Putative role of the adenosine A3 receptor in the antiproliferative action of N 6-(2-isopentenyl)adenosine

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N ⁶-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A₃ receptor (hA₃R) ligands with affinities in the high nanomolar range (K _i values of 159 and 649 nM, respectively). These values were comparable to the observed K _i value of adenosine on hA₃R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A₃R. In a functional assay in Chinese hamster ovary cells transfected with hA₃R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A₃R antagonist VUF5574. Both IPA and reference A₃R agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A₃R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A₃R-independent mechanism, as was previously reported for other A₃R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A₃R.     

Amperometric immunosensor based on polypyrrole/poly(m-pheylenediamine) multilayer on glassy carbon electrode for cytokinin N6-(Delta2-isopentenyl) adenosine assay

A novel immunosensor based on a multilayer-coated glassy carbon electrode was designed to determine isopentenyl adenosine (iPA) in plants. The multilayer consists of polypyrrole and poly(m-phenylenediamine) with K4Fe(CN)6 and horseradish peroxidase (HRP) entrapped during electropolymerization. The ferrocyanide doped in polypyrrole functions as the mediator. The glucose oxidase bound on the immunosensor by the competitive immunoreaction involving iPA catalyzed the oxidation of the added glucose with the formation of H2O2, which is in turn reduced in the presence of HRP entrapped in poly(m-phenylenediamine). The current of the oxidized production of ferrocyanide reduced at -50 mV is inversely proportional to the concentration of iPA in the competitive immunoreaction. This immunosensor is able to be used about 40 times; after that its surface can be regenerated for a new immunosensor assembly by washing with 0.1M citrate-phosphate buffer (pH 4.6). The percentage of current response reduction (CR%) (y) is linearly related to the logarithm of the concentration of iPA (x) in the 5-300 microg/ml range, with a regression equation of the form y = 42.13x - 27.79 and a correlation coefficient of 0.9861. Five hybrid rice grain samples were analyzed with results in satisfactory agreement to those obtained by high-performance liquid chromatography.     

Covalent flavinylation of L-aspartate oxidase from Escherichia coli using N6-(6-carboxyhexyl)-FAD succinimidoester

L-Aspartate oxidase is a flavoprotein catalyzing the first step in the de novo biosynthesis of pyridine nucleotides in E. coli. Binding of FAD to L-aspartate oxidase is relatively weak (K _d 6.7 × 10^–7 M), resulting in partial loss of the coenzyme under many experimental conditions. Only the three-dimensional structure of the apo-enzyme has been obtained so far. In order to probe the flavinbinding site of the enzyme, apo-L-aspartate oxidase has been reacted with N⁶-(6-carboxyhexyl)-FAD Succinimidoester. The structural characterization of the resulting N⁶-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows the covalent incorporation of 1 FAD-analog/ monomer. Residue Lys38 was identified as the target of the covalent modification. N⁶-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows only 2% catalytic activity as compared to the native enzyme. Comparison of some properties of the flavinylated and native enzymes suggests that, although the flavin is covalently bound to the former in the region predicted from molecular modeling studies, the microenvironment around the isoallossazine is different in the two forms.     

Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme

Each of the twelve enzymes for glycolytic fermentation, eleven from Escherichia coli and one from Saccharomyces cerevisiae, have been over-expressed in E. coli and purified with His-tags. Simple assays have been developed for each enzyme and they have been assembled for fermentation of glucose to ethanol. Phosphorus-31 NMR revealed that this in vitro reaction accumulates fructose 1,6-bisphosphate while recycling the cofactors NAD⁺ and ATP. This reaction represents a defined ATP-regeneration system that can be tailored to suit in vitro biochemical reactions such as cell-free protein synthesis. The enzyme from S. cerevisiae, pyruvate decarboxylase 1 (Pdc1; EC 4.1.1.1), was identified as one of the major ‘flux controlling’ enzymes for the reaction and was replaced with an evolved version of Pdc1 that has over 20-fold greater activity under glycolysis reaction conditions. This substitution was only beneficial when the ratio of glycolytic enzymes was adjusted to suit greater Pdc1 activity.     

Mono(ADP-ribosyl)ation of the N2 amino groups of guanine residues in DNA by pierisin-2, from the cabbage butterfly, Pieris brassicae

Pierisin-2 is a cytotoxic and apoptosis-inducing protein present in Pieris brassicae with a 91% homology in the deduced amino acid sequences to pierisin-1 from Pieris rapae. We earlier showed pierisin-1 to catalyze mono(ADP-ribosyl)ation of 2'-deoxyguanosine (dG) in DNA to form N2-(ADP-ribos-1-yl)-2'-deoxyguanosine, this DNA modification appearing linked to its cytotoxicity and ability to induce apoptosis in mammalian cell lines. In this paper, we documented evidence that pierisin-2 also catalyzed ADP-ribosylation of dG in DNA to give the same reaction product as demonstrated for pierisin-1, with similar efficiency. With oligonucleotides as substrates, ADP-ribosylation by pierisin-2 was suggested to occur by one-side attack of the carbon atom at 1 position of the ribose moiety in NAD toward N2 of dG. The presence of a unique ADP-ribosylation toxin targeting dG in DNA in two distinct species in a Pieris genus could be a quite important finding to better understand biological functions of pierisin-1 and -2 in Pieris butterflies and the generic evolution of these cabbage butterflies.     

The reactivation of DnaA(L366K) requires less acidic phospholipids supporting their role in the initiation of chromosome replication in Escherichia coli

DnaA(L366K), in concert with a wild-type DnaA (wtDnaA) protein, restores the growth of Escherichia coli cells arrested in the absence of adequate levels of cellular acidic phospholipids. In vitro and in vivo studies showed that DnaA(L366K) alone does not induce the initiation of replication, and wtDnaA must also be present. Hitherto the different behavior of wt and mutant DnaA were not understood. We now demonstrate that this mutant may be activated at significantly lower concentrations of acidic phospholipids than the wild-type protein, and this may explain the observed growth restoration in vivo.     

N(6)-Methyldeoxyadenosine, a nucleoside commonly found in prokaryotes, induces C2C12 myogenic differentiation

N(6)-methyl-2(')-deoxyadenosine (MedAdo) is a nucleoside naturally found in prokaryotic DNA. Interestingly, the N(6)-methylation of adenine in DNA seems to have been counter-selected during the course of evolution since MedAdo has not been detected in mammalian DNA until now. We show here that treatment with MedAdo induces myogenesis in C2C12 myoblasts. The presence of MedAdo in C2C12 DNA was investigated using a method based on HPLC coupled to electrospray ionization tandem mass spectrometry which is several thousand fold more sensitive than assays used previously. By this procedure, MedAdo is detected in the DNA from MedAdo-treated cells but remains undetectable in the DNA from control cells. Furthermore, MedAdo regulates the expression of p21, myogenin, mTOR, and MHC. Interestingly, in the pluripotent C2C12 cell line, MedAdo drives the differentiation towards myogenesis only. Thus, the biological effect of MedAdo is suppressed in the presence of BMP-2 which transdifferentiates C2C12 from myogenic into osteogenic lineage cells. Taken together these results point to MedAdo as a novel inducer of myogenesis and further extends the differentiation potentialities of this methylated nucleoside. Furthermore, these data raise the intriguing possibility that the biological effects of MedAdo on cell differentiation may have led to its counter-selection in eukaryotes.     

Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis

The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.     

Molecular docking of glucosamine-6-phosphate synthase in Rhizopus oryzae

Recent expansion of immunocompromised population has led to significant rise in zygomycosis caused by filamentous fungus Rhizopus oryzae. Due to emergence of fungal resistance and side-effects of antifungal drugs, there is increased demand for novel drug targets. The current study elucidates molecular interactions of peptide drugs with G-6-P synthase (catalyzing the rate-limiting step of fungal cell wall biosynthetic pathway) of R.oryzae by molecular docking studies. The PDB structures of enzyme in R.oryzae are not known which were predicted using I-TASSER server and validated with PROCHECK. Peptide inhibitors, FMDP and ADGP previously used against enzyme of E.coli (PDBid: 1XFF), were used for docking studies of enzyme in R.oryzae by SchrödingerMaestro v9.1. To investigate binding between enzyme and inhibitors, Glide and Induced Fit docking were performed. IFD results of 1XFF with FMDP yielded C1, R73, W74, T76, G99 and D123 as the binding sites. C379 and Q427 appear to be vital for binding of R.oryzae enzymes to inhibitors. The comparison results of IFD scores of enzyme in R.oryzae and E.coli (PDBid: 2BPL) yield appreciable score, hinting at the probable effectiveness of inhibitors FMDP and ADGP against R.oryzae, with ADGP showing an improved enzyme affinity. Moreover, the two copies of gene G-6-P synthase due to extensive fungal gene duplication, in R. oryzae eliminating the problem of drug ineffectiveness could act as a potential antifungal drug target in R. oryzae with the application of peptide ligands.     

基因组信息指导的星海链霉菌中2-甲硫基-N6-(4-羟基异戊烯基)-腺苷的分离鉴定

【目的】研究海洋链霉菌新种星海链霉菌Streptomyces xinghaiensis NRRL B24674~T次级代谢物中是否存在2-甲硫基-N~6-异戊烯基修饰的腺苷。【方法】通过生物信息学分析星海链霉菌S.xinghaiensis NRRL B24674~T基因组序列,寻找这类化合物的生物合成相关基因;采用正相硅胶柱色谱、反相硅胶柱色谱、凝胶柱色谱和高效液相色谱等分离技术对该菌株的发酵粗提物进行分离纯化;利用质谱与核磁共振等波谱技术鉴定化合物的结构。【结果】在星海链霉菌基因组中找到含有2-甲硫基-N~6-异戊烯基修饰的化合物生物合成途径中的2个同源蛋白;从该菌的发酵液中分离鉴定了2-甲硫基-N~6-(4-羟基异戊烯基)-腺苷(ms2io6A)。【结论】星海链霉菌S.xinghaiensis NRRL B24674~T存在此类腺苷修饰反应,并且是首次在链霉菌中发现此类腺苷修饰。生物信息学分析预示着链霉菌中可能普遍存在此类核酸或者核苷修饰。     

Toxoplasma gondii: in vitro and in vivo activities of the hydroxynaphthoquinone 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone alone or combined with sulfadiazine

The compound 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone (PHNQ6) was evaluated for activity against Toxoplasma gondii, alone or combined with sulfadiazine. Treatment with PHNQ6 combined with sulfadiazine protected at least 70 and 90% of mice infected with RH and EGS strains, respectively. Mice were treated with PHNQ6 (50 mg/kg/day) alone or combined with sulfadiazine (40 mg/L) 30 days after infection with P strain. The number of brain cysts was lower in mice treated with PHNQ6 alone or combined with sulfadiazine compared to that in control mice. Degenerated bradyzoites were observed in animals treated with PHNQ6. Infectivity of bradyzoites treated with PHNQ6 alone or combined with sulfadiazine was inhibited after in vitro incubation.     

Potato tuber isoapyrases: substrate specificity, affinity labeling, and proteolytic susceptibility

Apyrase/ATP-diphosphohydrolase hydrolyzes di- and triphosphorylated nucleosides in the presence of a bivalent ion with sequential release of orthophosphate. We performed studies of substrate specificity on homogeneous isoapyrases from two potato tuber clonal varieties: Desiree (low ATPase/ADPase ratio) and Pimpernel (high ATPase/ADPase ratio) by measuring the kinetic parameters K(m) and k(cat) on deoxyribonucleotides and fluorescent analogues of ATP and ADP. Both isoapyrases showed a broad specificity towards dATP, dGTP, dTTP, dCTP, thio-dATP, fluorescent nucleotides (MANT-; TNP-; ethene-derivatives of ATP and ADP). The hydrolytic activity on the triphosphorylated compounds was always higher for the Pimpernel apyrase. Modifications either on the base or the ribose moieties did not increase K(m) values, suggesting that the introduction of large groups (MANT- and TNP-) in the ribose does not produce steric hindrance on substrate binding. However, the presence of these bulky groups caused, in general, a reduction in k(cat), indicating an important effect on the catalytic step. Substantial differences were observed between potato apyrases and enzymes from various animal tissues, concerning affinity labeling with azido-nucleotides and FSBA (5'-p-fluorosulfonylbenzoyl adenosine). PLP-nucleotide derivatives were unable to produce inactivation of potato apyrase. The lack of sensitivity of both potato enzymes towards these nucleotide analogues rules out the proximity or adequate orientation of sulfhydryl, hydroxyl or amino-groups to the modifying groups. Both apyrases were different in the proteolytic susceptibility towards trypsin, chymotrypsin and Glu-C.     

Induction of gene mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.     

Induction of A:T to G:C transition mutations by 5-(2-chloroethyl)-2'-deoxyuridine (CEDU), an antiviral pyrimidine nucleoside analogue, in the bone marrow of Muta Mouse

5-(2-chloroethyl)-2'-deoxyuridine (CEDU) is a pyrimidine nucleoside analogue formerly in development for the treatment of herpes simplex virus infections. The compound proved clearly mutagenic in the mouse spot test and exhibited weak activity in the Salmonella reverse mutation test, which led to the termination of the compound's development. In another study, CEDU, administered orally to beta-galactosidase (lacZ) transgenic mice (Muta Mouse) for five days, induced a clear increase in lacZ mutant frequencies in spleen, lung, and bone marrow. In the present follow-up study, we analyzed 32 of those lacZ mutants isolated from the bone marrow of the Muta Mouse animals of the experiments mentioned above, in order to obtain further information on the type of mutations induced by CEDU. CEDU induced a pronounced increase in A:T to G:C transitions. The distribution of A:T to G:C transitions was clearly non-random, showing a bias towards T to C substitutions in the coding DNA strand and a preference to occur in the sequence motif 5'-(G or C)-T-G-3'. Our data support the hypothesis that CEDU, after being phosphorylated, is incorporated into cellular DNA in place of thymidine, which leads to mispairing with guanosine during subsequent DNA replication. As a result, the compound is thought to exert its mutagenicity by inducing mismatches leading to T to C transitions. Our findings point towards a mode of mutagenic action of CEDU that differs fundamentally from that of other antiviral antinucleosides whose clastogenic and recombinogenic activities prevail.     

The 2'-O- and 3'-O-Cy3-EDA-ATP(ADP) complexes with myosin subfragment-1 are spectroscopically distinct

Ribose-modified highly-fluorescent sulfoindocyanine ATP and ADP analogs, 2'(3')-O-Cy3-EDA-AT(D)P, with kinetics similar to AT(D)P, enable myosin and actomyosin ATPase enzymology with single substrate molecules. Stopped-flow studies recording both fluorescence and anisotropy during binding to skeletal muscle myosin subfragment-1 (S1) and subsequent single-turnover decay of steady-state intermediates showed that on complex formation, 2'-O- isomer fluorescence quenched by 5%, anisotropy increased from 0.208 to 0.357, and then decayed with turnover rate k(cat) 0.07 s(-1); however, 3'-O- isomer fluorescence increased 77%, and anisotropy from 0.202 to 0.389, but k(cat) was 0.03 s(-1). Cy3-EDA-ADP.S1 complexes with vanadate (V(i)) were studied kinetically and by time-resolved fluorometry as stable analogs of the steady-state intermediates. Upon formation of the 3'-O-Cy3-EDA-ADP.S1.V(i) complex fluorescence doubled and anisotropy increased to 0.372; for the 2'-O- isomer, anisotropy increased to 0.343 but fluorescence only 6%. Average fluorescent lifetimes of 2'-O- and 3'-O-Cy3-EDA-ADP.S1.V(i) complexes, 0.9 and 1.85 ns, compare with approximately 0.7 ns for free analogs. Dynamic polarization shows rotational correlation times higher than 100 ns for both Cy3-EDA-ADP.S1.V(i) complexes, but the 2'-O-isomer only has also a 0.2-ns component. Thus, when bound, 3'-O-Cy3-EDA-ADP's fluorescence is twofold brighter with motion more restricted and turnover slower than the 2'-O-isomer; these data are relevant for applications of these analogs in single molecule studies.     

Programmed cell death 6 (PDCD6) inhibits angiogenesis through PI3K/mTOR/p70S6K pathway by interacting of VEGFR-2

Programmed cell death 6 (PDCD6) was originally found as a pro-apoptotic protein, but its molecular mechanism is not well understood. In this study, we have attempted to investigate the effects of PDCD6 on the inhibition of angiogenesis-mediated cell growth as a novel anti-angiogenic protein. Purified recombinant human PDCD6 inhibited cell migration in a concentration-time-dependent manner. We also found that overexpressed PDCD6 suppressed vascular endothelial growth factor (VEGF)-induced proliferation, invasion, and capillary-like structure tube formation in vitro. PDCD6 suppressed phosphorylation of signaling regulators downstream from PI3K, including Akt, mammalian target of rapamycin (mTOR), glycogen synthase kinase-3β(GSK-3β), ribosomal protein S6 kinase (p70S6K), and also decreased cyclin D1 expression. We found binding PDCD6 to VEGFR-2, a key player in the PI3K/mTOR/P70S6K signaling pathway. Taken together, these data suggest that PDCD6 plays a significant role in modulating cellular angiogenesis.