Role of hyaluronidase inhibitors in the neutralization of toxicity of Egyptian horned viper Cerastes cerastes venom

Hyaluronidase “venom spreading factor” is a common component of snake venoms and indirectly potentiates venom toxicity. It may cause permanent local tissue destruction at the bite site/systemic collapse of the envenomated victim. The present study was performed to assess the benefits of inhibiting the hyaluronidase activity of Egyptian horned viper, Cerastes cerastes (Cc). The aqueous extracts of some medicinal plants were screened for their inhibitory effect on hyaluronidase activity of Cc venom. The results revealed that the Rosmarinus officinalis (Ro) extract is the most potent hyaluronidase inhibitor among the tested extracts. The Ro extract is more potent inhibitory effect on the hyaluronidase activity than the prepared rabbit monoclonal antiserum of previously purified hyaluronidase enzyme from Cc venom (anti-CcHaseII). In addition, the Ro extract is efficiently inhibited the activity of hemorrhagic toxin previously purified from Cc venom, and it also neutralized the edema inducing activity of the Cc venom in vivo. Furthermore, the Ro extract markedly increased the survival time of experimental mice injected with lethal dose of Cc venom up to 7 h in compared to mice injected with venom alone or with venom/anti-CcHaseII (15 ± 5, 75 ± 4 min), respectively. Our findings imply the significance of plant-derived hyaluronidase inhibitor in the neutralization of local effects of Cc venom and retardation of death time. Therefore, it may use as a therapeutic value in complementary snakebite therapy.     

Molecular and biochemical characterization of a novel intronic single point mutation in a Tunisian family with glycogen storage disease type III

Genetic deficiency of the glycogen debranching enzyme causes glycogen storage disease type III, an autosomal recessive inherited disorder. The gene encoding this enzyme is designated as AGL gene. The disease is characterized by fasting hypoglycemia, hepatomegaly, growth retardation, progressive myopathy and cardiomyopathy. In the present study, we present clinical features and molecular characterization of two consanguineous Tunisian siblings suffering from Glycogen storage disease type III. The full coding exons of the AGL gene and their corresponding exon–intron boundaries were amplified for the patients and their parents. Gene sequencing identified a novel single point mutation at the conserved polypyrimidine tract of intron 21 in a homozygous state (IVS21-8A>G). This variant cosegregated with the disease and was absent in 102 control chromosomes. In silico analysis using online resources showed a decreased score of the acceptor splice site of intron 21. RT-PCR analysis of the AGL splicing pattern revealed a 7 bp sequence insertion between exon 21 and exon 22 due to the creation of a new 3′ splice site. The predicted mutant enzyme was truncated by the loss of 637 carboxyl-terminal amino acids as a result of premature termination. This novel mutation is the first mutation identified in the region of Bizerte and the tenth AGL mutation identified in Tunisia. Screening for this mutation can improve the genetic counseling and prenatal diagnosis of GSD III.     

Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing,orientation, and enzyme identity

Biomass deconstruction to small simple sugars is a potential approach to biofuels production; however, the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large‐scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high‐yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyperstable α‐helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N‐terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation. Proteins 2016; 84:1043–1054. © 2016 Wiley Periodicals, Inc.     

Substrate specificities and availability of fucosyltransferase and beta-carotene hydroxylase for myxol 2'-fucoside synthesis in Anabaena sp. strain PCC 7120 compared with Synechocystis sp. strain PCC 6803

To elucidate the biosynthetic pathways of carotenoids, especially myxol 2'-glycosides, in cyanobacteria, Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120) and Synechocystis sp. strain PCC 6803 deletion mutants lacking selected proposed carotenoid biosynthesis enzymes and GDP-fucose synthase (WcaG), which is required for myxol 2'-fucoside production, were analyzed. The carotenoids in these mutants were identified using high-performance liquid chromatography, field desorption mass spectrometry, and (1)H nuclear magnetic resonance. The wcaG (all4826) deletion mutant of Anabaena sp. strain PCC 7120 produced myxol 2'-rhamnoside and 4-ketomyxol 2'-rhamnoside as polar carotenoids instead of the myxol 2'-fucoside and 4-ketomyxol 2'-fucoside produced by the wild type. Deletion of the corresponding gene in Synechocystis sp. strain PCC 6803 (sll1213; 79% amino acid sequence identity with the Anabaena sp. strain PCC 7120 gene product) produced free myxol instead of the myxol 2'-dimethyl-fucoside produced by the wild type. Free myxol might correspond to the unknown component observed previously in the same mutant (H. E. Mohamed, A. M. L. van de Meene, R. W. Roberson, and W. F. J. Vermaas, J. Bacteriol. 187:6883-6892, 2005). These results indicate that in Anabaena sp. strain PCC 7120, but not in Synechocystis sp. strain PCC 6803, rhamnose can be substituted for fucose in myxol glycoside. The beta-carotene hydroxylase orthologue (CrtR, Alr4009) of Anabaena sp. strain PCC 7120 catalyzed the transformation of deoxymyxol and deoxymyxol 2'-fucoside to myxol and myxol 2'-fucoside, respectively, but not the beta-carotene-to-zeaxanthin reaction, whereas CrtR from Synechocystis sp. strain PCC 6803 catalyzed both reactions. Thus, the substrate specificities or substrate availabilities of both fucosyltransferase and CrtR were different in these species. The biosynthetic pathways of carotenoids in Anabaena sp. strain PCC 7120 are discussed.     

Mutations in prickle orthologs cause seizures in flies, mice, and humans

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution.     

Xp22.3 interstitial deletion: A recognizable chromosomal abnormality encompassing VCX3A and STS genes in a patient with X-linked ichthyosis and mental retardation

X-linked ichthyosis is a genetic disorder affecting the skin and caused by a deficit in the steroid sulfatase enzyme (STS), often associated with a recurrent microdeletion at Xp22.31. Most of the STS deleted patients have X-linked ichthyosis as the only clinical feature and it is believed that patients with more complex disorders including mental retardation could be present as a result of contiguous gene deletion. In fact, VCX3A gene, a member of the VCX (variable charge, X chromosome) gene family, was previously proposed as the candidate gene for X-linked non-specific mental retardation in patients with X-linked ichthyosis.     

The Sleeping Beauty transposable element: evolution, regulation and genetic applications

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrate species are inactive due to the accumulation of mutations. A representative of a subfamily of fish elements estimated to be last active > 10 million years ago has been reconstructed, and named Sleeping Beauty(SB). This element opened up new avenues for studies on DNA transposition in vertebrates, and for the development of transposon tools for genetic manipulation in important model species and in humans. Multiple transposase binding sites within the terminal inverted repeats, a transpositional enhancer sequence, unequal affinity of the transposase to the binding sites and the activity of the cellular HMGB1 protein all contribute to a highly regulated assembly of SB synaptic complexes, which is likely a requirement for the subsequent catalytic steps. Host proteins involved in double-strand DNA break repair are limiting factors of SB transposition in mammalian cells, underscoring evolutionary, structural and functional links between DNA transposition, retroviral integration and V(D)J recombination. SB catalyzes efficient cut-and-paste transposition in a wide range of vertebrate cells in tissue culture, and in somatic tissues as well as the germline of the mouse and zebrafish in vivo, indicating its usefulness as a vector for transgenesis and insertional mutagenesis.     

Bio-inspired synthesis of AgNPs from lichen as potential antibacterial and mosquito larvicidal activity with negligible toxicity on Gambusia affinis

Mosquitoes serve as reservoirs for viruses and other microorganisms, posing a significant health-related issue for both humans as well as livestock. Control of these deadly disease-producing mosquito vectors is of paramount importance. The chemical analysis of Parmotrema reticulatum was examined by GC–MS. Further, lichen-mediated AgNPs were confirmed through UV–vis spectrophotometry, FTIR, TEM-EDX, and XRD. After 24 h post-treatment, the lichen-synthesized AgNPs showed considerable toxicity against distinct Aedes aegypti instars with LC₅₀ values of 44.61 (I instar), 51.27 (II instars), 61.34 (III instars), 72.95 (IV instar), and 89.84 (pupae) μg/mL, respectively. Further, both P. reticulatum extract and AgNPs greatly reduced the survival and reproductive efficiency of A. aegypti adults. Eventually, in conventional laboratory circumstances, the predatory effectiveness of Gambusia affinis against Ae. aegypti II and III instar larvae were 71.35% and 53.40%, respectively. In antibacterial assays, low concentrations of the P. reticulatum synthesized AgNPs inhibited the development of Pseudomonas aeruginosa and Citrobacter freundii. Surface damage, ROS production, and protein leakage are the antibacterial mechanisms of AgNPs. Overall, the lichen-derived AgNPs can be regarded as newer and safer Ae. aegypti control instruments.     

Gas chromatographic determination of amino acid enantiomers in bottled and aged wines

Free l- and d-amino acids were determined by chiral GC-MS in 26 wines, comprising white wines, red wines, ice wines and sparkling wines. The aim of the work was to investigate whether quantities and pattern of d-amino acids, in particular d-proline, correlate with the storage time of bottled wines. The relative quantities with respect to the corresponding l-enantiomer ranged in white wines from 0.4 to 3.9% d-Ala, 0.9–8.3% d-Asx, and 0.5–8.9% d-Glx, in red wines from 2.9 to 10.6% d-Ala, 2.2–10.9% d-Asx, and 3.9–7.4% d-Glx, and in sparkling wines from 2.2 to 9.8% d-Ala, 2.1–4.4% d-Asx and 1.3–6.1% d-Glx. Low relative quantities of 0.3–0.7% d-Pro were detected in three white wines stored for more than 20 years and did not exceed 0.2% d-Pro in two red wines stored for 10 and 20 years, respectively. An ice wine stored for 24 years contained 0.9% d-Pro, 6.4% d-Glx, 3.0% d-Asp and 1.5% d-Ala. The data confirm the presence of d-amino acids in wines. They do not provide evidence for a correlation between the storage time of bottled wines and quantities of d-amino acids.