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Biochemical Pharmacology (v.71, #6)
JAK/STAT signal transduction: Regulators and implication in hematological malignancies by Lyne Valentino; Josiane Pierre (pp. 713-721).
Signal transducers and activators of transcription (STATs) comprise a family of several transcription factors that are activated by a variety of cytokines, hormones and growth factors. STATs are activated through tyrosine phosphorylation, mainly by JAK kinases, which lead to their dimerization, nuclear translocation and regulation of target genes expression. Stringent mechanisms of signal attenuation are essential for insuring appropriate, controlled cellular responses. Among them phosphotyrosine phosphatases (SHPs, CD45, PTP1B/TC-PTP), protein inhibitors of activated STATs (PIAS) and suppressors of cytokine signaling (SOCS) inhibit specific and distinct aspects of cytokine signal transduction. SOCS proteins bind through their SH2 domain to phosphotyrosine residues in either cytokine receptors or JAK and thus can suppress cytokine signaling. Many recent findings indicate that SOCS proteins act, in addition, as adaptors that regulate the turnover of certain substrates by interacting with and activating an E3 ubiquitin ligase. Thus, SOCS proteins act as negative regulators of JAK/STAT pathways and may represent tumour suppressor genes. The discovery of oncogenic partner in this signaling pathway, more especially in diverse hematologic malignancies support a prominent role of deregulated pathways in the pathogenesis of diseases. Fusion proteins implicating the JH1 domain of JAK2 ( TEL- JAK2, BCR- JAK2), leading to deregulated activity of JAK2, have been described as the result of translocation. Somatic point mutation in JH2 domain of JAK2 (JAK2V617F), leading also to constitutive tyrosine phosphorylation of JAK2 and its downstream effectors was reported in myeloproliferative disorders. Furthermore, silencing of socs- 1 and shp- 1 expression by gene methylation is observed in some cancer cells.
Keywords: Abbreviations; BCR; break cluster region; EPO; erythropoietin; ETV6; ets translocation variant gene 6; GH; growth hormone; GM-CSF; granulocyte-macrophage colony-stimulating factor; IL; interleukin; JAK; Janus kinase; JH; JAK homology; PCM 1; pericentriolar material 1; PIAS; protein inhibitors of activated STAT; PRL; prolactin; PTP; protein tyrosine phosphatase; SHP; Src-homology 2 (SH2)-containing phosphatase; SOCS; suppressors of cytokine signaling; STAT; signal transducers and activators of transcription; TEL; translocated ETS leukaemia; TPO; thrombopoietin; TYK2; tyrosine kinase-2Signal transduction; JAK/STAT pathway; Regulation of STAT activity; Oncogenesis
Suppression of survival in human SKBR3 breast carcinoma in response to metal–chelator complexes is preferential for copper–dithiocarbamate by Maricela Viola-Rhenals; Mary Strasberg Rieber; Manuel Rieber (pp. 722-734).
Since diethyl dithiocarbamate (DEDTC) forms complexes with either zinc or copper, and 8-hydroxyquinoline (8-OHQ) also complexes with copper, we now compared the cytotoxic activity of Cu[DEDTC]2, Zn[DEDTC]2 and Cu[8-OHQ]2. This report shows that at nanomolar levels, only copper–[DEDTC]2, suppresses proliferation and clonogenicity of SKBR3 human breast carcinoma, concurrently with induction of apoptosis-associated PARP fragmentation. Susceptibility to these agents was paralleled by reactive oxygen generation (ROS) and greater expression of anti-oxidant enzymes like MnSOD and catalase, with no comparable effect on Cu/Zn superoxide dismutase. The lethal effects of Cu[DEDTC]2 manifested when adding the two separate aqueous components or the preformed synthetic complexes in DMSO, was prevented by N-acetyl cysteine or glutathione, with no comparable protection afforded by non-thiol anti-oxidants like mannitol or DMSO. Exogenously added catalase also protected cells from Cu[DEDTC]2, suggesting that this complex may kill after the levels of superoxide anion [O2−] dismutated by MnSOD increase hydrogen peroxide-related stress. Cu[DEDTC]2 also induced p21WAF1, a cdk inhibitor usually not inducible in mutant p53 tumors like SKBR3 carcinoma, correlating with dephosphorylation of the Sp1 transcription factor. Concentrations of Cu[DEDTC]2 cytotoxic for SKBR3 carcinoma did not induce comparable damage versus normal diploid human WI-38 fibroblasts. In contrast to the cytotoxic effect of nM levels of Cu[DEDTC]2 against SKBRR3 cells, no response was seen in the same cells exposed to 20μM cis-platin. Since neither DEDTC bound to zinc, nor copper bound to 8-OHQ showed comparable cytotoxicity, our results suggest that the greater activity of copper–DEDTC reflects a specific structure–activity relationship for the active complex. Since Cu[DEDTC]2 shows more effectiveness than other metal–chelator complexes, it may be worth further investigation as an alternative to cancer therapies.
Keywords: Abbreviations; Cu[DEDTC]; 2; bis-[diethyl dithiocarbamate] copper(II); Cu[8-OHQ]; 2; bis-[8-hydroxyquinoline] copper(II); Cu/ZnSOD; copper/zinc superoxide dismutase; H; 2; DCFDA; 2′,7′-dichlorodihydrofluorescein diacetate; MnSOD; manganese superoxide dismutase; ROS; reactive oxygen intermediates; Zn[DEDTC]; 2; bis-[diethyl dithiocarbamate] zinc(II); Zn[8-OHQ]; 2; bis-[8-hydroxyquinoline] zinc(II)ROS production; Anti-oxidant enzymes; Glutathione; cis; -Platin; H; 2; DCFDA
Inhibitory effect of dietary phenolic compounds on Chlamydia pneumoniae in cell cultures by J. Alvesalo; H. Vuorela; P. Tammela; M. Leinonen; P. Saikku; P. Vuorela (pp. 735-741).
Chlamydial infections are very common worldwide. All chlamydial species have a tendency to cause persistent infections, which have been associated to several chronic diseases including blinding trachoma, infertility and coronary heart disease (CHD). At present, no efficient treatment for the eradication of chronic chlamydial infections exists and, thus, new antichlamydial compounds are urgently needed. This study was designed to screen antichlamydial activity of natural flavonoids and other natural and structurally similar synthetic compounds against Chlamydia pneumoniae in human cell line (HL).HL cells were infected with C. pneumoniae and incubated 72h with studied compounds. Reduction in the number of inclusions was determined with immunofluorescence staining. In vitro minimum inhibitory concentration was also determined for some of the most active compounds. Thirty seven percentage of the studied compounds (57 in total) were highly active against C. pneumoniae and all the studied compounds were non-toxic to the host cells at studied concentrations.Our study revealed direct antichlamydial effect for selected polyphenolic compounds against C. pneumoniae, in vitro. We also demonstrated the ability of some of the investigated compounds to accumulate inside cells or into cell membranes and cause inhibition, even when present only prior to infection.
Keywords: Abbreviations; EB; elementary body; HL cells; human cell line; IFU; inclusion-forming unit; LDH; lactate dehydrogenase; RB; reticulate body; CHD; coronary heart disease; MIC; minimum inhibitory concentration Chlamydia pneumoniae; Flavonoids; Antichlamydial activity; Phenolic compounds
Resveratrol induces apoptosis of human malignant B cells by activation of caspase-3 and p38 MAP kinase pathways by Takayuki Shimizu; Tomonori Nakazato; Ming Ji Xian; Morihiko Sagawa; Yasuo Ikeda; Masahiro Kizaki (pp. 742-750).
Red wine polyphenol, trans-resveratrol (trans-3,4′,5-trihydroxy stilbene), has potent chemopreventive effects against various tumors. In this study, we found for the first time that resveratrol rapidly induces S phase cell cycle arrest of human malignant B cells including myeloma cells in dose- and time-dependent manners, followed by S phase cell cycle arrest through ATM/Chk pathway. Resveratrol-induced apoptosis occurs in association with the activation of caspase-3 and the loss of mitochondrial transmembrane potentials. In addition, resveratrol induces the phosphorylation of p38 MAP kinase, and specific inhibition of p38 MAP kinase abolishes the resveratrol-induced apoptosis, indicating that activation of the p38 MAP kinase pathway is required for inducing apoptosis in malignant B cells. These results suggest that resveratrol may have potential as a novel therapeutic agent for the patients with B cell malignancies including multiple myeloma.
Keywords: Resveratrol; B cell malignancy; Myeloma; Apoptosis; Caspase; p38 MAP kinase
Epidermal growth factor receptor inhibitor (PD168393) potentiates cytotoxic effects of paclitaxel against androgen-independent prostate cancer cells by Yeong-Shiau Pu; Min-Wei Hsieh; Chuang-Wei Wang; Guang-Yaw Liu; Chao-Yuan Huang; Chia-Chi Lin; Jing-Yi Guan; Shinne-Ren Lin; Tzyh-Chyuan Hour (pp. 751-760).
Recent data showed that epidermal growth factor receptor (EGFR) inhibitors, such as ZD1839, alone or in combination with chemotherapeutic agents for androgen-independent prostate cancer (AIPC) did not produce promising results in clinical settings. More effective regimens involving novel stronger inhibitor of EGFR and better combinations are needed. The anti-tumor activity of PD168393, an irreversible EGFR inhibitor, with or without chemotherapeutic agents for the treatment of AIPC was investigated in vitro. In results, both the androgen-independent cell lines PC-3 and DU145 expressed higher levels of EGFR than the androgen-dependent MDA PCa 2b and androgen-responsive LNCaP cells by Western blotting. DU145 was much more sensitive to PD168393 and ZD1839 than MDA PCa 2b. PD168393, but not ZD1839, significantly potentiated paclitaxel cytotoxicity against DU145 by MTT assay and median-effect analysis. The combination of PD168393 or ZD1839 with other cytotoxic agents including docetaxel and 5-fluorouracil, however, was either additive or antagonistic. Compared to paclitaxel alone, PD168393 significantly enhanced paclitaxel-induced DNA fragmentation, sub-G1 fraction accumulation, mitochondrial membrane dysfunction, cytochrome C release, caspase-3 activation and eventually apoptosis. These molecular events were accompanied by Bad up-regulation, p53 and p21Waf1/Cip1 induction, ERK1/2 inactivation and inhibition of EGFR phosphorylation in the presence of PD168393. These effects did not involve significant alteration in the Akt1/2 and STAT3 signaling pathway. In conclusion, the combination of paclitaxel and PD168393 produced a profound synergistic growth inhibition of AIPC cells. Combining PD168393 with paclitaxel may have clinical benefits and warrants further investigation.
Keywords: Docetaxel; Synergism; Quinazoline; Chemotherapeutic agents; Hormone refractory
MZF1 possesses a repressively regulatory function in ERCC1 expression by Qing-Wu Yan; Eddie Reed; Xiao-Song Zhong; Keith Thornton; Yi Guo; Jing Jie Yu (pp. 761-771).
ERCC1 is a critical gene within the nucleotide excision repair pathway. Overexpression of ERCC1 through promoter-mediating transcriptional regulation is associated with repair of cisplatin-induced DNA damage and clinical resistance to platinum-chemotherapy. Several transcriptional repressors and activators within the 5′-flanking region of the ERCC1 gene may be involved in the up-regulation of this gene. Minimal sequence within the promoter region required for ERCC1 transcription was analyzed by CAT assay and demonstrated that the region of −220 to −110 is essential to constitutive expression of ERCC1 gene in ovarian cancer cell line A2780/CP70. A more forward upstream region seems to be responsible for cisplatin-induced expression. Study of the functional cis-element in this region by electrophoretic mobility shift assay indicates that a MZF1-like site as well as an AP1-like site responded in a time-dependent manner to cisplatin stimulation with altered binding activities. EMSA with MZF1 ZN1–4 consensus oligonucleotides suggests that the MZF1 N-terminal domain of zinc finger cluster may bind to the MZF1-like site of the ERCC1 promoter region. MZF1 mRNA in A2780/CP70 cells decreased upon cisplatin exposure as analyzed by quantitative PCR, suggesting that MZF1 may mediate cisplatin-invoked gene expression in these cells. Overexpression of MZF1 repressed the ERCC1 promoter activity as determined in co-transfection assay, suggesting that MZF1 might be a repressor of ERCC1 transcription upon cisplatin exposure. In summary, our studies revealed a core promoter region and adjacent drug-responsible region within the ERCC1 promoter. The drug-responsible region contains cis-elements of activator, AP1 and repressor, MZF1. In response to cisplatin treatment, decreased MZF1 and increased AP1 binding activities appear to be the leading mechanism of up-regulation of ERCC1 expression. Our findings imply potential therapeutic strategies to antagonize drug resistant mechanisms in treatment of human ovarian cancer.
Keywords: Abbreviations; CDDP; cisplatin or; cis; -diamminedichloroplatinum(II); NER; nucleotide excision repair; ERCC1; excision repair cross-complementation group 1; EMSA; electrophoretic mobility shift assay; RT-PCR; reverse transcriptase-polymerase chain reaction; CAT; chloramphenicol acetyltransferase; ELISA; enzyme-linked immunoassay; CMV; cytomegalovirus; AP1; activator protein 1; MZF1; myeloid zinc finger gene 1MZF1; AP1; ERCC1; Repressor; Cisplatin resistance; NER
Methionine dependence of tumours: A biochemical strategy for optimizing paclitaxel chemosensitivity in vitro by Valerie Pavillard; Anna Nicolaou; John A. Double; Roger M. Phillips (pp. 772-778).
Methionine dependence is a unique feature of cancer cells characterized by growth and cell cycle arrest (typically in S and G2/M) under conditions of methionine depletion. Following replenishment of media with methionine, the cell cycle blockade is reversible and during this recovery period, cells may become more susceptible to the action of cell cycle specific drugs. The response of a panel of methionine dependent (HTC, Phi-1, PC3 and 3T3) cells to vinblastine and paclitaxel was compared to methionine independent Hs-27 cells under conditions of methionine depletion (M−H+; methionine depleted media supplemented with homocysteine) and starvation (M−H−; media without methionine or homocysteine). All cell lines were significantly more resistant to both agents under M−H+ and M−H− conditions compared to controls under normal culture conditions [M+H−]; however, the magnitude of resistance was reduced in the methionine independent Hs-27 cells. During recovery from methionine depletion and starvation, the response of the methionine dependent cells to vinblastine and paclitaxel was significantly enhanced compared to controls. Although the activity of vinblastine on the Hs-27 cell line was comparable to controls, these methionine independent cells became significantly more resistant to paclitaxel during recovery studies (IC50=2.13±0.5μM) compared to control cultures (IC50=0.13±0.15μM). Whilst the mechanism responsible for this remains uncertain, the increased activity of paclitaxel against methionine dependent cells in conjunction with the decreased activity against Hs-27 cells suggests that methionine depletion strategies may enhance the therapeutic index of paclitaxel.
Keywords: Methionine-dependence; Paclitaxel; Chemosensitivity; Vinblastine; Homocysteine
Selective lysis of erythrocytes infected with the trophozoite stage of Plasmodium falciparum by polyene macrolide antibiotics by Ursula I.M. Wiehart; Marina Rautenbach; Heinrich C. Hoppe (pp. 779-790).
The continuous increase in strains of the human malaria parasite Plasmodium falciparum resistant to most front-line antimalarial compounds is reason for grave clinical concern. The search for new drugs led us to investigate a number of membrane active polyene macrolide antibiotics, such as amphotericin B, nystatin, filipin and natamycin. The interaction of these compounds with sterols in bilayer cell membranes can lead to cell damage and ultimately cell lysis. The malaria parasite modifies the host erythrocyte membrane by changing the protein and lipid composition and thus the infected cell could be a selective target for membrane active compounds. We found that erythrocytes infected with the trophozoite stage of P. falciparum were particularly susceptible to lysis by amphotericin B (Fungizoneâ„¢) and, to a lesser extent, nystatin, as determined by ELISA and various microscopy assays. Liposomal amphotericin B (AmBisomeâ„¢) displayed a similar specificity for parasitised erythrocytes, but complete lysis required a longer incubation period. In contrast, filipin and natamycin did not distinguish between normal and parasite-infected erythrocytes, but lysed both at similar concentrations. In addition, when added to ring-stage cultures, the amphotericin B preparations and nystatin produced a marked disruption in parasite morphology in less than 2h without an accompanying permeabilisation of the infected host cell, suggesting a second plasmodicidal mode of action. The results imply that selected polyene macrolide antibiotics or their derivatives could find application in the treatment of severe malaria caused by of P. falciparum.
Keywords: Malaria; Erythrocyte; Amphotericin; Nystatin; Filipin; Haemolysis
Sensitization to gimatecan-induced apoptosis by tumor necrosis factor-related apoptosis inducing ligand in prostate carcinoma cells by Paola Perego; Emilio Ciusani; Laura Gatti; Nives Carenini; Elisabetta Corna; Franco Zunino (pp. 791-798).
Since the intrinsic resistance of prostate carcinoma likely reflects a low susceptibility to drug-induced apoptosis, in this study we explored the possibility of sensitizing prostate carcinoma cells to apoptosis by combination of TRAIL with camptothecins. Indeed, these agents are known to activate different pathways of apoptosis. Topotecan- and gimatecan induced moderate up-regulation of TRAIL-R1 and -R2 which resulted in a different cell response to the combination in androgen-independent cells (DU-145 and PC-3). In DU-145 cells apoptosis was increased by lower TRAIL concentrations and was earlier than in PC-3 cells, as shown using Annexin V-binding assay. The relative resistance of PC-3 cells to drug-induced apoptosis was associated with constitutive Akt activation, higher levels of cFLIP-L and Bcl-2, and lower levels of Bax. The different expression/activation of apoptosis-related factors appears to influence the sensitization of prostate carcinoma cells by TRAIL. Potentiation of camptothecin-induced apoptosis by TRAIL appears dependent on cooperation between extrinsic and intrinsic pathways, as documented by loss of the sensitization to apoptosis following reduction of caspase 8 after small interfering RNA transfection. The efficacy of the approach may be critically dependent on the intrinsic susceptibility to apoptosis of different tumors. These observations support that the activation of multiple signals could enhance apoptotic response and suggest the therapeutic interest of the TRAIL/camptothecin combination.
Keywords: Abbreviations; TRAIL; TNF-related apoptosis inducing ligand; TPT; topotecan; ST1481 (gimatecan); 7-butoxyiminomethylcamptothecin; siRNA; small interfering RNAGimatecan; TRAIL; Prostate carcinoma; Apoptosis; Cell sensitivity; Camptothecins
Vitamin E inhibits hemolysis induced by hemin as a membrane stabilizer by Fang Wang; Tinghua Wang; Jianhua Lai; Ming Li; Chenggang Zou (pp. 799-805).
Hemin is a potential cytolytic agent. To test the effect of vitamin E on hemin-mediated permeability in cell membranes, sheep erythrocytes were chosen as an appropriate model to study hemolysis induced by hemin. Hemin-induced hemolysis but did not elicit lipid peroxidation in sheep erythrocytes. Vitamin E was effective in inhibiting hemin-mediated hemolysis. Both chromanol ring and the isoprenoid side chain of tocopherols were essential for inhibition of hemin-induced hemolysis. There was a strong correlation between the inhibitory effects of tocopherols on hemin-induced erythrocyte hemolysis and their effects on fluorescence anisotropy of cell membranes. Our results suggested that, in contrast to its antioxidant activity, vitamin E inhibits hemolysis induced by hemin as a membrane stabilizing agent.
Keywords: Erythrocyte; Hemin; Hemolysis; Vitamin E; Membrane stabilizer; Membrane fluidity
Berberine suppresses MEK/ERK-dependent Egr-1 signaling pathway and inhibits vascular smooth muscle cell regrowth after in vitro mechanical injury by Kae-Woei Liang; Chih-Tai Ting; Sui-Chu Yin; Ying-Tsung Chen; Shing-Jong Lin; James K. Liao; Shih-Lan Hsu (pp. 806-817).
Vascular smooth muscle cell (SMC) proliferation plays an important role in the pathogenesis of atherosclerosis and post-angioplasty restenosis. Berberine is a well-known component of the Chinese herb medicine Huanglian ( Coptis chinensis), and is capable of inhibiting SMC contraction and proliferation, yet the exact mechanism is unknown. We therefore investigated the effect of berberine on SMC growth after mechanic injury in vitro. DNA synthesis and cell proliferation assay were performed to show that berberine inhibited serum-stimulated rat aortic SMC growth in a concentration-dependent manner. Mechanical injury with sterile pipette tip stimulated the regrowth of SMCs. Treatment with berberine prevented the regrowth and migration of SMCs into the denuded trauma zone. Western blot analysis showed that activation of the MEK1/2 (mitogen-activated protein kinase kinase 1/2), extracellular signal-regulated kinase (ERK), and up-regulation of early growth response gene (Egr-1), c-Fos and Cyclin D1 were observed sequentially after mechanic injury in vitro. Semi-quantitative reverse-transcription PCR assay further confirmed the increase of Egr-1, c-Fos, platelet-derived growth factor (PDGF) and Cyclin D1 expression in a transcriptional level. However, berberine significantly attenuated MEK/ERK activation and downstream target (Egr-1, c-Fos, Cyclin D1 and PDGF-A) expression after mechanic injury in vitro. Our study showed that berberine blocked injury-induced SMC regrowth by inactivation of ERK/Egr-1 signaling pathway thereby preventing early signaling induced by injury in vitro. The anti-proliferative properties of berberine may be useful in treating disorders due to inappropriate SMC growth.
Keywords: Berberine; Early growth response gene (Egr-1); Extracellular signal-regulated kinase (ERK); MEK1/2 (mitogen-activated protein kinase kinase 1/2); Platelet-derived growth factor (PDGF); Vascular smooth muscle cell
Differential activity of kaempferol and quercetin in attenuating tumor necrosis factor receptor family signaling in bone cells by Jian L. Pang; Dennis A. Ricupero; Su Huang; Nigar Fatma; Dhirendra P. Singh; Jose R. Romero; Naibedya Chattopadhyay (pp. 818-826).
Increasing data from epidemiological and in vitro studies show that the isoflavonoids, genistein and daidzein, and the flavonols, quercetin and kaempferol, are protective against postmenopausal bone loss. However, the physiological mechanisms for these effects are not well understood. We now report that kaempferol exerts profound antiosteoclastogenic effects by acting on both osteoblasts and osteoclasts. Kaempferol but not quercetin dose-dependently inhibited tumor necrosis factor α (TNFα)-induced production of the osteoclastogenic cytokines interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1/CCL2) in osteoblasts. The effect on IL-6 was posttranscriptional, whereas kaempferol reduced MCP-1 mRNA levels. In addition, in mouse primary calvarial osteoblasts, kaempferol but not quercetin blocked TNFα-induced translocation of the nuclear factor κB (NF-κB) subunit p65 from the cytoplasm to the nucleus. However, TNFα-stimulated intracellular ROS production was unaltered by kaempferol. In RAW264.7 cells, a monocyte/macrophage precursor for osteoclasts, both kaempferol and quercetin dose-dependently inhibited the receptor activator of NF-κB ligand (RANKL)-induced immediate-early oncogene c-fos expression at 6h. After 3–5 days, both flavonols robustly inhibited RANKL-induced expression of the osteoclastic differentiation markers, RANK and calcitonin receptor. Consistent with down regulation of these osteoclastic differentiation markers, both flavonols strongly attenuated the RANKL-induced formation of multinucleated osteoclasts. However, kaempferol was more potent than quercetin in inhibiting RANKL-stimulated effects on RAW264.7 cells. Thus, our data indicate that kaempferol exerts profound antiosteoclastogenic effects by specifically antagonizing TNF receptor family action on bone cells at two distinct levels, by disrupting production of osteoclastogenic cytokines from osteoblasts and attenuating osteoclast precursor cell differentiation.
Keywords: Bone loss; c-Fos; Calcitonin receptor; RANK ligand; Monocyte; Osteoblast
Agonist potency differentiates G protein activation and Ca2+ signalling by the orexin receptor type 1 by Johanna Magga; Genevieve Bart; Christian Oker-Blom; Jyrki P. Kukkonen; Karl E.O. Åkerman; Johnny Näsman (pp. 827-836).
The G protein coupling characteristics of a flag epitope-tagged orexin receptor type 1 (OX1R) was investigated in HEK293 cells. Immunoprecipitation of the OX1R and immunoblotting revealed interactions with Gq/G11 proteins as well as with Gs and Gi proteins. Stimulation with orexin-A did not affect the ability of the OX1R to coprecipitate Gq/G11 proteins, but it robustly elevated the intracellular concentration of Ca2+, [Ca2+]i. No changes in cAMP levels could be detected upon receptor stimulation. To get further insight into the functional correlation of G protein activation and Ca2+ signalling, we used baculovirus transduction to express chimeric G proteins, containing the Gαs protein backbone with various Gα donor sequences (Gαs/x) at the N and C termini, and measured cAMP as functional output. The Gαs/x chimeric proteins with Gα11(Gαq) and Gα16 structure in the C terminus were stimulated by the OX1R. Concentration–response curves with Gαs/16 revealed an agonist potency correlation between G protein activation and the elevation of [Ca2+]i via discharge of intracellular Ca2+ stores, a feature also recognized for the muscarinic M3 receptor. However, in contrast to the M3 receptor, the OX1R elevated [Ca2+]i via influx from extracellular space at about 30-fold lower agonist concentration. The results suggest that the OX1R is linked to influx of Ca2+ through a signal pathway independent of Gq/G11 protein activation.
Keywords: Abbreviations; [Ca; 2+; ]; i; intracellular free Ca; 2+; concentration; CMV; cytomegalovirus promoter; EGFP; enhanced green fluorescent protein; OX; 1; R; orexin receptor type 1; MAPK; mitogen-activated protein kinaseOrexin receptor; Calcium; G protein; Baculovirus; Cyclic AMP; HEK293 cells
Characterization of the inhibitory activity of tenatoprazole on the gastric H+,K+-ATPase in vitro and in vivo by Jai Moo Shin; Michel Homerin; Florence Domagala; Hervé Ficheux; George Sachs (pp. 837-849).
Tenatoprazole is a prodrug of the proton pump inhibitor (PPI) class, which is converted to the active sulfenamide or sulfenic acid by acid in the secretory canaliculus of the stimulated parietal cell of the stomach. This active species binds to luminally accessible cysteines of the gastric H+,K+-ATPase resulting in disulfide formation and acid secretion inhibition. Tenatoprazole binds at the catalytic subunit of the gastric acid pump with a stoichiometry of 2.6nmolmg−1 of the enzyme in vitro. In vivo, maximum binding of tenatoprazole was 2.9nmolmg−1 of the enzyme at 2h after IV administration. The binding sites of tenatoprazole were in the TM5/6 region at Cys813 and Cys822 as shown by tryptic and thermolysin digestion of the ATPase labeled by tenatoprazole. Decay of tenatoprazole binding on the gastric H+,K+-ATPase consisted of two components. One was relatively fast, with a half-life 3.9h due to reversal of binding at cysteine 813, and the other was a plateau phase corresponding to ATPase turnover reflecting binding at cysteine 822 that also results in sustained inhibition in the presence of reducing agents in vitro. The stability of inhibition and the long plasma half-life of tenatoprazole should result in prolonged inhibition of acid secretion as compared to omeprazole. Further, the bioavailability of tenatoprazole was two-fold greater in the ( S)-tenatoprazole sodium salt hydrate form as compared to the free form in dogs which is due to differences in the crystal structure and hydrophobic nature of the two forms.
Keywords: Abbreviations; DTT; 1,4-dithio-; dl; -threitol; F-MI; fluorescein-5-maleimide; GERD; gastro-esophageal reflux disease; GSH; glutathione; H2RA; H; 2; -receptor antagonist; NEM; N; -ethyl maleimide; PPI; proton pump inhibitor; TM; transmembrane segment; TPZ; tenatoprazoleThe gastric H,K-ATPase; Tenatoprazole; Binding stoichiometry; Inhibition of acid secretion; Proton pump inhibitor; Omeprazole
Ceramide-1-phosphate activates cytosolic phospholipase A2α directly and by PKC pathway by Hiroyuki Nakamura; Tetsuya Hirabayashi; Masaya Shimizu; Toshihiko Murayama (pp. 850-857).
Ceramide-1-phosphate (C1P), a novel bioactive sphingolipid, is implicated in the vital cellular processes such as cell proliferation and inflammation. The role of C1P on activity of cytosolic phospholipase A2α (cPLA2α), a key enzyme for the release of arachidonic acid (AA) and prostanoids, has not been well elucidated. In this study, we investigated the effect of C1P on the release of AA from L929 cells and a variant, which lacks cPLA2α expression, C12 cells. C1P at 30μM alone induced AA release from L929 cells without an increase in intracellular Ca2+ concentration. C1P-induced AA release was marginal in C12 cells, and treatment with an intracellular Ca2+ chelator (BAPTA-AM) or an inhibitor of cPLA2α (2μM pyrrophenone) decreased C1P-induced AA release in L929 cells. C1P increased the enzymatic activity of cPLA2α over two-fold in the presence of Ca2+. C1P triggered the translocation of cPLA2α and its C2 domain from the cytosol to the perinuclear region in CHO-K1 cells. Interestingly, C1P at 10μM synergistically enhanced ionomycin-induced AA release from L929 cells. The AA release induced by C1P with and without ionomycin decreased by treatment with protein kinase C (PKC) inhibitor (10μM GF109203X) and in the PKC-depleted cells. C1P at 10μM stimulated the translocation of PKC (α and δ) from the soluble to the membrane fractions. We propose that C1P stimulates AA release via two mechanisms; direct activation of cPLA2α, and the PKC-dependent pathway.
Keywords: Ceramide-1-phosphate; Sphingolipids; Cytosolic phospholipase A2α; Arachidonic acid; Protein kinase C; L929 cells
Drug–protein recognition processes investigated by NMR relaxation data by Silvia Martini; Claudia Bonechi; Mario Casolaro; Gianfranco Corbini; Claudio Rossi (pp. 858-864).
In this paper we investigated the interaction processes occurring at the protein–solvent interface for prednisolone–albumin and prednisone–albumin systems, using an approach based on the analysis of proton selective relaxation rate enhancements of the ligand in the presence of the macromolecule. The contribution from the bound ligand fraction to the observed relaxation rate in relation to protein concentration allowed the calculation of the affinity index[A]LT and the normalized affinity index[AIN]LT which removes the effects of motional anisotropies and different proton densities, and isolates the contribution due to a decrease in the ligand dynamics caused by the binding with the protein. This approach allowed the comparison of the binding ability of prednisolone and prednisone towards albumin.
Keywords: 1; H NMR; Spin–lattice relaxation rate; Ligand–macromolecules interaction; Affinity index; Prednisolone; Prednisone
Role of the second transmembrane domain of rat adenosine A1 receptor in ligand–receptor interaction by Ke-Qiang Xie; Yan Cao; Xing-Zu Zhu (pp. 865-871).
Initial mutagenesis studies exploring the ligand recognition model of A1 adenosine receptor (A1R) mainly focused on the residues in the 5th–7th transmembrane domains (TMs5–7). Little is known about the role of residues in TM2. To explore the importance of reserved hydrophobic region in TM2 of A1R, we mutated the hydrophobic residues at positions 65 and 69 to hydrophilic residues (L65T, Leu-65 to Thr-65; I69T, Ile-69 to Thr-69; I69S, Ile-69 to Ser-69) to change the hydrophobicity at the outer end of TM2. Binding assays showed that the affinities of mutant receptors were significantly decreased for ribose group-containing agonists (2-chloro- N6-cyclopentyladenosine (CCPA) and 5′- N-ethyl-carboxamidoadenosine (NECA)) but not for antagonists, N6-cyclopentyl-9-methyladenine (N-0840), an adenine derivative lacking ribose group, and 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), a xanthine derivative. This observation suggests that the hydrophobic region at the outer end of TM2 may mediate the recognition of the ribose group of CCPA and NECA.
Keywords: Abbreviations; ADA; adenosine deaminase; A; 1; R; adenosine A; 1; receptor; CCPA; 2-chloro-; N; 6; -cyclopentyladenosine; CPA; N; 6; -cyclopentyladenosine; DMEM; Dulbecco's modified Eagle's medium; DPCPX; 8-cyclopentyl-1,3-dipropylxanthine; FCS; fetal calf serum; GPCR; G protein-coupled receptor; HA; hemagglutinin A; I; isoleucine; L; leucine; N-0840; N; 6; -cyclopentyl-9-methyladenine; NECA; 5′-; N; -ethyl-carboxamidoadenosine; PCR; polymerase chain reaction; S; serine; T; threonine; TM; transmembrane domain; V; valine; WT; wild typeAdenosine A; 1; receptor; Transmembrane domain; Hydrophobic; Hydrophilic; Mutagenesis; Ribose group
Antioxidative function and biodistribution of [Gd@C82(OH)22] n nanoparticles in tumor-bearing mice by Jiangxue Wang; Chunying Chen; Bai Li; Hongwei Yu; Yuliang Zhao; Jin Sun; Yufeng Li; Gengmei Xing; Hui Yuan; Jun Tang; Zhen Chen; Huan Meng; Yuxi Gao; Chang Ye; Zhifang Chai; Chuanfeng Zhu; Baocheng Ma; Xiaohong Fang; Lijun Wan (pp. 872-881).
Oxidative stress is considered to be one of the important mechanisms involved in carcinogenesis. In our previous study, gadolinium endohedral metallofullerenol ([Gd@C82(OH)22] n nanoparticles) have shown high inhibitory activity on hepatoma cell (H22) growth in mice. To explore the antioxidative functions of nanoparticles, we investigated the biodistribution of [Gd@C82(OH)22] n nanoparticles, the changes of blood coagulation profiles, the metabolism of reactive oxygen species (ROS) in the tumor-bearing mice as well as the possible relationships between nanoparticles treatment and ROS production in this paper. The activities of hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and catalase (CAT) as well as the levels of reduced glutathione (GSH), protein-bound thiols and malondialdehyde (MDA) were compared between the tumor-bearing mice and normal mice. Transplanted tumors were grown in mice by subcutaneous injection of murine hepatoma cells in the mice. The comparison of the above parameters between nanoparticles and cyclophosphamide (CTX) therapy were also investigated. [Gd@C82(OH)22] n administration can efficiently restore the damaged liver and kidney of the tumor-bearing mice. All the activities of enzymes and other parameters related to oxidative stress were reduced after [Gd@C82(OH)22] n treatment and tended closely to the normal levels. The results suggest that [Gd@C82(OH)22] n nanoparticle treatment could regulate ROS production in vivo.
Keywords: Abbreviations; CAT; catalase; CDNB; 1-chlorinechloro-2,4-binitrobenzenedinitrobenzene; CTX; cyclophosphamide; DTNB; 5,5′-dithiobis 2-nitrobenzoic acid; GSH; glutathione; GSH-px; glutathione peroxidase; GST; glutathione; S; -transferase; OH; hydroxyl radical; H; 2; O; 2; hydrogen peroxide; ICP-MS; inductively coupled plasma-mass spectrometry; MDA; malondialdehyde; NTP; 2-nitro-5-thiobenzoate anion; 1; O; 2; singlet oxygen; O; 2; −; superoxide anions; PBS; phosphate buffer solution; ROS; reactive oxygen species; –SH; protein thiols; SOD; superoxidase dismutase; TBARS; thiobarbituric acid-reactive substance[Gd@C; 82; (OH); 22; ]; n; nanoparticles; Oxidative stress; Antioxidant; Biodistribution; In vivo
The pattern of deoxycytidine- and deoxyguanosine kinase activity in relation to messenger RNA expression in blood cells from untreated patients with B-cell chronic lymphocytic leukemia by Kourosh Lotfi; Karin Karlsson; Anna Fyrberg; Gunnar Juliusson; Viggo Jonsson; Curt Peterson; Staffan Eriksson; Freidoun Albertioni (pp. 882-890).
Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) catalyze the first step in the intracellular cascade of fludarabine (2-fluoroadenine-β-d-arabinofuranoside) and cladribine (2-chlorodeoxyadenosine) phosphorylation, which leads to activation of these prodrugs, commonly used for treatment of chronic lymphocytic leukemia (CLL). Thus, resistance to nucleoside analogues may primarily be due to low levels of deoxynucleoside kinase activity.The purpose of this study was to investigate the activity profiles of dCK and dGK and characterize the possible relationship between the levels of dCK enzymatic activities and mRNA levels in B-CLL cells from untreated patient samples in an attempt to determine the best approach for predicting sensitivity to nucleoside analogues and thereby optimizing treatment of CLL. For this purpose, dCK and dGK analyses were done in blood cells from 59 untreated symptomatic patients with CLL. The dGK activity towards 2-chlorodeoxyadenosine was significantly lower than of dCK (median 73pmol/mgprotein/min (85–121, 95% CI) versus 353pmol/mgprotein/min (331–421)). The median dCK mRNA level was 0.107 (0.096–0.120, 95% CI). There was a lack of correlation between the activities of dCK and dGK, which indicates that these proteins are regulated independently. We also found that the dCK and dGK activity measurement towards their endogenous substrates were comparable to the nucleoside analogues tested. Such variations in enzyme activities and mRNA levels may well explain differences in clinical responses to treatment.There was no correlation between the levels of dCK mRNAs and enzymatic activities using a quantitative real-time PCR procedure. Sequencing of dCK mRNA did not reveal alternate splicing or mutations in the coding region. The relation between activity and mRNA levels was studied by short interfering RNA (siRNA) method, which showed that in the siRNA treated cells the down-regulation of dCK expression, and activity followed each other. However, in control cells the mRNA levels remained stable but the protein activity markedly decreased. These data demonstrate that the dCK activity is not reflected by dCK mRNA expression that indicates a post-translational mechanism(s).
Keywords: Purine analogues; Deoxycytidine kinase; Deoxyguanosine kinase; Chronic lymphocytic leukemia