Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes

Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.     

Synthesis and biological activity of 5'-capped derivatives of 5'-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine

The oligonucleotides A5'pp5'A2'p5'A2'p5'A and A5'ppp5'A2'p5'A2'p5'A were prepared by reaction of AMP or ADP, respectively, with the 5'-(phosphoimidazolidate) of A2'p5'A2'p5'A. A5'pppp5'A2'(p5'A)n (n = 1-3) were synthesized by reaction of p5'A2'(p5'A)n (n = 1-3) with adenosine 5'-trimetaphosphate. All structures were confirmed by enzyme digestion and 1H and 31P nuclear magnetic resonance (NMR). The products A5'pppp5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were found to be identical with two of the products of the 2-5A synthetase catalyzed reaction of Ap4A with ATP, thus confirming the structural assignments made by earlier investigators. In extracts of mouse L cells programmed with encephalomyocarditis virus RNA, A5'pppp5'A2'p5'A2'p5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were equipotent with 2-5A itself as inhibitors of translation. The oligomers A5'ppp5'A2'p5'A2'p5'A and A2'pppp5'A2'p5'A were about 100 times less active than 2-5A, and A5'pp5'A2'p5'A2'p5'A was without translational inhibitory activity. When affinity for the 2-5A-dependent endonuclease was determined (by displacement of 2-5A[32P]pCp from endonuclease), all of the analogues, as well as 2-5A itself, had similar affinities for the endonuclease except for A5'pppp5'A2'p5'A, which was bound approximately 100 times less effectively. Under conditions of the radiobinding assay, A5'pppp5'A2'p5'A2'p5'A was degraded (t1/2 = 2 h) to ATP, ADP, AMP, ppp5'A2'p5'A2'p5'A, and p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)     

2',5'-Phosphodiesterase activity depends upon the presence of a 3'-hydroxyl moiety in the penultimate position of the oligonucleotide substrate

3'-Deoxyadenosine (3'dA, cordycepin)-substituted analogs of 2-5A core 5'-monophosphate (p5'A2'p5'A2'p5'A) were examined for their sensitivity toward degradation by the 2'-phosphodiesterase activity in cytoplasmic extracts of mouse L cells. The analogs, p5'(3'dA)-2'p5'A2'p5'A, p5'(3'dA)2'p5'A2'p5'(3'dA) and p5'A2'p5'A2'p5'(3'dA) were degraded at a rate comparable to p5'A2'p5'A2'p5'A itself. On the other hand, under the assay conditions examined p5'A2'p5'(3'dA)2'p5'A, like p5'(3'dA)2'p5'(3'dA)2'p5'(3'dA), was completely resistant to degradation. The data imply that sensitivity to the 2',5'-phosphodiesterase activity of mouse L cells requires the presence of 3'-hydroxyl moiety in the penultimate nucleotide.     

The comparative effects of 5'-methylthioadenosine and some of its analogs on cells containing, and deficient in, 5'-methylthioadenosine phosphorylase

The antiproliferative effects of 5'-methylthioadenosine and the 5'-methylthioadenosine analogs, 5'-isobutylthioadenosine, 5'-deoxyadenosine and 5'-methylthiotubercidin were examined using two mouse cell lines, one 5'-methylthioadenosine phosphorylase-deficient the other containing 5'-methylthioadenosine phosphorylase. All of the compounds were found to be growth inhibitory to both cell lines, demonstrating that these compounds need not be degraded to exert their inhibitory effects. A correlation was observed between the potency of the growth inhibitory effect and the ability of the cells to degrade these compounds. 5'-Methylthioadenosine, 5'-deoxyadenosine and 5'-isobutylthioadenosine, all of which are substrates for the 5'-methylthioadenosine phosphorylase in vitro, were more growth inhibitory to the 5'-methylthioadenosine phosphorylase-deficient cells than to the 5'-methylthioadenosine phosphorylase-containing cells, whereas, the 7-deaza analog, 5'-methylthiotubercidin, a nondegradable inhibitor of the 5'-methylthioadenosine phosphorylase, was a more potent inhibitor of the 5'-methylthioadenosine phosphorylase-containing cell line. Due to the inhibition by 5'-methylthiotubercidin on 5'-methylthioadenosine phosphorylase in vitro the disposition of cellularly-synthesized 5'-methylthioadenosine was explored using both cell types. 5'-Methylthiotubercidin inhibited the accumulation of exogenous 5'-methylthioadenosine from 5'-methylthioadenosine phosphorylase-deficient cells with no effect on intracellular 5'-methylthioadenosine. In contrast, 5'-methylthiotubercidin caused a large accumulation of extracellular 5'-methylthioadenosine with a concomitant smaller increase intracellularly in 5'-methylthioadenosine phosphorylase-containing cells. That cellularly-synthesized 5'-methylthioadenosine as well as the cellular excretion of this nucleoside are altered in response to treatment with 5'-methylthiotubercidin suggests two possible sites at which 5'-methylthiotubercidin may exert its effect.     

The comparative effects of 5′-methylthioadenosine and some of its analogs on cells containing,and deficient in, 5′-methylthioadenosine phosphorylase

The antiproliferative effects of 5′-methylthioadenosine and the 5′-methylthioadenosine analogs, 5′-isobutylthioadenosine, 5′-deoxyadenosine and 5′-methylthiotubercidin were examined using two mouse cell lines, one 5′-methylthioadenosine phosphorylase-deficient the other containing 5′-methylthioadenosine phosphorylase. All of the compounds were found to be growth inhibitory to both cell lines, demonstrating that these compounds need not be degraded to exert their inhibitory effects. A correlation was observed between the potency of the growth inhibitory effect and the ability of the cells to degrade these compounds. 5′-Methylthioadenosine, 5′-deoxyadenosine and 5′-isobutylthioadenosine, all of which are substrates for the 5′-methylthioadenosine phosphorylase in vitro, were more growth inhibitory to the 5′-methylthioadenosine phosphorylase-deficient cells than to the 5′-methylthioadenosine phosphorylase-containing cells, whereas, the 7-deaza analog, 5′-methylthiotubercidin, a nondegradable inhibitor of the 5′-methylthioadenosine phosphorylase, was a more potent inhibitor of the 5′-methylthioadenosine phosphorylase-containing cell line. Due to the inhibition by 5′-methylthiotubercidin on 5′-methylthioadenosine phosphorylase in vitro the disposition of cellularly-synthesized 5′-methylthioadenosine was explored using both cell types. 5′-Methylthiotubercidin inhibited the accumulation of exogenous 5′-methylthioadenosine from 5′-methylthioadenosine phosphorylase-deficient cells with no effect on intracellular 5′-methylthioadenosine. In contrast, 5′-methylthiotubercidin caused a large accumulation of extracellular 5′-methylthioadenosine with a concomitant smaller increase intracellularly in 5′-methylthioadenosine phosphorylase-containing cells. That cellularly-synthesized 5′-methylthioadenosine as well as the cellular excretion of this nucleoside are altered in response to treatment with 5′-methylthiotubercidin suggests two possible sites at which 5′-methylthiotubercidin may exert its effect.     

The identification of (3R,4S)-5-fluoro-5-deoxy-D-ribulose-1-phosphate as an intermediate in fluorometabolite biosynthesis in Streptomyces cattleya

(3R,4S)-5-Fluoro-5-deoxy-D-ribulose-1-phosphate (5-FDRulP) has been identified as the third fluorinated intermediate on the biosynthetic pathway to fluoroacetate and 4-fluorothreonine in Streptomyces cattleya. 5-FDRulP is generated after formation of 5'-fluoro-5'-deoxyadenosine (5'-FDA) and then phosphorolysis of 5'-FDA to 5-fluoro-5-deoxy-D-ribose-1-phosphate (5-FDRP) by the action of a purine nucleoside phosphorylase. An isomerase mediates the conversion of 5-FDRP to 5-FDRulP. The identity of the (3R,4S) diastereoisomer of 5-FDRulP was established by comparative (19)F{(1)H} NMR studies whereby 5-FDRulP that accumulated in a cell free extract of S. cattleya, was treated with a phytase to generate the non-phosphorylated sugar, 5-fluoro-5-deoxy-D-ribulose (5-FDRul). This S. cattleya product was compared to the product of an in-vitro biotransformation where separately 5-fluoro-5-deoxy-D-ribose and 5-fluoro-5-deoxy-D-xylose were converted to 5-fluoro-5-deoxy-D-ribulose and 5-fluoro-5-deoxy-D-xylulose respectively by the action of glucose isomerase. It was demonstrated that 5-fluoro-5-deoxy-D-ribose gave the identical diastereoisomer to that observed from 5-FDRulP.     

2-Methyladenosine-Substituted 2',5'-oligoadenylates: conformations, 2-5A binding and catalytic activities with human ribonuclease L

2-Methyladenosine-substituted analogues of 2-5A, p5'A2'p5'A2'p5'(me2A), p5'(me2A)2'p5'A2'p5'A, and p5'(me2A) 2'p5'(me2A)2'pS'(me2A), were prepared via a modification of a lead ion-catalyzed ligation reaction. These 5'-monophosphates were subsequently converted into the corresponding 5'-triphosphates. Both binding and activation of human recombinant RNase L by various 2-methyladenosine-substituted 2-5A analogues were examined. Among the 2-5A analogues, p5'A2'p5'A2'p5'(me2A) showed the strongest binding affinity and was as effective as 2-5A itself as an activator of RNase L. The CD spectra of both p5'(me2A)2'p5'A2'p5'A and p5'A2'p5'A2'p5'(me2A) were superimposable on that of p5'A2'p5'A2'p5'A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2-5A.     

Effect of analogues of 5'-methylthioadenosine on cellular metabolism. Inactivation of S-adenosylhomocysteine hydrolase by 5'-isobutylthioadenosine.

The effects of a number of nucleosides related to 5'-methylthioadenosine on the activities of S-adenosylhomocysteine hydrolase, 5'-methylthioadenosine phosphorylase, spermidine synthase and spermine synthase were investigated. Both 5'-methylthioadenosine and 5'-isobutylthioadenosine gave rise to an enzyme-activated irreversible inhibition of S-adenosylhomocysteine hydrolase, but 5'-methylthiotubercidin (5'-methylthio-7-deaza-adenosine), 5'-deoxy-5'-chloroformycin, 5'-ethylthio-2-fluoro-adenosine and 1,N6-etheno-5'-methylthioadenosine were totally ineffective in producing this inactivation. Of the nucleosides tested, only 5'-methylthioadenosine, 5'-methylthiotubercidin and 5'-isobutylthioadenosine were inhibitory towards the aminopropyltransferases responsible for the synthesis of spermine and spermidine. 5'-Methylthiotubercidin, 5'-deoxy-5'-chloroformycin and 5'-isobutylthioadenosine were inhibitors of the degradation of 5'-methylthioadenosine by 5'-methylthioadenosine phosphorylase, but only 5'-isobutylthioadenosine was also a substrate for this enzyme. These results suggest that the effects of 5'-isobutylthioadenosine of the cell may result from the combination of inhibitory actions on polyamine synthesis, 5'-methylthioadenosine degradation and S-adenosylhomocysteine degradation. The resulting increased concentrations of S-adenosylhomocysteine could bring about inhibition of methyltransferase reactions. A new convenient method for the assay of S-adenosylhomocysteine hydrolase in the direction of synthesis is described.     

Expression and in vitro properties of guinea pig IL-5: comparison to human and murine orthologs

Interleukin-5 (IL-5) is a key mediator of eosinophilic inflammation. The biological role of this cytokine in an allergic airway inflammatory response has been widely demonstrated in guinea pigs, yet the interaction of guinea pig IL-5 (gpIL-5) with its receptor has not been studied. Experiments were performed to quantitate the interaction of gpIL-5 with gpIL-5r and to compare this affinity with that of hIL-5 and mIL-5 and their cognate receptors. The cross-species affinity and agonist efficacy were evaluated to see if gpIL-5r had a restricted species reactivity (as is the case with mIL-5r) or did not distinguish between IL-5 orthologs (similar to hIL-5r). gpIL-5 was cloned using mRNA isolated from cells obtained by bronchoalveolar lavage. Recombinant gpIL-5 was expressed in T. ni insect cells and purified from spent media. Binding assays were performed using insect cells expressing hIL-5ralphabeta or gpIL-5ralphabeta1 as previously described (Cytokine, 12:858-866, 2000) or using B13 cells which express mIL-5r. The agonist potency and efficacy properties of each IL-5 ortholog were evaluated by quantitating the proliferative response of human TF-1 cells and murine B13 cells. gpIL-5 bound with high affinity to recombinant gpIL-5r as demonstrated by displacing [125I]hIL-5 (Ki = 160 pM). gpIL-5 also bound to hIL-5r with high affinity (Ki = 750 pM). hIL-5 and mIL-5 showed similar, high-affinity binding profiles to both gpIL-5r and hIL-5r. In contrast, gpIL-5 and hIL-5 did not bind to the mIL-5r as demonstrated by an inability to displace [125I]mIL-5, even at 1000-fold molar excess. These differences in affinity for IL-5r orthologs correlated with bioassay results: human TF-1 cells showed roughly comparable proliferative responses to guinea pig, human and murine IL-5 whereas murine B13 cells showed a strong preference for murine over guinea pig and human IL-5 (EC50 = 1.9, 2200 and 720 pM, respectively). Recombinant gpIL-5 binds to the gpIL-5r with high affinity, similar to that seen with the human ligand-receptor pair. gpIL-5r and hIL-5r do not distinguish between the three IL-5 orthologs whereas mIL-5r has restricted specificity for its cognate ligand.     

Functional characterization of alternatively spliced 5-HT2 receptor isoforms from the pharynx and muscle of the parasitic nematode,Ascaris suum

Serotonin (5-HT) receptors play key regulatory roles in nematodes and alternatively spliced 5-HT2 receptor isoforms have been identified in the parasitic nematode, Ascaris suum. 5-HT2As1 and 5-HT2As2 contain different C-termini, and 5-HT2As1Delta4 lacks 42 amino acids at the C-terminus of the third intracellular loop. 5-HT2As1 and 5-HT2As2 exhibited identical pharmacological profiles when stably expressed in human embryonic kidney (HEK) 293 cells. Both 5-HT2As isoforms had higher affinity for 5-HT than their closely related Caenorhabditis elegans homolog (5-HT2Ce). This increased 5-HT affinity was not related to the substitution in 5-HT2As1 of F120 for Y in the highly conserved DRY motif found in the second intracellular loop of other 5-HT receptors, since a 5-HT2As1F120Y mutant actually exhibited increased 5-HT affinity compared with that of 5-HT2As1. As predicted, cells expressing either 5-HT2As1 or 5-HT2As2 exhibited a 5-HT-dependent increase in phosphatidylinositol (PI) turnover. In contrast, although 5-HT2As1Delta4 displayed a 10-fold higher affinity for 5-HT and 5-HT agonists than either 5-HT2As1 or 5-HT2As2, 5-HT2As1Delta4 did not couple to either PI turnover or adenyl cyclase activity. Based on RT-PCR, 5-HT2As1 and 5-HT2As2 were more highly expressed in pharynx and body wall muscle and 5-HT2As1Delta4 in nerve cord/hypodermis. This is the first report of different alternatively spliced 5-HT2 receptor isoforms from any system.     

Biological activities of phosphodiester linkage isomers of 2-5A

To determine the relative importance of the 2',5'-phosphodiester bond of 2-5A in its binding to and activation of the 2-5A-dependent ribonuclease (RNase L, RNase F), a number of phosphodiester linkage isomers of 2-5A were prepared. These isomers were obtained either by lead ion-catalyzed polymerization of adenosine 5'-phosphorimidazolidate or by T4 polynucleotide kinase-catalyzed 5'-phosphorylation of adenylyl(3' leads to 5')adenylyl(3' leads to 5')adenosine followed by reaction of the corresponding phosphorimidazolidates with tri(n-butylammonium)pyrophosphate. The following 2-5A isomers thus were prepared: ppp5'A2'p5'A3'p5'A, ppp5'A3'p5'A2'p5'A, ppp5'A3'p5'A3'p5'A("3-5A"), ppp5'A2'p5'A3'p5'A2'p5'A,and ppp5'A3'p5'A2'p5'-A2'p5'A. The ability of these isomeric 2-5As to interact with the 2-5A-dependent endonuclease was ascertained by three different criteria: (i) ability to prevent the protein synthesis inhibitory effects of 2-5A, (ii) activity as an inhibitor of translation in encephalomyocarditis RNA-programmed L cell extracts, and (iii) ability to prevent binding of the radiolabeled probe, ppp5'A2'p5'A2'p5'A2'p5'A3'[32P]p5'Cp, to the endonuclease of L cell extracts. In certain experiments, degradation of oligonucleotide was minimized or eliminated by altering assay conditions, providing alternate phosphodiesterase substrates, or by using purified endoribonuclease of Ehrlich ascites cells. By all criteria, replacement of 2',5'-bond by a 3',5'-bond led to a substantial decrease in biological activity. Generally, replacement of just one 2',5'-phosphodiester bond with a 3',5'-linkage led to at least a one order of magnitude loss of activity. In accord with this trend, ppp5'A3'p5'A3'p5'A(3-5A) was greater than 10,000 less active than 2-5A in binding to the endonuclease or as an inhibitor of protein synthesis.     

Generation and replication-dependent dilution of 5fC and 5caC during mouse preimplantation development

One of the recent advances in the epigenetic field is the demonstration that the Tet family of proteins are capable of catalyzing conversion of 5-methylcytosine (5mC) of DNA to 5-hydroxymethylcytosine (5hmC). Interestingly, recent studies have shown that 5hmC can be further oxidized by Tet proteins to generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), which can be removed by thymine DNA glycosylase (TDG). To determine whether Tet-catalyzed conversion of 5mC to 5fC and 5caC occurs in vivo in zygotes, we generated antibodies specific for 5fC and 5caC. By immunostaining, we demonstrate that loss of 5mC in the paternal pronucleus is concurrent with the appearance of 5fC and 5caC, similar to that of 5hmC. Importantly, instead of being quickly removed through an enzyme-catalyzed process, both 5fC and 5caC exhibit replication-dependent dilution during mouse preimplantation development. These results not only demonstrate the conversion of 5mC to 5fC and 5caC in zygotes, but also indicate that both 5fC and 5caC are relatively stable and may be functional during preimplantation development. Together with previous studies, our study suggests that Tet-catalyzed conversion of 5mC to 5hmC/5fC/5caC followed by replication-dependent dilution accounts for paternal DNA demethylation during preimplantation development.     

Peripheral 5-carboxamidotryptamine induces hindlimb scratching by stimulating 5-HT1A receptors in rats.

Treatment of rats with 5-carboxamidotryptamine (5-CT) or 5-methoxy-tryptamine (5-MeOT) induces a hindlimb scratch response. These compounds have high affinity for 5-HT1A and 5-HT1D receptors. The selective 5-HT1A receptor agonist N,N-dipropyl-5-CT (DP-5-CT) also induced hindlimb scratching while the selective 5-HT1D receptor agonist, sumatriptan, did not. 5-CT-induced hindlimb scratching was inhibited dose-dependently by several 5-HT1A antagonists (BMY 7378, NAN-190, MDL 73005EF and pindobind-5-HT1A) as well as the non-selective 5-HT antagonist, methiothepin. Pretreatment of rats with the serotonin (5-HT) synthesis inhibitor, p-chlorophenylalanine (PCPA) or the 5-HT depleting agent, reserpine, markedly attenuated 5-CT-induced hindlimb scratching. These data suggest that hindlimb scratching induced by 5-HT agonists may not be centrally mediated but rather may be mediated by a neuronal 5-HT1A receptor localized outside the blood-brain barrier.     

Circular dichroism spectra of DNA quadruplexes [d(G(5)T(5))](4) as formed with G(4) and T(4) tetrads and [d(G(5)T(5)). d(A(5)C(5))]2 as formed with Watson-Crick-like (G-C)(2) and (T-A)(2) tetrads

We utilize electrophoresis and find that a thermally treated equimolar mixture of the oligonucleotide d(G(5)T(5)) and its complementary oligonucleotide d(A(5)C(5)) exhibits either two bands or a single band in one lane, depending on the conditions of the incubation solutions. The thermally treated d(G(5)T(5)) solution loaded in a different lane exhibits a single band of the parallel quadruplex [d(G(5)T(5))](4), which is composed of homocyclic hydrogen-bonded G(4) and T(4) tetrads previously proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(A(5)C(5)), the fast band is assigned to a Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex, so that the slow band with the same low mobility as that of [d(G(5)T(5))](4) may be assigned to either [d(G(5)T(5))](4) itself or a [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex. If the latter compound is true, this may be the antiparallel quadruplex composed of the heterocyclic hydrogen-bonded G-C-G-C and T-A-T-A tetrads proposed previously. After removing these three bands for the duplex and two kinds of hypothetical quadruplexes, we electrophoretically elute the corresponding compounds in the same electrophoresis buffer using an electroeluter. The eluted compounds are ascertained to be stable by electrophoresis. The circular dichroism (CD) and UV absorption spectra measured for the three isolated compounds are found to be clearly different. For the electrophoretic elution of the hypothetical [d(G(5)T(5))](4) quadruplex, the result of the molecularity of n = 4 obtained from the CD melting curve analysis provides further support for the formation of the parallel [d(G(5)T(5))](4) quadruplex already proposed. For the thermally treated equimolar mixture of d(G(5)T(5)) and d(C(5)A(5)), the fast band with a molecularity of n = 2 corresponds to the Watson-Crick duplex, d(G(5)T(5)). d(A(5)C(5)). The slow band with a molecularity of n = 4 indicates the antiparallel quadruplex [d(G(5)T(5)). d(A(5)C(5))](2), whose observed CD and UV spectra are different from those of [d(G(5)T(5))](4). By electrophoresis, after reannealing the eluted compound [d(G(5)T(5)). d(A(5)C(5))](2), a distinct photograph showing the band splitting of this quadruplex band into the lower duplex and upper quadruplex bands is not possible; but by a transilluminator, we occasionally observe this band splitting with the naked eye. The linear response polarizability tensor calculations for the thus determined structures of the [d(G(5)T(5))](4) quadruplex, the McGavin-like [d(G(5)T(5)). d(A(5)C(5))](2) quadruplex, and the Watson-Crick d(G(5)T(5)). d(A(5)C(5)) duplex are found to qualitatively predict the observed CD and UV spectra.     

Contrasting use of CCR5 structural determinants by R5 and R5X4 variants within a human immunodeficiency virus type 1 primary isolate quasispecies

Macrophagetropic R5 human immunodeficiency virus type 1 (HIV-1) isolates often evolve into dualtropic R5X4 variants during disease progression. The structural basis for CCR5 coreceptor function has been studied in a limited number of prototype strains and suggests that R5 and R5X4 Envs interact differently with CCR5. However, differences between unrelated viruses may reflect strain-specific factors and do not necessarily represent changes resulting from R5 to R5X4 evolution of a virus in vivo. Here we addressed CCR5 domains involved in fusion for a large set of closely related yet functionally distinct variants within a primary isolate swarm, employing R5 and R5X4 Envs derived from the HIV-1 89.6(PI) quasispecies. R5 variants of 89.6(PI) could fuse using either N-terminal or extracellular loop CCR5 sequences in the context of CCR5/CXCR2 chimeras, similar to the unrelated R5 strain JRFL, but R5X4 variants of 89.6(PI) were highly dependent on the CCR5 N terminus. Similarly, R5 89.6(PI) variants and isolate JRFL tolerated N-terminal CCR5 deletions, but fusion by most R5X4 variants was markedly impaired. R5 89.6(PI) Envs also tolerated multiple extracellular domain substitutions, while R5X4 variants did not. In contrast to CCR5 use, fusion by R5X4 variants of 89.6(PI) was largely independent of the CXCR4 N-terminal region. Thus, R5 and R5X4 species from a single swarm differ in how they interact with CCR5. These results suggest that R5 Envs possess a highly plastic capacity to interact with multiple CCR5 regions and support the concept that viral evolution in vivo results from the emergence of R5X4 variants with the capacity to use the CXCR4 extracellular loops but demonstrate less-flexible interactions with CCR5 that are strongly dependent on the N-terminal region.     

Identification of regions within the third FnIII-like domain of the IL-5Ralpha involved in IL-5 interaction

Previously, two binding sites for interleukin 5 (IL-5) were identified on the IL-5 receptor alpha chain (IL-5Ralpha). They are located within the CD loop of the first fibronectin type III (FnIII)-like domain and the EF loop of the second FnIII-like domain. The first binding site was identified by exploiting the different abilities of human IL-5Ralpha (hIL-5Ralpha) and mouse IL-5Ralpha (mIL-5Ralpha) to bind hIL-5. Here we show that ovine IL-5 (oIL-5) has the ability to activate the hIL-5Ralpha but not the mIL-5Ralpha. By using chimeras of the mIL-5Ralpha and hIL-5Ralpha we demonstrate that residues within the first and third FnIII-like domains of mIL-5Ralpha are responsible for this lack of activity. Furthermore, mutation of residues on hIL-5Ralpha to mIL-5Ralpha within the predicted DE and FG loop regions of the third FnIII domain reduces oIL-5 activity. These results show that regions of the third FnIII domain of IL-5Ralpha are involved in binding, in addition to the regions in domains one and two of the IL-5Ralpha that were identified in an earlier study.     

Synthesis and biological activities of beta-alanyltyrosine derivative of 2',5'-oligoadenylate, and its use in radiobinding assay for 2',5-oligoadenylate

beta-Alanyltyrosine derivative of 2',5'-tetraadenylate 5'-triphosphate, pppA2'p5'A2'-p5'A2'p5'A-beta-Ala-Tyr was prepared by coupling of periodate-oxidized pppA2'p5'-A2'p5'A2'p5'A with beta-alanyltyrosine methyl ester, followed by reduction with sodium cyanoborohydride. Its stability to 2',5'-phosphodiesterase and phosphatase was investigated in mouse L cell extract. The 5'-triphosphate of the compound was cleaved gradually to form the 5'-dephosphorylated derivative, A2'p5'A2'p5'A2'p5'A-beta-Ala-Tyr, followed by slow degradation of the 2',5'-phosphodiester bond. On the other hand, pppA2'p5'A2'p5'A2'p5'A was hydrolyzed very quickly under the same conditions. The tetramer derivative bound tightly to the 2',5'-oligoadenylate-dependent endoribonuclease in rabbit reticulocyte lysate or mouse L cell extract and inhibited protein synthesis of mouse L cells more effectively than the unmodified 2',5'-tetraadenylate 5'-triphosphate. The corresponding trimer derivative had slightly weaker activities than the unmodified trimer for binding to the endoribonuclease and for inhibition of protein synthesis. The compound, pppA2'p5'A2'p5'-A2'p5'A-beta-Ala-Tyr, was iodinated easily at the tyrosine residue with 125I, giving a high-specific-radioactivity derivative which was used as a radio-labeled probe in a radiobinding assay for 2',5'-oligoadenylate.     

Synthesis and antioxidant activities of novel 4-Schiff base-7-benzyloxy-coumarin derivatives

4-Schiff base-7-benzyloxy-coumarins 5a(1)-5h(2) and its derivative 6 were designed and synthesized based on the 7-benzyloxy-coumarin structure as novel antioxidants. The in vitro antioxidant activities screening revealed that 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of compounds 5b(1), 5d(1), 5f(1), 5f(2), 5g(1) and 5g(2), and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) cation (ABTS(+)) radical scavenging activities of compounds 5a(1), 5b(1), 5c(1), 5c(2), 5d(1), 5e(1), 5e(2), 5f(2), 5g(1), 5g(2) and 5h(1) were better than that of the commercial antioxidant butylated hydroxytoluene (BHT), while the superoxide anion radical scavenging activities of 5a(2) and 5g(2) were stronger than that of the commercial antioxidant butylated hydroxyanisole (BHA), and the hydroxyl radical scavenging activity of 5e(1) was much better than that of the common antioxidant ascorbic acid.     

Synthesis and structure determination of some sugar amino acids related to alanine and 6-deoxymannojirimycin.

(5'R)-5'-Methyl-5'-[methyl (4S)-2,3-O-isopropylidene-beta-L-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione was synthesised starting from methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosid-5-ulose applying the Bucherer-Bergs reaction. Its 5'-R configuration was confirmed by X-ray crystallography. Corresponding alpha-amino acid-methyl (5R)-5-amino-5-C-carboxy-5,6-dideoxy-alpha-D-lyxo-hexofuranoside (alternative name: 2-[methyl (4S)-2,3-O-isopropylidene-beta-L-erythrofuranosid-4-C-yl]-D-alanine) was obtained from the above hydantoin by acid hydrolysis of the isopropylidene group followed by basic hydrolysis of the hydantoin ring. Total deprotection afforded 5-C-carboxy-6-deoxymannojirimycin. Analogously, methyl (5S)-5-amino-5-C-carboxy-5,6-dideoxy-alpha-L-lyxo-hexofuranoside and 5-C-carboxy-6-deoxy-L-mannojirimycin were prepared from the corresponding (5'S)-5'-methyl-5'-[methyl (4R)-2,3-O-isopropylidene-beta-D-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione starting from methyl 6-deoxy-2,3-O-isopropylidene-alpha-L-lyxo-hexofuranosid-5-ulose.