Role of C5a-C5aR axis in the development of atherosclerosis

Complement component 5a (C5a) is a 74 amino acid glycoprotein and an important proinflammatory mediator that is cleaved enzymatically from its precursor, C5, on activation of the complement cascade. C5a is quickly metabolised by carboxypeptidases, forming the less-potent C5a desArg. C5a and C5a desArg interact with their receptors (C5aR and C5L2), which results in a number of effects which are essential to the immune response. C5a has a broad range of biological effects throughout the human body because the widespread expression of C5a receptors throughout the human organs enables C5a and C5a desArg to elicit a broad range of biological effects. Recently, accumulating evidence in humans and experimental animal models shows that the C5a-C5aR axis is involved in the development of atherosclerosis lesions. The absence or blockade of C5aRs greatly reduces the formation of atherosclerotic lesions or wire-injury-induced neointima formation in atherosclerosis-prone mice. Serum C5a level was related to the major adverse cardiovascular events in patients with advanced atherosclerosis and those with drug-eluting stent implantation. Thus, the C5a-C5aR axis may be a significant pathogenic driver of arteriosclerotic vascular disease, making C5a-C5aR inhibition an attractive therapeutic strategy.     

The role of the N-terminal domain of the complement fragment receptor C5L2 in ligand binding

C5L2 is a new cellular receptor found to interact with the human anaphylatoxins complement factor C5a and its C-terminal cleavage product C5a des Arg. The classical human C5a receptor (C5aR) preferentially binds C5a, with a 10-100-fold lower affinity for C5a des Arg. In contrast, C5L2 binds both ligands with nearly equal affinity. C5aR presents acidic and tyrosine residues in its N terminus that interact with the core of C5a while a hydrophobic pocket formed by the transmembrane helices interacts with residues in the C terminus of C5a. Here, we have investigated the molecular basis for the increased affinity of C5L2 for C5a des Arg. Rat and mouse C5L2 preferentially bound C5a des Arg, whereas rodent C5aR showed much higher affinity for intact C5a. Effective peptidic and non-peptidic ligands for the transmembrane hydrophobic pocket of C5aR were poor inhibitors of ligand binding to C5L2. An antibody raised against the N terminus of human C5L2 did not affect the binding of C5a to C5L2 but did inhibit C5a des Arg binding. A chimeric C5L2, containing the N terminus of C5aR, had little effect on the affinity for C5a des Arg. Mutation of acidic and tyrosine residues in the N terminus of human C5L2 revealed that 3 residues were critical for C5a des Arg binding but had little involvement in C5a binding. C5L2 thus appears to bind C5a and C5a des Arg by different mechanisms, and, unlike C5aR, C5L2 uses critical residues in its N-terminal domain for binding only to C5a des Arg.     

Function of small hydrophobic proteins of paramyxovirus

Mumps virus (MuV), a rubulavirus of the paramyxovirus family, causes acute infections in humans. MuV has seven genes including a small hydrophobic (SH) gene, which encodes a type I membrane protein of 57 amino acid residues. The function of the SH protein is not clear, although its expression is not necessary for growth of MuV in tissue culture cells. It is speculated that MuV SH plays a role in viral pathogenesis. Simian virus 5 (SV5), a closely related rubulavirus, encodes a 44-amino-acid-residue SH protein. Recombinant SV5 lacking the SH gene (rSV5DeltaSH) is viable and has no growth defect in tissue culture cells. However, rSV5DeltaSH induces apoptosis in tissue culture cells and is attenuated in vivo. Neutralizing antibodies against tumor necrosis factor alpha (TNF-alpha) and TNF-alpha receptor 1 block rSV5DeltaSH-induced apoptosis, suggesting that SV5 SH plays an essential role in blocking the TNF-alpha-mediated apoptosis pathway. Because MuV is closely related to SV5, we hypothesize that the SH protein of MuV has a function similar to that of SV5, even though there is no sequence homology between them. To test this hypothesis and to study the function of MuV SH, we have replaced the open reading frame (ORF) of SV5 SH with the ORF of MuV SH in a SV5 genome background. The recombinant SV5 (rSV5DeltaSH+MuV-SH) was analyzed in comparison with SV5. It was found that rSV5DeltaSH+MuV-SH was viable and behaved like wild-type SV5, suggesting that MuV SH has a function similar to that of SV5 SH. Furthermore, both ectopically expressed SV5 SH and MuV SH blocked activation of NF-kappaB by TNF-alpha in a reporter gene assay, suggesting that both SH proteins can inhibit TNF-alpha signaling.     

Analgesic properties of a systemically-administered synthetic dipeptide of 5-hydroxytryptophan

Synthetic peptides of 5-hydroxytryptophan (5-HTP), including N-acetyl-5-HTP-5-HTP amide (5-HTP-ACETYL-DP), specifically inhibit the binding of serotonin to serotonin binding protein. 5-HTP-ACETYL-DP also produces a long-lasting, opiate-sensitive analgesia following central, but not systemic administration. The present study evaluated an apolar derivative of 5-HTP dipeptide, N-hexanoyl-5-HTP-5-HTP amide (5-HTP-HEX-DP), for its analgesic properties in rats following systemic administration. 5-HTP-HEX-DP (5–50 mg/kg) significantly increased jump thresholds in a dose-dependent manner with peak analgesia occurring at 2.5 hr after injection, and lasting up to 5 hr. In the tail-flick assay, 5-HTP-HEX-DP (20 mg/kg) produced a significant antinociceptive effect at 1 hr post-injection using both high and low intensity levels of radiant heat. While 5-HTP-HEX-DP and morphine each elicited analgesia following acute administration, chronic (14 days) incremental dosing with 5-HTP-HEX-DP or morphine resulted in persistent analgesia in 5-HTP-HEX-DP-treated animals, and a loss of analgesia in morphine-treated rats. Thus, significant tolerance to morphine, but not 5-HTP-HEX-DP analgesia developed using this protocol. Hence, 5-HTP-HEX-DP is a systemically-active analgesic which fails to develop tolerance when administered daily over 14 days.     

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.     

Serotonergic regulation of blood glucose levels in the crayfish, Procambarus clarkii: site of action and receptor characterization

The present study investigated the site of action of 5-hydroxytryptamine (5-HT) and pharmacologically characterized the receptors involved in regulating blood glucose levels in the crayfish, Procambarus clarkii. Injection of 5-HT into intact animals increased glucose levels in a dose-dependent manner. In contrast, 5-HT failed to elicit a hyperglycemic response in eyestalk-ablated animals. Effects of several 5-HT receptor agonists and antagonists were examined. 5-CT, oxymetazoline (both 5-HT(1) receptor agonists) and alpha-methyl-5-HT (a 5-HT(2) receptor agonist), but not 1-phenylbiguanide, m-CPBG (both 5-HT(3) receptor agonists), or RS 67333 (a 5-HT(4) receptor agonist), induced hyperglycemic responses in a dose-dependent manner. In addition, 8-OH-DPAT (a 5-HT(1A) receptor agonist), L-694,247 (a 5-HT(1B/1D) receptor agonist), and DOI (a 5-HT(2A) receptor agonist) were effective in significantly increasing the glucose levels, whereas both BW 723C86 (a 5-HT(2B) receptor agonist) and m-CPP (a 5-HT(2C) receptor agonist) were ineffective. Finally, ketanserin (a 5-HT(2A) receptor antagonist), but not p-MPPF (a 5-HT(1A) receptor antagonist), GR 55562 (a 5-HT(1B/1D) receptor antagonist), SB 206553 (a 5-HT(2B/2C) receptor antagonist), or tropisetron (a 5-HT(3) receptor antagonist), was able to block 5-HT-induced hyperglycemia. The combined results support the hypothesis that 5-HT exerts its hyperglycemic effect by enhancing the release of hyperglycemic factor(s) from the eyestalks, and suggest that 5 HT-induced hyperglycemia is mediated by 5-HT(1)- and 5-HT(2)-like receptors.     

The metabolism of the epoxide of testosterone by human placental microsomes.

Although 19-hydroxy-4beta,5-oxido-5beta-androstane-3,17 dione (2a) is converted to estradiol-17beta by human placental microsomes, the incubation of 17beta-hydroxy-4beta,5-oxido-5beta-androstan-3-one (2b) under the same conditions produces only metabolites which are more polar than 17beta-estradiol. The metabolites have been isolated and identified as 3alpha-hydroxy-4beta,5-oxido-5beta-androstan-17-one (4a), 4beta,5-oxido-5beta-androstane-3beta, 17beta-diol (5a) and 4beta,5-oxido-5beta-androstane-3alpha,17beta-diol (6a). These results indicate that functionalization at C-19 is a prerequisite for the biological aromatization of such androgen epoxides.     

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.     

Preferential elimination of chromosome 5R of rye in the progeny of 5R5D dimonosomics

Transmission of chromosome 5R of rye (Secale cereale L.) and chromosome 5D of common wheat (Triticum aestivum L.) through gametes of 5R5D dimonosomics (2n = 42, 20W″ + 5R′ + 5D′) was studied. Chromosome 5R was found to have lower competitiveness as compared to 5D. Gametes with the rye chromosome were two times less often involved in the formation of a progeny. The combined frequency of the karyotypes of wheat (5D5D) and wheat monosomics (5D) was 11.6-fold higher than the frequency of the karyotypes of substitution lines (5R5R) and monosomics for the rye chromosome (5R). The karyotypes of 10.38% of hybrid plants had aberrant 5R chromosomes with different translocations formed as a result of breakages in the centromere and in the proximal region of the long arm. Telocentrics for the short arm t5RS, i5RS isochromosomes, and chromosomes with a terminal deletion T5RS.5RL-del were identified. The absence of amplification of SSR markers mapped on 5RS and the detection of PCR products for a number of 5RL markers (including the genome-specific rye marker Xrms115) permitted nine plants carrying only the long arm of chromosome 5R to be revealed. Since t5RL telocentrics were not detected by the cytological analysis, the results obtained allow us to suggest the presence of small intercalary translocations of the long arm of chromosome 5R in chromosome 5D or in other wheat chromosomes.     

Functional analyses of two alternative isoforms of the transcription factor Pax-5

The Pax-5 gene plays a central role in B cell development, activation, and differentiation. At least four different isoforms have been identified, of which isoform Pax-5a has been extensively studied, while functions for alternative isoforms were previously unknown. Here, using a transient transfection system, we provide evidence that alternative isoform Pax-5d acts as a dominant-negative regulator by suppressing activity of Pax-5a in a dose-dependent manner. In contrast, co-expression in the presence of alternative isoform Pax-5e causes an increase in Pax-5a activity. Protein studies on Pax-5e using Western blot analysis revealed that this 19-kDa isoform migrates as a 27-kDa species on SDS-polyacrylamide electrophoresis gels, while a mutant Pax-5e form in which a C-terminal cysteine residue has been mutated, runs at the expected 19 kDa. Using both Western blot and immunoprecipitation assays, we further provide evidence that this size discrepancy may be caused by a tight association between Pax-5e and a thioredoxin-like factor. Comparison of various B cell lines as well as resting and lipopolysaccharide-activated mature B lymphocytes shows that increased B cell proliferation correlates with increased levels of Pax-5e/thioredoxin, whereas increased Pax-5d amounts correlate with inhibition of cell growth. Together, our results suggest that during activation and differentiation of B lymphocytes, Pax-5a function is modulated by two alternative spliced isoforms: the dominant negative Pax-5d isoform may mediate inhibition of Pax-5a activity in resting B cells, while alternative isoform Pax-5e associated with thioredoxin may increase Pax-5a activity through an unknown (redox) mechanism.     

An analysis of the 5-hydroxytryptamine (serotonin) receptor subtypes of central neurones of Helix aspersa.

1. Intracellular recordings were made from identified neurones in the central nervous system of Helix aspersa. Two types of cell were used, those excited by 5-hydroxytryptamine (5-HT) and acetylcholine and those inhibited by 5-HT and dopamine. The actions of a range of 5-HT agonists and antagonists were tested for their ability to interact with 5-HT receptors. 2. 5-Carboxyamidotryptamine, alpha-methyl-5-HT and N-methyl-5-HT were active on cells excited by 5-HT, with similar potencies to 5-HT. Only 5-carboxyamidotryptamine and 5-methoxytryptamine were equiactive with 5-HT on cells inhibited by 5-HT. Most of the non-indole analogues were inactive or very weak agonists on both receptors. 3. MDL 72222 was the most active antagonist tested against 5-HT excitation, showing some selectivity for 5-HT over acetylcholine. Cinanserin and ketanserin also showed selectivity for 5-HT over acetylcholine. 4. Tryptamine was inhibitory on both cell types and was a potent antagonist of 5-HT excitation, showing selectivity for 5-HT over acetylcholine. 5. It is concluded that the 5-HT excitatory receptor recognizes the indole nucleus with substitution on position 5, save for 5-fluorotryptamine which was inhibitory. It does not appear that these 5-HT receptors can be classified in terms of the vertebrate subtypes of 5-HT receptor. However, it should be noted that only two receptor subtypes located on a small number of neurones were studied in these experiments and other 5-HT receptor suptypes may be located on other groups of neurones and peripheral tissues. These receptors may recognize other 5-HT receptor ligands including non-indoles.     

Identification of the Isoform-specific Interactions between the Tail and the Head of Class V Myosin

Vertebrates have three isoforms of class V myosin (Myo5), Myo5a, Myo5b, and Myo5c, which are involved in transport of multiple cargoes. It is well established that the motor functions of Myo5a and Myo5b are regulated by a tail inhibition mechanism. Here we found that the motor function of Myo5c was also inhibited by its globular tail domain (GTD), and this inhibition was abolished by high Ca²⁺, indicating that the tail inhibition mechanism is conserved in vertebrate Myo5. Interestingly, we found that Myo5a-GTD and Myo5c-GTD were not interchangeable in terms of inhibition of motor function, indicating isoform-specific interactions between the GTD and the head of Myo5. To identify the isoform-specific interactions, we produced a number of Myo5 chimeras by swapping the corresponding regions of Myo5a and Myo5c. We found that Myo5a-GTD, with its H11-H12 loop being substituted with that of Myo5c, was able to inhibit the ATPase activity of Myo5c and that Myo5a-GTD was able to inhibit the ATPase activity of Myo5c-S1 and Myo5c-HMM only when their IQ1 motif was substituted with that of Myo5a. Those results indicate that the H11-H12 loop in the GTD and the IQ1 motif in the head dictate the isoform-specific interactions between the GTD and head of Myo5. Because the IQ1 motif is wrapped by calmodulin, whose conformation is influenced by the sequence of the IQ1 motif, we proposed that the calmodulin bound to the IQ1 motif interacts with the H11-H12 loop of the GTD in the inhibited state of Myo5.     

An analysis of the 5-hydroxytryptamine (serotonin) receptor subtypes of central neurones of Helix aspersa

1. Intracellular recordings were made from identified neurones in the central nervous system of Helix aspersa. Two types of cell were used, those excited by 5-hydroxytryptamine (5-HT) and acetylcholine and those inhibited by 5-HT and dopamine. The actions of a range of 5-HT agonists and antagonists were tested for their ability to interact with 5-HT receptors.2. 5-Carboxyamidotryptamine, α-methyl-5-HT and N-methyl-5-HT were active on cells excited by 5-HT, with similar potencies to 5-HT. Only 5-carboxyamidotryptamine and 5-methoxytryptamine were equiactive with 5-HT on cells inhibited by 5-HT. Most of the non-indole analogues were inactive or very weak agonists on both receptors.3. MDL 72222 was the most active antagonist tested against 5-HT excitation, showing some selectivity for 5-HT over acetylcholine. Cinanserin and ketanserin also showed selectivity for 5-HT over acetylcholine.4. Tryptamine was inhibitory on both cell types and was a potent antagonist of 5-HT excitation, showing selectivity for 5-HT over acetylcholine.5. It is concluded that the 5-HT excitatory receptor recognizes the indole nucleus with substitution on position 5, save for 5-fluorotryptamine which was inhibitory. It does not appear that these 5-HT receptors can be classified in terms of the vertebrate subtypes of 5-HT receptor. However, it should be noted that only two receptor subtypes located on a small number of neurones were studied in these experiments and other 5-HT receptor subtypes may be located on other groups of neurones and peripheral tissues. These receptors may recognize other 5-HT receptor ligands including non-indoles.     

Novel functional dissection of the localization-specific roles of budding yeast polo kinase Cdc5p

Budding yeast polo kinase Cdc5p localizes to the spindle pole body (SPB) and to the bud-neck and plays multiple roles during M-phase progression. To dissect localization-specific mitotic functions of Cdc5p, we tethered a localization-defective N-terminal kinase domain of Cdc5p (Cdc5pDeltaC) to the SPB or to the bud-neck with components specifically localizing to one of these sites and characterized these mutants in a cdc5Delta background. Characterization of a viable, SPB-localizing, CDC5DeltaC-CNM67 mutant revealed that it is defective in timely degradation of Swe1p, a negative regulator of Cdc28p. Loss of BFA1, a negative regulator of mitotic exit, rescued the lethality of a neck-localizing CDC5DeltaC-CDC12 or CDC5DeltaC-CDC3 mutant but yielded severe defects in cytokinesis. These data suggest that the SPB-associated Cdc5p activity is critical for both mitotic exit and cytokinesis, whereas the bud neck-localized Cdc5p is required for proper Swe1p regulation. Interestingly, a cdc5Delta bfa1Delta swe1Delta triple mutant is viable but grows slowly, whereas cdc5Delta cells bearing both CDC5DeltaC-CNM67 and CDC5DeltaC-CDC12 grow well with only a mild cell cycle delay. Thus, SPB- and the bud-neck-localized Cdc5p control most of the critical Cdc5p functions and downregulation of Bfa1p and Swe1p at the respective locations are two critical factors that require Cdc5p.     

The release of C5a in complement-activated serum does not require C6

The influence of terminal complement components on the generation and release of the complement C5a fragment was investigated by comparing the levels of C5a in complement-activated serum with the levels of C5a produced in serum depleted of complement C6. In order to investigate the release of C5a, a modified C5a assay was developed that utilizes an anti-C5b monoclonal antibody to remove C5, C5b, and C5b-C5a complexes from samples prior to C5a assay. The modified assay was developed because the standard methodology, which includes an acid-precipitation step designed to dissociate C5a and C5b, cannot distinguish free C5a from the C5a that is bound to C5b. Therefore, the standard methodology is not capable of monitoring the influence of terminal components on C5a/C5b dissociation. Levels of C5a were measured in complement-activated whole human serum, in serum depleted of C6, and in serum containing inhibitory levels of anti-C6 Fab using both the modified C5a assay and the standard methodology. Sera were complement-activated with either zymosan to activate the alternative complement pathway or with antibody-coated sheep erythrocytes to activate the classical pathway. The levels of free C5a in C6-depleted sera after activation were equivalent to the C5a levels in activated whole serum, indicating that C6 is not required for the release of C5a from C5b. In addition, the quantity of C5a detected in zymosan-activated sera using the standard acid-precipitation methodology was greater than C5a levels when assayed using the modified immunoadsorption technique, confirming that acid-treatment enhances the C5a dissociation and promotes C5a recovery. Since the other terminal components, C7, C8, and C9, bind to C5b only after C5b only after C6 is bound, these results indicate that none of the terminal components are required for the release of C5a. Although the terminal components could influence the rate of C5a release, the quantity of C5a released in serum was entirely independent of terminal components.     

Enhanced anti-angiogenic effect of a deletion mutant of plasminogen kringle 5 on neovascularization

Kringle 5 (K5), a proteolytic fragment of plasminogen, has been proved to be an angiogenic inhibitor. Previously, we have evaluated the effect of K5 on the vascular leakage and neovascularization in a rat model of oxygen-induced retinopathy. In this study, we expressed K5 and a deletion mutant of K5 (K5 mutant) in a prokaryocyte expression system and purified them by affinity chromatography. K5 mutant was generated by deleting 11 amino acids from K5 while retaining the three disulfide bonds. The anti-angiogenic activity of intact K5 and K5 mutant were compared in endothelial cells and retinal neovascularization rat model. K5 mutant inhibited the proliferation of primary human retinal capillary endothelial cells (HRCEC) in a concentration-dependent manner, with an apparent EC50 of approximate 35 nmol/L, which is twofold more potent than intact K5. In the even higher concentration range, K5 mutant did not inhibit pericytes from the same origin of HRCEC, which suggested an endothelial cell-specific inhibition. K5 mutant had no effect on normal liver cells and Bel7402 hepatoma cells even at high concentration range either. Intravitreal injection of the K5 and mutant in the oxygen-induced retinopathy rat model both resulted in significantly fewer neovascular tufts and nonperfusion area than controls with PBS injection, as shown by fluorescein angiography. Furthermore, K5 mutant exhibited more strong inhibition effect on neovascularization than intact K5 by quantification of vascular cells. These results suggest that this K5 deletion mutant is a more potent angiogenic inhibitor than intact K5 and may have therapeutic potential in the treatment of those disorders with neovascularization, such as solid tumor, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and hyperplasia of prostate.     

Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA

Recent studies showed that Ten-eleven translocation (Tet) family dioxygenases can oxidize 5-methyl-2’-deoxycytidine (5-mdC) in DNA to yield the 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2’-deoxycytidine (5-HmdC, 5-FodC and 5-CadC). 5-HmdC in DNA may be enzymatically deaminated to yield 5-hydroxymethyl-2’-deoxyuridine (5-HmdU). After their formation at CpG dinucleotide sites, these oxidized pyrimidine nucleosides, particularly 5-FodC, 5-CadC, and 5-HmdU, may be cleaved from DNA by thymine DNA glycosylase, and subsequent action of base-excision repair machinery restores unmethylated cytosine. These processes are proposed to be important in active DNA cytosine demethylation in mammals. Here we used a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) method, along with the use of stable isotope-labeled standards, for accurate measurements of 5-HmdC, 5-FodC, 5-CadC and 5-HmdU in genomic DNA of cultured human cells and multiple mammalian tissues. We found that overexpression of the catalytic domain of human Tet1 led to marked increases in the levels of 5-HmdC, 5-FodC and 5-CadC, but only a modest increase in 5-HmdU, in genomic DNA of HEK293T cells. Moreover, 5-HmdC is present at a level that is approximately 2–3 and 3–4 orders of magnitude greater than 5-FodC and 5-CadC, respectively, and 35–400 times greater than 5-HmdU in the mouse brain and skin, and human brain. The robust analytical method built a solid foundation for dissecting the molecular mechanisms of active cytosine demethylation, for measuring these 5-mdC derivatives and assessing their involvement in epigenetic regulation in other organisms and for examining whether these 5-mdC derivatives can be used as biomarkers for human diseases.     

Identification of a new class of small molecule C5a receptor antagonists

C5a is a terminal product of the complement cascade that activates and attracts inflammatory cells including granulocytes, mast cells and macrophages via a specific GPCR, the C5a receptor (C5aR). Inhibition of C5a/C5aR interaction has been shown to be efficacious in several animal models of autoimmune diseases, including RA, SLE and asthma. This account reports the discovery of a new class of C5aR antagonists through high-throughput screening. The lead compounds in this series are selective and block C5a binding, C5a-promoted calcium flux in human neutrophils with nanomolar potency.     

Respective role of each of the purine N7 nitrogens of 5'-O-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine in binding to and activation of the RNase L of mouse cells

Through a combination of chemical and enzymatic approaches a series of sequence-specific tubercidin-substituted ppp5'A2'p(5'A2'p)n5'A (n = 1 to about 10; 2-5A) analogues were generated. In addition to the previously developed methodology of Imai and Torrence [Imai, J., & Torrence, P.F. (1985) J. Org. Chem. 50, 1418-1420], a new approach to synthesis of 2',5'-linked oligonucleotides utilized adenosine in 3',5' linkage as a precursor to the targeted 5'-terminus of the desired product. For instance, A3'p5'A could be condensed under conditions of lead ion catalysis with tubercidin 5'-phosphate to give A3'p5'A2'p5'(c7A). Treatment with the 3',5'-specific nuclease P1 led to p5'A2'p5'(c7A). The combined use of the above procedures led to the synthesis of p5'(c7A)2'p5'A2'p5'A, p5'A2'p5'(c7A)2'p5'A, p5'A2'p5'A2'p5'(c7A), and p5'A2p5'(c7A)2'p5'(c7A), which were converted to their corresponding 5'-triphosphates by the usual methods. Evaluation of these analogues for their ability to bind to and activate the 2-5A-dependent endonuclease (RNase L) of mouse L cells showed that there were small changes (less than or equal to 10-fold) in the ability of the four tubercidin analogues to bind to RNase L. However, whenever the first and/or third adenosine nucleotide units were replaced by tubercidin, a dramatic decrease in ability to activate RNase L occurred. Only the second (from the 5'-terminus) adenosine residue could be replaced by tubercidin without any effect on RNase L activation ability.