123-132).
Accumulation of methotrexate (MTX) and its polyglutamates (PGs) has been recognized as an important factor in MTX efficacy. We have previously described a multidrug-resistant human breast cancer cell line, MCF7/MX, that exhibits reduced accumulation of total MTX as well as MTX-PGs, and that is resistant to continuous MTX exposure [Volk EL, Rohde K, Rhee M, McGuire JJ, Doyle LA, Ross DD, et al. Methotrexate cross-resistance in a mitoxantrone selected multidrug-resistant MCF7 breast cancer cell line is due to enhanced energy-dependent drug efflux. Cancer Res 2000;60:3514–21]. These cells express high levels of the breast cancer resistance protein (BCRP/ABCG2) that has been shown to actively transport MTX and short-chain MTX-PGs in vitro. However, the effect of BCRP on MTX-PG accumulation in intact cells was unclear. Here, we show that MTX transport by BCRP is required for the observed lower levels of MTX-PGs in the resistant cells. When BCRP was inhibited with fumitremorgin C, or in cells expressing a mutated form of BCRP that is unable to transport MTX, MTX-PG accumulation was similar or even higher than that in the parental cells that do not express BCRP. Concomitantly, there was increased inhibition of thymidylate synthase. It had previously been suggested that BCRP-mediated efflux of MTX-PGs contributed to the reduced MTX-PG accumulation. However, we found no evidence of BCRP-mediated efflux of MTX-PGs from intact cells, suggesting that direct efflux of MTX-PGs does not play a major role in MTX resistance. Together, these data show that BCRP overexpression can cause a reduction in total MTX accumulation as well as a reduction in the proportion of long-chain MTX-PGs. In contrast, BCRP overexpression did not affect natural folate accumulation or the relative distribution of folylpolyglutamates in the resistant, as compared to the parental, cells. Thus, it appears that BCRP overexpression affects the metabolism of the antifolate MTX, but not that of natural folates, although indirect effects cannot be excluded.
Keywords: Abbreviations; BCRP; breast cancer resistance protein; FPGS; folylpolyglutamate synthetase; FTC; fumitremorgin C; gGH; gamma glutamyl hydrolase; MRP; multidrug-resistance protein; MTX; methotrexate; PG; polyglutamate; UdR; deoxyuridineDrug resistance; Methotrexate; Breast cancer resistance protein; Polyglutamylation; Folate metabolism
Activation of protein kinase Cδ by proteolytic cleavage contributes to manganese-induced apoptosis in dopaminergic cells: protective role of Bcl-2 by Masashi Kitazawa; Vellareddy Anantharam; Yongjie Yang; Yoko Hirata; Arthi Kanthasamy; Anumantha G. Kanthasamy (pp. 133-146).
Chronic inorganic manganese exposure causes selective toxicity to the nigrostriatal dopaminergic system, resulting in a Parkinsonian-like neurological condition known as Manganism. Apoptosis has been shown to occur in manganese-induced neurotoxicity; however, the down-stream cellular target of caspase-3 that contributes to DNA fragmentation is not established. Herein, we demonstrate that proteolytic activation of protein kinase Cδ (PKCδ) by caspase-3 plays a critical role in manganese-induced apoptotic cell death. Treatment of PC12 cells with manganese caused a sequential activation of mitochondrial-dependent pro-apoptotic events, including mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, and DNA fragmentation. Overexpression of Bcl-2 in PC12 cells remarkably attenuated each of these events, indicating that the mitochondrial-dependent apoptotic cascade contributes to manganese-induced apoptosis. Furthermore, PKCδ was proteolytically cleaved by caspase-3, causing a persistent activation of the kinase. The manganese-induced proteolytic cleavage of PKCδ was significantly blocked by Bcl-2-overexpression. Administration of active recombinant PKCδ induced DNA fragmentation in PC12 cells, suggesting a pro-apoptotic role of PKCδ. Furthermore, expression of catalytically inactive mutant PKCδK376R via a lentiviral gene delivery system effectively attenuated manganese-induced apoptosis. Together, these results suggest that the mitochondrial-dependent caspase cascade mediates apoptosis via proteolytic activation of PKCδ in manganese-induced neurotoxicity.
Keywords: Abbreviations; DMEM; Dulbecco's modified Eagle's medium; ELISA; enzyme-linked immuno-sorbent assay; MnCl; 2; manganese chloride; PC12; rat pheochromocytoma cells; PKC; protein kinase C; ROS; reactive oxygen species; Z-DEVD-FMK; benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone; Z-VAD-FMK; benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketoneMitochondria; Oxidative stress; PKC; Gene delivery; Manganese; Parkinson's disease
BCL-xL overexpression effectively protects against tetrafluoroethylcysteine-induced intramitochondrial damage and cell death by Han K. Ho; Zhong-Hua Hu; Shie-Pon Tzung; David M. Hockenbery; Nelson Fausto; Sidney D. Nelson; Sam A. Bruschi (pp. 147-157).
S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine (TFEC), a major metabolite of the industrial gas tetrafluoroethylene, has been shown to mediate nephrotoxicity by necrosis. TFEC-induced cell death is associated with an early covalent modification of specific intramitochondrial proteins; including aconitase, α-ketoglutarate dehydrogenase (KGDH) subunits, HSP60 and HSP70. Previous studies have indicated that the TAMH line accurately models TFEC-induced in vivo cell death with dose- and time-dependent inhibitions of both KGDH and aconitase activities. Here, we show that the molecular pathway leading to TFEC-mediated cell death is associated with an early cytosolic to mitochondrial translocation of BAX, a pro-apoptotic member of the BCL-2 family. Immunoblot analyses indicated movement of BAX (21kDa) to the mitochondrial fraction after exposure to a cytotoxic concentration of TFEC (250μM). Subsequent cytochrome c release from mitochondria was also demonstrated, but only a modest increase in caspase activities was observed, suggesting a degeneration of early apoptotic signals into secondary necrosis. Significantly, TAMH cells overexpressing BCL-xL preserved cell viability even to supratoxicological concentrations of TFEC (≤600μM), and this cytoprotection was associated with decreased HSP70i upregulation, indicating suppression of TFEC-induced proteotoxicity. Hence, TFEC-induced necrotic cell death in the TAMH cell line is mediated by BAX and antagonized by the anti-apoptotic BCL-2 family member, BCL-xL.
Keywords: Abbreviations; AMC; 7-amino-4-methylcoumarin; ATF3; activating transcription factor-3; BSS; buffered saline solution; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; FBS; fetal bovine serum; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; HSP; heat shock protein; INT; iodonitrotetrazolium chloride; KGDH; α-ketoglutarate dehydrogenase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; PIPES; piperazine-N,N′-bis(2-ethanesulfonic acid); TNF; tumor necrosis factor; VDAC; voltage-dependent anion-selective channelTFEC; BAX translocation; BCL-xL; Cytotoxicity; Intramitochondrial damage; Necrosis
Glycine taken up through GLYT1 and GLYT2 heterotransporters into glutamatergic axon terminals of mouse spinal cord elicits release of glutamate by homotransporter reversal and through anion channels by Luca Raiteri; Sara Stigliani; Antonella Siri; Mario Passalacqua; Edon Melloni; Maurizio Raiteri; Giambattista Bonanno (pp. 159-168).
Glycine concentration-dependently elicited [3H]d-aspartate ([3H]d-ASP) release from superfused mouse spinal cord synaptosomes. Glycine effect was insensitive to strychnine or 5,7-dichlorokynurenic acid, but was prevented by the glycine transporter blocker glycyldodecylamide. Glycine also evoked release of endogenous glutamate, which was sensitive to glycyldodecylamide and abolished in low-Na+ medium. Experiments with purified synaptosomes and gliasomes show that the glycine-evoked [3H]d-ASP release largely originates from glutamatergic nerve terminals. The glycine-evoked [3H]d-ASP release was halved by NFPS, a selective blocker of GLYT1 transporters, or by Org 25543, a selective GLYT2 blocker, and almost abolished by a mixture of the two, suggesting that activation of GLYT1 and GLYT2 present on glutamatergic terminals triggers the release of [3H]d-ASP. Accordingly, confocal microscopy experiments show localization of GLYT1 and GLYT2 in purified synaptosomes immuno-stained for the vesicular glutamate transporter vGLUT1. The glycine effect was independent of extra- and intraterminal Ca2+ ions. It was partly inhibited by the glutamate transporter blockerdl-TBOA and largely prevented by the anion channel blockers niflumic acid and NPPB. To conclude, transporters for glycine (GLYT1 or/and GLYT2) and for glutamate coexist on the same spinal cord glutamatergic terminals. Activation of glycine heterotransporters elicits glutamate release partly by homotransporter reversal and largely through anion channels.
Keywords: Abbreviations; [; 3; H]; d; -ASP; [; 3; H]; d; -aspartate; BAPTA; 1,2-; bis; -(2-aminophenoxy)ethane-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid; 5,7-DCK; 5,7-dichlorokynurenic acid; GDA; glycyldodecylamide; GFAP; glial fibrillary acidic protein; NFPS; N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine; NPPB; 5-nitro-2-(3-phenylpropylamino)benzoic acid; Org 25543; 4-benzyloxy-3,5-dimethoxy-; N; -[1-(dimethylaminocyclopentyl)methyl] benzamide; PSD-95; 95; kDa postsynaptic density protein; SDS–PAGE; SDS–polyacrylamide gel electrophoresis; dl; -TBOA; dl-threo-β-benzyloxyaspartic acid; vGLUT1; vesicular glutamate transporter type 1Glutamate release; Glycine transporter types; Glycine heterotransporters; Glycine–glutamate interactions; Carrier-mediated release; Anion channels
Binding affinity and agonist activity of putative endogenous cannabinoids at the human neocortical CB1 receptor by Marc Steffens; Josef Zentner; Jürgen Honegger; Thomas J. Feuerstein (pp. 169-178).
We investigated the affinity of putative endocannabinoids (2-arachidonylglycerol, 2-AG; noladin ether, virodhamine) for the human neocortical CB1 receptor. Functional activity of these compounds (including anandamide, AEA) was determined by examining basal and forskolin-stimulated cAMP formation. Assays were performed with synaptosomes, prepared from fresh human neocortical tissue. Receptor affinity was assessed from competition binding experiments with the CB1/2 agonist [3H]-CP55.940 in absence or presence of a protease inhibitor to assess enzymatic stability. Noladin ether and virodhamine inhibited [3H]-CP55.940 binding ( Ki: 98, 1740nM, respectively). Protease inhibition decreased the Ki value of virodhamine ( Ki: 912nM), but left that of noladin ether unchanged. 2-AG almost lacked affinity ( Ki>10μM). Basal cAMP formation was unaffected by AEA and noladin ether, but strongly enhanced by 2-AG and virodhamine. Forskolin-stimulated cAMP formation was inhibited by AEA and noladin ether (IC50: 69, 427nM, respectively) to the same extent as by CP55.940 ( Imax each ∼30%). Inhibitions by AEA or noladin ether were blocked by the CB1 receptor antagonist AM251. Virodhamine increased forskolin-stimulated cAMP formation, also in presence of AM251, by ∼20%. 2-AG had no effect; in presence of AM251, however, 10μM 2-AG stimulated cAMP formation by ∼15%. Our results suggest, that AEA and noladin ether are full CB1 receptor agonists in human neocortex, whereas virodhamine may act as a CB1 receptor antagonist/inverse agonist. Particularly the (patho)physiological role of 2-AG should be further investigated, since its CB1 receptor affinity and agonist activity especially in humans might be lower than generally assumed.
Keywords: Abbreviations; AEA; anandamide; 2-AG; 2-arachidonylglycerol; FAAH; fatty acid amidohydrolase; PMSF; phenylmethylsulfonylfluoride2-Arachidonylglycerol; Anandamide; Endocannabinoid; Human brain; Noladin ether; Virodhamine
Characterization of antinociceptive activity of novel endomorphin-2 and morphiceptin analogs modified in the third position by Jakub Fichna; Jean-Claude do-Rego; Piotr Kosson; Jean Costentin; Anna Janecka (pp. 179-185).
In the present study we investigated and compared the in vivo analgesia of centrally administered endomorphin-2 and morphiceptin, and their analogs modified in position 3. Two series of analogs were synthesized by introducing unnatural aromatic amino acids in thed configuration: 3-(1-naphthyl)-d-alanine (d-1-Nal), 3-(2-naphthyl)-d-alanine (d-2-Nal), 3-(4-chlorophenyl)-d-alanine (d-ClPhe), 3-(3,4-dichlorophenyl)-d-alanine (d-Cl2Phe). Antinociceptive activity of endomorphin-2, morphiceptin, and their analogs was compared in the mouse hot-plate test, performed after i.c.v. administration of the peptides at a dose of 10μg/animal. The best results were obtained for two morphiceptin analogs, [d-Phe3]morphiceptin and [d-1-Nal3]morphiceptin, which showed greatly improved analgesic activity, as compared to morphiceptin. In the endomorphin-2 series none of the modifications produced analogs more potent than the parent compound, but [d-1-Nal3]endomorphin-2 was the best analog. Antinociception induced by endomorphin-2 was reversed by concomitant i.c.v. administration of [d-Phe3]endomorphin-2, [d-2-Nal3]endomorphin-2, and [d-2-Nal3]morphiceptin, indicating that these analogs were weak μ-opioid antagonists.
Keywords: μ-Opioid receptor; Hot-plate test; Peptide analogs; Agonists; Antagonists; Naloxone
Bidirectional transfer of methadone across human placenta by Ilona A. Nekhayeva; Tatiana N. Nanovskaya; Sujal V. Deshmukh; Olga L. Zharikova; Gary D.V. Hankins; Mahmoud S. Ahmed (pp. 187-197).
Methadone maintenance programs are considered the standard of care for the pregnant opiate addict. However, data on changes in methadone pharmacokinetics (PK) during pregnancy are limited and do not include its disposition by the placenta due to obvious ethical and safety considerations. Accordingly, investigations in our laboratory are focusing on human placental disposition of opiates including methadone. Recently, we reported on methadone metabolism by placental aromatase and provide here data on its bidirectional transfer across the tissue utilizing the technique of dual perfusion of placental lobule. The concentrations of the opiate transfused into the term placental tissue were those reported for its in vivo levels in the maternal serum of women under treatment with the drug. Data obtained indicated that the opiate has no adverse effects on placental viability and functional parameters and that it is retained by the tissue. Also, methadone transfer and its clearance index in the fetal to maternal direction (0.97±0.05) was significantly higher ( P<0.05) than in the maternal to fetal (0.83±0.09). The observed asymmetry in methadone transfer could be explained by the unidirectional activity of the efflux transporter P glycoprotein (P-gp) that is highly expressed in variable amounts in trophoblast tissue. Therefore, placental disposition of methadone might be an important contributor to the regulation of its concentration in the fetal circulation and consequently may affect the incidence and intensity of neonatal abstinence syndrome for women treated with the drug during pregnancy.
Keywords: Abbreviations; PK; pharmacokinetics; P-gp; P glycoprotein; NAS; neonatal abstinence syndrome; BUP; bupremorphine; LAAM; l; -α-acetylmethadol; AP; antipyrine; M; →; F; maternal to fetal; F; →; M; fetal to maternal; EDDP; 2-ethyliolene-1,5-dimethyl-3,3-diphenylpyrrolidine; EMDP; 2-ethyl-5-methyl-3,3-diphenyl-1-pyrrolineMethadone; Human placenta; Pregnancy; Transplacental transfer