Synthesis and Antiviral Activities of 5-Substituted 1-(2-Deoxy-2-C-methylene-4-thio-β-D-erythro-pentofuranosyl)uracilst†

Abstract

Various 5-substituted 1-(2-deoxy-2-C-methylene-4-thio-β-D-erythropentofuranosyl)uracils (4′-thioDMDUs) were synthesized from D-glucose via sila-Pummerer-type glycosylation. All of the β-anomers of 5-substituted 4′-thioDMDU, except the 5-hydroxyethyl derivative, showed potent anti-HSV-1 activity (ED₅₀ = 0.016–0.096 μg/mL). 5-Ethyl- and 5-iodo-4′-thioDMDUs were also active against HSV-2 (ED₅₀ = 0.17 and 0.86 μg/mL, respectively). 5-Bromovinyl-4′-thioDMDU was particularly active against VZV (ED₅₀ = 0.013 μg/mL).

     

Synthesis of (2′S)-1-(2-C-Azidomethyl-2-deoxy and 2-C-Cyanomethyl-2-deoxy-β-D-arabinofuranosyl)cytosines

Abstract

2′-C-Cyanomethyl-2′-deoxy-arabinosylcytosine 3 and 2′-C-azidomethyl-2′-deoxy-arabinosylcytosine 4 were synthesized from uridine. The antineoplastic activities of these compounds were evaluated.     

Lipid peroxidation modification of protein generates Nepsilon-(4-oxononanoyl)lysine as a pro-inflammatory ligand

4-Oxo-2(E)-nonenal (ONE), a peroxidation product of ω-6 polyunsaturated fatty acids, covalently reacts with lysine residues to generate a 4-ketoamide-type ONE-lysine adduct, N(ε)-(4-oxononanoyl)lysine (ONL). Using an ONL-coupled protein as the immunogen, we raised the monoclonal antibody (mAb) 9K3 directed to the ONL and conclusively demonstrated that the ONL was produced during the oxidative modification of a low density lipoprotein (LDL) in vitro. In addition, we observed that the ONL was present in atherosclerotic lesions, in which an intense immunoreactivity was mainly localized in the vascular endothelial cells and macrophage- and vascular smooth muscle cell-derived foam cells. Using liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for quantification of the ONL and confirmed that the ONL was indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. To evaluate the biological implications for ONL formation, we examined the recognition of ONL by the scavenger receptor lectin-like oxidized LDL receptor-1 (LOX-1). Using CHO cells stably expressing LOX-1, we evaluated the ability of ONL to compete with the acetylated LDL and found that both the ONE-modified and ONL-coupled proteins inhibited the binding and uptake of the modified LDL. In addition, we demonstrated that the ONL-coupled protein was incorporated into differentiated THP-1 cells via LOX-1. Finally, we examined the effect of ONL on the expression of the inflammation-associated gene in THP-1 and observed that the ONL-coupled proteins significantly induced the expression of atherogenesis-related genes, such as the monocyte chemoattractant protein-1 and tumor necrosis factor-α, in a LOX-1-dependent manner. Thus, ONL was identified to be a potential endogenous ligand for LOX-1.     

Adiponectin ameliorates doxorubicin-induced cardiotoxicity through Akt protein-dependent mechanism

Accumulating evidence shows that obesity is associated with doxorubicin cardiac toxicity in the heart, but the molecular mechanisms that contribute to this pathological response are not understood. Adiponectin is an adipose-derived, cardioprotective factor that is down-regulated in obesity. Here, we investigated the effect of adiponectin on doxorubicin (DOX)-induced cardiotoxicity and assessed the mechanisms of this effect. A single dose of DOX was intraperitoneally injected into the abdomen of adiponectin knock-out (APN-KO) and wild-type (WT) mice. APN-KO mice had increased mortality and exacerbated contractile dysfunction of left ventricle compared with WT mice. APN-KO mice also showed increased apoptotic activity and diminished Akt signaling in the failing myocardium. Systemic delivery of adenoviral vector expressing adiponectin improved left ventricle dysfunction and myocardial apoptosis following DOX injection in WT and APN-KO mice but not in Akt1 heterozygous KO mice. In cultured rat neonatal cardiomyocytes, adiponectin stimulated Akt phosphorylation and inhibited DOX-stimulated apoptosis. Treatment with sphingosine kinase-1 inhibitor or sphingosine 1-phosphate receptor antagonist diminished adiponectin-induced Akt phosphorylation and reversed the inhibitory effects of adiponectin on myocyte apoptosis. Pretreatment with anti-calreticulin antibody reduced the binding of adiponectin to cardiac myocytes and blocked the adiponectin-stimulated increase in Akt activation and survival in cardiomyocytes. Interference of the LRP1/calreticulin co-receptor system by siRNA or blocking antibodies diminished the stimulatory actions of adiponectin on Akt activation and myocyte survival. These data show that adiponectin protects against DOX-induced cardiotoxicity by its ability to promote Akt signaling.     

Influence of Ser/Pro-rich domain and kinase domain of double cortin-like protein kinase on microtubule-binding activity

Doublecortin-like protein kinase (DCLK) is a Ser/Thr protein kinase predominantly expressed in brain. DCLK is composed of three functional domains; the N-terminal doublecortin-like (DC) domain, the C-terminal kinase domain and Ser/Pro-rich (SP) domain in between DC and kinase domains. Although the DC domain is known to mediate microtubule association, functional roles of the SP domain and the kinase domain on microtubule association is not known. In this study, we investigated the microtubule-binding activity of zebrafish DCLK (zDCLK) using various deletion mutants and chimeric proteins. The microtubule-binding activity of various mutants of zDCLK was assessed both by immunocytochemical analysis and by biochemical analysis using detergent extraction method. When the kinase domain was removed from zDCLK, the microtubule-binding activity was significantly enhanced. Although the zDCLK(DC + SP) mutant showed a strong microtubule-binding activity, the DC domain alone showed much lower microtubule-binding activity, indicating that the SP domain of zDCLK plays a role in enhancing microtubule-binding activity of the DC domain. These results suggest that both the kinase domain and the SP domain are involved in regulating the microtubule-binding activity of DCLK.     

Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement

The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.     

Cyp26b1 within the growth plate regulates bone growth in juvenile mice

Retinoic acid (RA) is an active metabolite of vitamin A and plays important roles in embryonic development. CYP26 enzymes degrade RA and have specific expression patterns that produce a RA gradient, which regulates the patterning of various structures in the embryo. However, it has not been addressed whether a RA gradient also exists and functions in organs after birth. We found localized RA activities in the diaphyseal portion of the growth plate cartilage were associated with the specific expression of Cyp26b1 in the epiphyseal portion in juvenile mice. To disturb the distribution of RA, we generated mice lacking Cyp26b1 specifically in chondrocytes (Cyp26b1^Δchon cKO). These mice showed reduced skeletal growth in the juvenile stage. Additionally, their growth plate cartilage showed decreased proliferation rates of proliferative chondrocytes, which was associated with a reduced height in the zone of proliferative chondrocytes, and closed focally by four weeks of age, while wild-type mouse growth plates never closed. Feeding the Cyp26b1 cKO mice a vitamin A-deficient diet partially reversed these abnormalities of the growth plate cartilage. These results collectively suggest that Cyp26b1 in the growth plate regulates the proliferation rates of chondrocytes and is responsible for the normal function of the growth plate and growing bones in juvenile mice, probably by limiting the RA distribution in the growth plate proliferating zone.     

NADH‐dependent glutamate synthase plays a crucial role in assimilating ammonium in the Arabidopsis root

Plant roots under nitrogen deficient conditions with access to both ammonium and nitrate ions, will take up ammonium first. This preference for ammonium rather than nitrate emphasizes the importance of ammonium assimilation machinery in roots. Glutamine synthetase (GS) and glutamate synthase (GOGAT) catalyze the conversion of ammonium and 2‐oxoglutarate to glutamine and glutamate. Higher plants have two GOGAT species, ferredoxin‐dependent glutamate synthase (Fd‐GOGAT) and nicotinamide adenine dinucleotide (NADH)‐GOGAT. While Fd‐GOGAT participates in the assimilation of ammonium, which is derived from photorespiration in leaves, NADH‐GOGAT is highly expressed in roots and its importance needs to be elucidated. While ammonium as a minor nitrogen form in most soils is directly taken up, nitrate as the major nitrogen source needs to be converted to ammonium prior to uptake. The aim of this study was to investigate and quantify the contribution of NADH‐GOGAT to the ammonium assimilation in Arabidopsis (Arabidopsis thaliana Columbia) roots. Quantitative real‐time polymerase chain reaction (PCR) and protein gel blot analysis showed an accumulation of NADH‐GOGAT in response to ammonium supplied to the roots. In addition the localization of NADH‐GOGAT and Fd‐GOGAT did not fully overlap. Promoter–β‐glucuronidase (GUS) fusion analysis and immunohistochemistry showed that NADH‐GOGAT was highly accumulated in non‐green tissue like vascular bundles, shoot apical meristem, pollen, stigma and roots. Reverse genetic approaches suggested a reduction in glutamate production and biomass accumulation in NADH‐GOGAT transfer DNA (T‐DNA) insertion lines under normal CO₂ condition. The data emphasize the importance of NADH‐GOGAT in the ammonium assimilation in Arabidopsis roots.