Synthesis of 2-bromomethyl-3-hydroxy-2-hydroxymethyl-propyl pyrimidine and theophylline nucleosides under microwave irradiation. Evaluation of their activity against hepatitis B virus

Alkylation of 2-methylthiopyrimidin-4(1H)-one (1a) and its 5(6)-alkyl derivatives 1b-d as well as theophylline (7) with 2,2-bis(bromomethyl)-1,3-diacetoxypropane (2) under microwave irradiation gave the corresponding acyclonucleosides 1-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]-2-methyl-thio pyrmidin-4(1H)-ones 3a-d and 7-[(3-acetoxy-2-acetoxymethyl-2-bromomethyl)prop-1-yl]theophylline (8), which upon further irradiation gave the double-headed acyclonucleosides 1,1 '-[(2,2-diacetoxymethyl)-1,3-propylidene]-bis[(2-(methylthio)-pyrimidin-4(1H)-ones] 4a-c, and 7,7 '-[(2,2-diacetoxymethyl)-1,3-propylidene]-bis(theophylline) (9). The deacetylated derivatives were obtained by the action of sodium methoxide. The activity of deacetylated nucleosides against Hepatitis B virus was evaluated. Compound 5b showed moderate inhibition activity against HBV with mild cytotoxicity.     

Spectroscopic identification of new ellagitannins and a trigalloyl-glucosylkaempferol from an extract of Euphorbia cotinifolia L. with antitumour and antioxidant activity

From an extract of leaves and small branches of Euphorbia cotinifolia L., 17 polyphenols were isolated including two new ellagitannins and a trigalloyl-glucosylkaempferol. Based on extensive spectral data (UV, ESI-MS, 1H NMR, DEPT and 1D/2D NMR) and chemical studies, their structures were characterized as 1-O-galloyl-3,6-hexahydroxydiphenoyl-D-B1,4-glucopyranose (5), 1-O-galloyl-3,6-valoneoyl-D-B1,4-glucopyranose (6), and kaempferol 3-O-(2",3",6"-tri-O-galloyl)-beta-D-glucopyranoside (13). Biological evaluation indicated that the 80% aqueous methanol extract (AME), chloroform extract (CE), and some pure compounds have potent scavenging activity in the DPPH assay with SC50 values lower than that of ascorbic acid, especially 5, 7-9, and a mixture of hyperin 6"-gallate (11) and isoquercitrin 6"-gallate (12). Moreover, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cell viability assay, 6 and 8 exhibited the highest inhibition of human hepatocellular carcinoma cells (Hep-G2), while AME, CE, 5, 7, 9, and the mixture of 11 and 12 were found to be moderate growth inhibitors according to their IC50 values. In addition, AME, 5, and 8 exhibited significant antiproliferative activity against colon carcinoma cells (HCT-116); however, CE and the other examined compounds displayed moderate to low antitumour activity against HCT-116 cells.     

Evaluation of vitamin E in the treatment of erectile dysfunction in aged rats

AimsThe present study aims to elucidate the role of oxidative stress in erectile dysfunction associated with aging and to investigate the effect of treatment with vitamin E in this respect.Main methodsRats were divided into four groups: young (3-month-old), aged rats (18-month-old), aged rats given 80 IU of vitamin E/rat/day for 21-days, aged rats given 5 mg/kg of sildenafil/day for 21-days. Intracavernosal pressure/mean arterial pressure (ICP/MAP), nitric oxide production, TBARS, GSH levels and SOD activity in corpus cavernosum were measured.Key findingsSignificant decrease in ICP/MAP was observed in aged rats at both low and high frequency of stimulation. Significant increase in ICP/MAP was observed in aged rats treated with vitamin E over the range of 0.8 to 5 Hz but young control values were not restored. Percentage potentiation of ICP/MAP than aged group at 0.8 Hz was 326 ± 41.3% and 897 ± 72.2% for vitamin E and sildenafil respectively. Decreased levels of NO₂/NO₃ and SOD activity in the penile tissue observed with aging were elevated back to control by either vitamin E or sildenafil. Penile concentration of TBARS was 20.86 ± 0.83 for aged rats vs. 11.39 ± 0.79 nmol/g tissue for young controls. Both vitamin E and sildenafil reduced penile TBARS in aged rats.SignificanceThis study proves that antioxidant therapy with vitamin E ameliorates the age-associated erectile dysfunction. Sildenafil may exert some antioxidant properties which add to the advantages of its long-term use. The effect of combinations of low-dose sildenafil and vitamin E on age-associated erectile dysfunction merits to be studied.     

Analysis of IL28B variants in an Egyptian population defines the 20 kilobases minimal region involved in spontaneous clearance of hepatitis C virus

Spontaneous clearance of hepatitis C virus (HCV) occurs in ~30% of acute infections. Host genetics play a major role in HCV clearance, with a strong effect of single nucleotide polymorphisms (SNPs) of the IL28B gene already found in different populations, mostly infected with viral genotypes 1 and 3. Egypt has the highest prevalence of HCV infection in the world, which is mostly due to viral genotype 4. We investigated the role of several IL28B SNPs in HCV spontaneous clearance in an Egyptian population. We selected nine SNPs within the IL28B genomic region covering the linkage disequilibrium (LD) block known to be associated with HCV clearance in European populations. These SNPs were genotyped in 261 HCV-infected Egyptian subjects (130 with spontaneous clearance and 131 with chronic infection). The most associated SNPs were rs12979860 (P = 1.6 × 10(-7)) and the non-synonymous IL28B SNP, rs8103142 (P = 1.6 × 10(-7)). Interestingly, three SNPs at the two bounds of the region were monomorphic, reducing the size of the LD block in which the causal variants are potentially located to ～20 kilobases. HCV clearance in Egypt was associated with a region of IL28B smaller than that identified in European populations, and involved the non-synonymous IL28B SNP, rs8103142.     

Behavioural analyses of quinine processing in choice, feeding and learning of larval Drosophila

Gustatory stimuli can support both immediate reflexive behaviour, such as choice and feeding, and can drive internal reinforcement in associative learning. For larval Drosophila, we here provide a first systematic behavioural analysis of these functions with respect to quinine as a study case of a substance which humans report as "tasting bitter". We describe the dose-effect functions for these different kinds of behaviour and find that a half-maximal effect of quinine to suppress feeding needs substantially higher quinine concentrations (2.0 mM) than is the case for internal reinforcement (0.6 mM). Interestingly, in previous studies (Niewalda et al. 2008, Schipanski et al 2008) we had found the reverse for sodium chloride and fructose/sucrose, such that dose-effect functions for those tastants were shifted towards lower concentrations for feeding as compared to reinforcement, arguing that the differences in dose-effect function between these behaviours do not reflect artefacts of the types of assay used. The current results regarding quinine thus provide a starting point to investigate how the gustatory system is organized on the cellular and/or molecular level to result in different behavioural tuning curves towards a bitter tastant.     

A novel G protein-coupled receptor of Schistosoma mansoni (SmGPR-3) is activated by dopamine and is widely expressed in the nervous system

Schistosomes have a well developed nervous system that coordinates virtually every activity of the parasite and therefore is considered to be a promising target for chemotherapeutic intervention. Neurotransmitter receptors, in particular those involved in neuromuscular control, are proven drug targets in other helminths but very few of these receptors have been identified in schistosomes and little is known about their roles in the biology of the worm. Here we describe a novel Schistosoma mansoni G protein-coupled receptor (named SmGPR-3) that was cloned, expressed heterologously and shown to be activated by dopamine, a well established neurotransmitter of the schistosome nervous system. SmGPR-3 belongs to a new clade of "orphan" amine-like receptors that exist in schistosomes but not the mammalian host. Further analysis of the recombinant protein showed that SmGPR-3 can also be activated by other catecholamines, including the dopamine metabolite, epinine, and it has an unusual antagonist profile when compared to mammalian receptors. Confocal immunofluorescence experiments using a specific peptide antibody showed that SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and the peripheral innervation of the body wall muscles. In addition, we show that dopamine, epinine and other dopaminergic agents have strong effects on the motility of larval schistosomes in culture. Together, the results suggest that SmGPR-3 is an important neuronal receptor and is probably involved in the control of motor activity in schistosomes. We have conducted a first analysis of the structure of SmGPR-3 by means of homology modeling and virtual ligand-docking simulations. This investigation has identified potentially important differences between SmGPR-3 and host dopamine receptors that could be exploited to develop new, parasite-selective anti-schistosomal drugs.     

Adverse Drug Reactions in Hospitalised Children in Germany Are Decreasing: Results of a Nine Year Cohort-Based Comparison

Background

In recent years, efforts have been made to improve paediatric drug therapy. The aim of this research was to investigate any changes regarding the frequency and nature of adverse drug reactions (ADRs) in hospitalized children in one paediatric general medical ward over a 9-year period.

Methodology

Two prospective observational cohort studies were conducted at a large University hospital in Germany in 1999 and 2008, respectively. Children aged 0–18 years admitted to the study ward during the study periods were included. ADRs were identified using intensive chart review. Uni- and multivariable regression has been used for data analysis.

Results

A total of 520 patients (574 admissions) were included [1999: n = 144 (167); 2008: n = 376 (407)]. Patients received a total of 2053 drugs [median 3, interquartile range (IQR) 2–5]. 19% of patients did not receive any medication. Median length of stay was 4 days (IQR 3–7; range 1–190 days) with a significantly longer length of stay in 1999. The overall ADR incidence was 13.1% (95% CI, 9.8–16.3) varying significantly between the two study cohorts [1999: 21.9%, 95% CI, 14.7–29.0; 2008: 9.2%, 95% CI, 5.9–12.5 (p<0.001)]. Antibacterials and corticosteroids for systemic use caused most of the ADRs in both cohorts (1999; 2008). Exposure to systemic antibacterials decreased from 62.9% to 43.5% whereas exposure to analgesics and anti-inflammatory drugs increased from 17.4% to 45.2%, respectively. The use of high risk drugs decreased from 75% to 62.2%. In 1999, 45.7% and in 2008 96.2% of ADRs were identified by treating clinicians (p<0.001).

Conclusions

Between 1999 and 2008, the incidence of ADRs decreased significantly. Improved treatment strategies and an increased awareness of ADRs by physicians are most likely to be the cause for this positive development. Nevertheless further research on ADRs particularly in primary care and the establishment of prospective pharmacovigilance systems are still needed.     

Alpha-actinin-4 is selectively required for insulin-induced GLUT4 translocation

Insulin induces GLUT4 translocation to the muscle cell surface. Using differential amino acid labeling and mass spectrometry, we observed insulin-dependent co-precipitation of actinin-4 (ACTN4) with GLUT4 (Foster, L. J., Rudich, A., Talior, I., Patel, N., Huang, X., Furtado, L. M., Bilan, P. J., Mann, M., and Klip, A. (2006) J. Proteome Res. 5, 64-75). ACTN4 links F-actin to membrane proteins, and actin dynamics are essential for GLUT4 translocation. We hypothesized that ACTN4 may contribute to insulin-regulated GLUT4 traffic. In L6 muscle cells insulin, but not platelet-derived growth factor, increased co-precipitation of ACTN4 with GLUT4. Small interfering RNA-mediated ACTN4 knockdown abolished the gain in surface-exposed GLUT4 elicited by insulin but not by platelet-derived growth factor, membrane depolarization, or mitochondrial uncoupling. In contrast, knockdown of alpha-actinin-1 (ACTN1) did not prevent GLUT4 translocation by insulin. GLUT4 colocalized with ACTN4 along the insulin-induced cortical actin mesh and ACTN4 knockdown prevented GLUT4-actin colocalization without impeding actin remodeling or Akt phosphorylation, maintaining GLUT4 in a tight perinuclear location. We propose that ACTN4 contributes to GLUT4 traffic, likely by tethering GLUT4 vesicles to the cortical actin cytoskeleton.     

Selective regulation of the perinuclear distribution of glucose transporter 4 (GLUT4) by insulin signals in muscle cells

Insulin regulates glucose transporter 4 (GLUT4) availability at the surface of muscle and adipose cells. In L6 myoblasts, stably expressed GLUT4myc is detected mostly in a perinuclear region. In unstimulated cells, about half of perinuclear GLUT4myc colocalizes with the transferrin receptor (TfR). Insulin stimulation selectively decreased the perinuclear colocalization of GLUT4myc with TfR determined by 3D-reconstruction of fluorescence images. Perinuclear GLUT4myc adopted two main distributions defined morphometrically as 'conical' and 'concentric'. Insulin rapidly reduced the proportion of cells with conical in favor of concentric perinuclear GLUT4myc distributions in association with the gain in surface GLUT4myc. Upon removal of insulin, the GLUT4myc perinuclear distribution and surface levels reversed in parallel. In contrast, hypertonicity (which like insulin elevates surface GLUT4myc) did not elicit perinuclear GLUT4myc redistribution. Insulin also caused redistribution of perinuclear vesicle-associated membrane protein-2 (VAMP2), without alteration of perinuclear TfR and VAMP3. Inhibitory mutants of phosphatidylinositol-3 kinase (Deltap85) or Akt substrate AS160 (AS160-4P) prevented insulin-mediated perinuclear GLUT4myc redistribution. Tetanus toxin expression did not prevent the perinuclear GLUT4myc redistribution, suggesting that redistribution is independent of GLUT4myc fusion with the plasma membrane. We propose that insulin causes selective, dynamic relocalization of perinuclear GLUT4myc and VAMP2 and perinuclear GLUT4myc redistribution is a direct target of insulin-derived signals.