Medicinal plants used in the management of diabetes mellitus 2015

Diabetes mellitus is one of the common endocrine disorders prevalent in almost all of the countries. This chronic pathology is characterized by hyperglycemia caused by defective insulin action, insulin secretion, or the combination of both. Prolonged persistence of elevated blood glucose level consequently caused a series of complications such as nephropathy, retinopathy, and cardiomyopathy. Currently available synthetic drugs for treating this disease are found to be associated with many adverse effects. The use of plants in medicine is an age-long practice in various parts of the globe for both preventive and curative purposes. Several warnings have been issued over lack of quality control, scientific evidence for the efficacy, and potential adverse effects of herbal remedies including hepatotoxicity, nephrotoxicity, cardiotoxicity, and reproductive toxicity among others. Despite all of these, reliance on herbs as medicine for the management of diabetes mellitus is still much practiced by a large proportion of the world population because they are readily available and affordable with perceived reduced toxicity. Therefore, with the upsurge of interests in medicinal plants, there is a need for thorough scientific investigations of these plants for both efficacy and potential toxicity. In this issue, we present some recent advances in the use of medicinal plants for treating diabetes mellitus. B. Pang et al. (“Innovative Thoughts of Treating Diabetes from the Perspective of Traditional Chinese Medicine”)presented a review article on the contribution of traditional Chinese medicine to the development of alternative and complementary medicine for the treatment and prevention of diabetes mellitus. In another paper (“Effect of Rhizoma Coptidis (Huang Lian) on Treating Diabetes Mellitus”), B. Pang et al. discussed the efficacy and safety of Rhizoma Coptidis in the treatment of diabetes mellitus. In another study (“Evaluation of the Effects of Cornus mas L. Fruit Extract on Glycemic Control and Insulin Level in Type 2 Diabetic Adult Patients: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial”), R. Soltani et al. reported the results of a clinical trial on the effect of Cornus mas L. fruit extract on hyperglycemia in type 2 diabetic patients. In addition, W. Liu et al. (“The Effects of Chinese Medicine on Activation of Wnt/β-Catenin Signal Pathway under High Glucose Condition”) present a valuable review on some compounds implicated in the regulation of Wnt/β-catenin signal pathway as a mechanism of action involved in the antihyperglycemic activity from Chinese medicine. Furthermore, A. O. T. Ashafa and M. I. Kazeem (“Toxicopathological Evaluation of Hydroethanol Extract of Dianthus basuticus in Wistar Rats”) reported on the effects of Dianthus basuticus (a Basotho plant with acclaimed antidiabetic activity) on some biochemical parameters and histology of Wistar rats. Finally, X.-J. Li et al. (“TCM Formula Xiaoyaosan Decoction Improves Depressive-Like Behaviors in Rats with Type 2 Diabetes”) evaluated the effect of traditional medicine formula, Xiaoyaosan, on the cognitive function of diabetic rats. After the first volume of this special issue that was published in 2014, we hope that this issue will present additional valuable information for scientists and clinicians

Distinct Metal Ion Requirements for the Phosphomonoesterase and Phosphodiesterase Activities of Calf Intestinal Alkaline Phosphatase

The roles of Mg2+ and Zn2+ ions in promoting phosphoryl transfer catalysed by alkaline phosphatase are yet to be fully characterised. We investigated the divalent metal ion requirements for the monoesterase and diesterase activities of calf intestinal alkaline phosphatase. The synergistic effect of Mg2+ and Zn2+ in promoting the hydrolysis of para-nitrophenyl phosphate (monoesterase reaction) by alkaline phosphatase is not observed in the hydrolysis of the diesterase substrate, bis-para-nitrophenyl phosphate. Indeed, the diesterase reaction is inhibited by concentrations of Mg2+ that were optimal for the monoesterase reaction. This study reveals that the substrate specificities of alkaline phosphatases and related bimetalloenzymes are subject to regulation by changes in the nature and availability of cofactors, and the different cofactor requirements of the monoesterase and diesterase reactions of mammalian alkaline phosphatases could have significance for the biological functions of the enzymes

Control of serine integrase recombination directionality by fusion with the directionality factor

Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The ‘reverse’ reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase–RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase–RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle

Catalytic cofactors (Mg2+ and Zn2+ ions) influence the pattern of vanadate Inhibition of the monoesterase activity of calf intestinal alkaline phosphatase

The mechanism of modulation of vanadate inhibition of alkaline phosphatase activity by catalytic cofactors has not been fully characterized. We investigated the effect of the interaction of catalytic cofactors (Mg2+ and Zn2+) and vanadate (an active site inhibitor) on the rate of hydrolysis of para-nitrophenyl phosphate (pNPP) (monoesterase reaction) by calf intestinal alkaline phosphatase (CIAP). The results showed that vanadate significantly inhibited \u2018cofactor-free\u2019 CIAP, and the inhibition was relieved by the presence of the catalytic cofactors in the reaction. Our results show that the absence of the cofactors did not significantly alter the Km of the reaction, but caused a decrease in the Vmax. Kinetic analyses showed that vanadate inhibited CIAP-catalyzed hydrolysis of pNPP by decreasing the Vmax and increasing the Km of the reaction. The presence of cofactors in the reaction alleviated the effect of vanadate by increasing the Vmax and decreasing the Km. The activity of the dialyzed CIAP was increased by the addition of catalytic cofactors to vanadate-inhibited enzyme. This study provides preliminary data that reversible inhibition of CIAP is subject to the influence of catalytic cofactors. Further studies will reveal detailed mechanistic aspects of this observation and its significance in the biological system

Nicked-site substrates for a serine recombinase reveal enzyme-DNA communications and an essential tethering role of covalent enzyme-DNA linkages

To analyse the mechanism and kinetics of DNA strand cleavages catalysed by the serine recombinase Tn3 resolvase, we made modified recombination sites with a single-strand nick in one of the two DNA strands. Resolvase acting on these sites cleaves the intact strand very rapidly, giving an abnormal half-site product which accumulates. We propose that these reactions mimic second-strand cleavage of an unmodified site. Cleavage occurs in a synapse of two sites, held together by a resolvase tetramer; cleavage at one site stimulates cleavage at the partner site. After cleavage of a nicked-site substrate, the half-site that is not covalently linked to a resolvase subunit dissociates rapidly from the synapse, destabilizing the entire complex. The covalent resolvase–DNA linkages in the natural reaction intermediate thus perform an essential DNA-tethering function. Chemical modifications of a nicked-site substrate at the positions of the scissile phosphodiesters result in abolition or inhibition of resolvase-mediated cleavage and effects on resolvase binding and synapsis, providing insight into the serine recombinase catalytic mechanism and how resolvase interacts with the substrate DNA

Suicide inactivation of horseradish peroxidase by excess hydrogen peroxide: The effects of reaction pH, buffer ion concentration, and redox mediation

The inactivation of peroxidases by its oxidant substrate H2O2 limits the usefulness of these versatile enzymes. Here, we investigated the effect of reaction conditions on inactivation of horseradish peroxidase by excess H2O2. Inactivation was more pronounced at pH extremes, indicating that reactions in which the oxidation products induce significant changes in reaction pH could accentuate the loss of peroxidase activity. In reactions carried out in sodium acetate buffer, higher inactivation rates were observed when the buffer ion concentration was increased, an indication that peroxidase might be generating reactive radicals from the buffer molecules. Promethazine exerted a modest protective effect against inactivation; however, higher concentrations of the redox mediator caused a slight increase in inactivation, likely due to the formation of reactive promethazine radicals, which in turn attack the protein via a mechanism different from that caused by excess H2O2. These findings will help in defining the optimal reaction conditions that preserve the activity of the peroxidase molecules

Control of ϕC31 integrase-mediated site-specific recombination by protein trans-splicing.

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices

Zinc-finger recombinase activities in vitro

Zinc-finger recombinases (ZFRs) are chimaeric proteins comprising a serine recombinase catalytic domain linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse ‘Z-sites’, which each comprise a central core sequence flanked by zinc-finger domain-binding motifs. Here, we show that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro. No off-site activity was detected. Under different reaction conditions, ZFRs can catalyse Z-site-specific double-strand DNA cleavage. ZFR recombination activity in Escherichia coli and in vitro is highly dependent on the length of the Z-site core sequence. We show that this length effect is manifested at reaction steps prior to formation of recombinants (binding, synapsis and DNA cleavage). The design of the ZFR protein itself is also a crucial variable affecting activity. A ZFR with a very short (2 amino acids) peptide linkage between the catalytic and zinc-finger domains has high activity in vitro, whereas a ZFR with a very long linker was less recombination-proficient and less sensitive to variations in Z-site length. We discuss the causes of these phenomena, and their implications for practical applications of ZFRs

Site-specific recombinases: molecular machines for the genetic revolution

The fields of molecular genetics, biotechnology and synthetic biology are demanding ever more sophisticated molecular tools for programmed precise modification of cell genomic DNA and other DNA sequences. This review presents the current state of knowledge and development of one important group of DNA-modifying enzymes, the site-specific recombinases (SSRs). SSRs are Nature's ‘molecular machines’ for cut-and-paste editing of DNA molecules by inserting, deleting or inverting precisely defined DNA segments. We survey the SSRs that have been put to use, and the types of applications for which they are suitable. We also discuss problems associated with uses of SSRs, how these problems can be minimized, and how recombinases are being re-engineered for improved performance and novel applications