Inhibition of enterovirus 71 (EV-71) infections by a novel antiviral peptide derived from EV-71 capsid protein VP1

Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50) values ranging from 6-9.3 μM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.     

Chemical reactivity and microbicidal action of bethoxazin

Bethoxazin is a new broad spectrum industrial microbicide with applications in material and coating preservation. However, little is known of its reactivity profile and mechanism of action. In this study, we examined the reactivity of bethoxazin toward biologically important nucleophilic groups using UV-vis spectroscopy and LC-MS/MS techniques and found the molecule to be highly electrophilic. Bethoxazin reacted with molecules containing free sulfhydryl groups such as GSH and human serum albumin to form covalent adducts that were detectable by MS, but did not react with amino, carboxylic, phenolic, amino oxo, alcoholic, and phosphate functional groups. Bethoxazin potently inhibited the catalytic activity of yeast DNA topoisomerase II and the growth of yeast BY4742 cells at low micromolar concentrations. However, the reduced form of bethoxazin and GSH-treated bethoxazin were both inactive in these assays. The experimentally determined relative reactivity of bethoxazin and its reduced form analog correlated with their biological activities as well as their quantum-mechanically calculated electrophilicity properties. Taken together, the results suggest that bethoxazin may exert its microbicidal action by reacting with sensitive endogenous sulfhydryl biomolecules of microbial cells. Consistent with this view, the inhibitory activity of bethoxazin on topoisomerase II may be due to its ability to react with critical free cysteine sulfhydryl groups on the enzyme. Our studies have provided for the first time a better understanding of the reactivity of bethoxazin, as well as some insights into the mechanism by which the compound exerts its microbicidal action.     

Role of multi-drug resistance-associated protein-1 transporter in statin-induced myopathy

This study investigated the effects of probenecid to inhibit the multi-drug resistance-associated protein-1 (MRP-1) in mediating the efflux and myotoxicity in rat skeletal muscles, with administration of rosuvastatin. Male Sprague-Dawley rats were administered daily, for 15 days, with either rosuvastatin (50, 100 or 200 mg/kg) or probenecid (100 mg/kg) alone, or with a combination of rosuvastatin (50, 100 or 200 mg/kg) and probenecid (100 mg/kg). Skeletal muscle toxicity was elevated with probenecid administered with 200 mg/kg/day of rosuvastatin, with the elevation of creatine kinase by 12-fold, alanine aminotrasferase by 10-fold and creatinine by 9-fold at day 15, with no adverse effects observed when probenecid was given alone. Mitochondria ultrastructural damage with enlargement, disruption, cristolysis and vaculation was seen in the soleus and plantaris of animals administered with probenecid and high dosages of statin. These muscles were also expressing more succinic dehydrogenase (SDH)-positive and cytochrome oxidase (CyOX)-positive fibers. Although generally well-tolerated, statins produce a variety of adverse skeletal muscle events. Hydrophilic statins, with reduced levels of non-specific passive diffusion rates into extra-hepatic tissues, are still seen to produce myopathy. This highlights the important roles of transport mechanisms in statin transport at the skeletal muscles. Excessive influx, reduced efflux or the combination of the two could result in elevated cellular levels of statins at the skeletal muscles, resulting in toxicity. This study provides preliminary evidence that the MRP-1 transporter and efflux at skeletal muscles possibly play significant roles in statin-induced myopathy.     

The third intracellular loop stabilizes the inactive state of the neuropeptide Y1 receptor

Constitutively active G-protein-coupled receptors (GPCRs) can signal even in the absence of ligand binding. Most Class I GPCRs are stabilized in the resting conformation by intramolecular interactions involving transmembrane domain (TM) 3 and TM6, particularly at loci 6.30 and 6.34 of TM6. Signaling by Gi/Go-coupled receptors such as the Neuropeptide Y1 receptor decreases already low basal metabolite levels. Thus, we examined constitutive activity using a biochemical assay mediated by a Gi/Gq chimeric protein and a more direct electrophysiological assay. Wild-type (WT-Y1) receptors express no measurable, agonist-independent activation, while mu-opioid receptors (MOR) and P2Y12 purinoceptors showed clear evidence of constitutive activation, especially in the electrophysiological assay. Neither point mutations at TM6 (T6.30A or N6.34A) nor substitution of the entire TM3 and TM6 regions from the MOR into the Y1 receptor increased basal WT-Y1 activation. By contrast, chimeric substitution of the third intracellular loop (ICL3) generated a constitutively active, Y1-ICL3-MOR chimera. Furthermore, the loss of stabilizing interactions from the native ICL3 enhanced the role of surrounding residues to permit basal receptor activation; because constitutive activity of the Y1-ICL3-MOR chimera was further increased by point mutation at locus 6.34, which did not alter WT-Y1 receptor activity. Our results indicate that the ICL3 stabilizes the Y1 receptor in the inactive state and confers structural properties critical for regulating Y receptor activation and signal transduction. These studies reveal the active participation of the ICL3 in the stabilization and activation of Class I GPCRs.     

Response of the dinoflagellate Alexandrium tamarense to a range of nitrogen sources and concentrations: growth rate, chemical carbon and nitrogen, and pigments

The responses of cellular C and N, pigments and growth rates of Alexandrium tamarense to different sources of N at high concentrations (6, 12, 25, 50 and 100 μM-N) were examined. Nitrate induced the highest concentration of cellular C (an indicator of biovolume) and cellular N, followed by ammonium and then urea. Cellular C to N ratio (an indicator of physiological status) also varied between N sources. Nitrate grown cells had lower range of C:N ratios and ammonium grown cells had highly variable range. Urea cultures had the highest range of cellular C:N ratio. Pigment composition remained unchanged with all N substrates. The pigments decreased with increasing nitrate concentrations, but with ammonium pigments increased. On the other hand, urea concentrations had no clear relationship with cellular pigments. Variability in the growth of cells was due to both the physiological condition and pigments. When the cells are exposed to different N environments and concentration, they exhibit a physiological acclimation by regulating their cellular materials which is associated with growth.     

Dockers at the crossroads.

The family of docker proteins containing phosphotyrosine-binding (PTB) domains appears to represent a family of critically positioned and exquisitely controlled signalling proteins that relay signals from the activated receptors to downstream pathways. These proteins all have a membrane attachment domain, a PTB domain that targets the protein to a subset of receptors and a number of phosphorylatable tyrosines that dock other signalling proteins. Evidence is accruing that suggests that the PTB domain has evolved from a pleckstrin homology (PH) domain to bind to a range of sequences that, while bestowing specificity, allows switching of the docker protein between receptors or signalling systems. The history of the PTB domain and how it influences the participation of docker protein in various signalling pathways are discussed.     

L1 (LINE-1) retrotransposable elements provide a "fossil" record of the phylogenetic history of murid rodents

The single most difficult problem in phylogenetic analysis is deciding whether a shared taxonomic character is due to common ancestry or one that appeared independently due to convergence, parallelism, or reversion to an ancestral state. Mammalian L1 retrotransposons undergo periodic amplifications in which multiple copies of the elements are interspersed in the genome. Because these elements apparently are transmitted only by inheritance and are retained in the genome, a shared L1 amplification event can only be an inherited ancestral character. We propose that L1 amplification events can be an excellent tool for analyzing mammalian evolution and demonstrate here how we addressed several refractory problems in rodent systematics using L1 DNA as a taxonomic character.      

Molecular and allergenic characterization of recombinant tropomyosin from mud crab Scylla olivacea

Molecular Biology Reports - Tropomyosin is a major allergen in crustaceans, including mud crab species, but its molecular and allergenic properties in Scylla olivacea are not well known. Thus, this...