Functional interaction of diphenols with polyphenol oxidase. Molecular determinants of substrate/inhibitor specificity

Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to their respective quinones. The quinones autopolymerize to form dark pigments, an undesired effect. PPO is therefore the target for the development of antibrowning and antimelanization agents. A series of phenolic compounds experimentally evaluated for their binding affinity and inhibition constants were computationally docked to the active site of catechol oxidase. Docking studies suggested two distinct modes of binding, dividing the docked ligands into two groups. Remarkably, the first group corresponds to ligands determined to be substrates and the second group corresponds to reversible inhibitors. Analyses of the complexes provide structural explanations for correlating subtle changes in the position and nature of the substitutions on diphenols to their functional properties as substrates and inhibitors. Higher reaction rates and binding are reckoned by additional interactions of the substrates with key residues that line the hydrophobic cavity. The docking results suggest that inhibition of oxidation stems from an interaction between the aromatic carboxylic acid group and the apical His109 of the four coordinates of the trigonal pyramidal coordination polyhedron of CuA. The spatial orientation of the hydroxyl in relation to the carboxylic group either allows a perfect fit in the substrate cavity, leading to inhibition, or because of a steric clash flips the molecule vertically, facilitating oxidation. This is the first study to explain, at the molecular level, the determinants of substrate and inhibitor specificity of a catechol oxidase, thereby providing a platform for the design of selective inhibitors useful to both the food and pharmaceutical industries.     

Pulmonary and systemic endotoxin tolerance in preterm fetal sheep exposed to chorioamnionitis

In a model of human chorioamnionitis, fetal sheep exposed to a single injection, but not repeated injections, of intra-amniotic endotoxin develop lung injury responses. We hypothesized that repeated exposure to intra-amniotic endotoxin induces endotoxin tolerance. Fetal sheep were given intra-amniotic injections of saline (control) or Escherichia coli LPS O55:B5 (10 mg) either 2 days (2-day group, single exposure), 7 days (7-day group, single exposure), or 2 plus 7 days (2- and 7-day repeat exposure) before preterm delivery at 124 days gestation (term=150 days). Endotoxin responses were assessed in vivo in the lung and liver, and in vitro in monocytes from the blood and the lung. Compared with the single 2-day LPS exposure group, the (2 plus 7 days) repeat LPS-exposed lambs had: 1) decreased lung neutrophil and monocyte inducible NO synthase (NOSII) expression, and 2) decreased lung cytokine and liver serum amyloid A3 mRNA expression. In the lung, serum amyloid A3 mRNA expression decreased in the airway epithelial cells but not in the lung inflammatory cells. Unlike the single 7-day LPS exposure group, peripheral blood and lung monocytes from the repeat-LPS group did not increase IL-6 secretion or hydrogen peroxide production in response to in vitro LPS. Compared with controls, TLR4 expression did not change but IL-1R-associated kinase M expression increased in the monocytes from repeat LPS-exposed lambs. These results are consistent with the novel finding of endotoxin tolerance in preterm fetal lungs exposed to intra-amniotic LPS. The findings have implications for preterm infants exposed to chorioamnionitis for both responses to lung injury and postnatal nosocomial infections.     

Metalloids in plants: A systematic discussion beyond description

Metalloids represent a wide range of elements with intermediate physiochemical properties between metals and non-metals. Many of the metalloids, like boron, selenium, and silicon are known to be essential or quasi-essential for plant growth. In contrast, metalloids viz. arsenic and germanium are toxic to plant growth. The toxicity of metalloids largely depends on their concentration within the living cells. Some elements, at low concentration, may be beneficial for plant growth and development; however, when present at high concentration, they often exert negative effects. In this regard, understanding the molecular mechanisms involved in the uptake of metalloids by roots, their subsequent transport to different tissues and inter/intra-cellular redistribution has great importance. The mechanisms of metalloids' uptake have been well studied in plants. Also, various transporters, as well as membrane channels involved in these processes, have been identified. In this review, we have discussed in detail the aspects concerning the positive/negative effects of different metalloids on plants. We have also provided a thorough account of the uptake, transport, and accumulation, along with the molecular mechanisms underlying the response of plants to these metalloids. Additionally, we have brought up the previous theories and debates about the role and effects of metalloids in plants with insightful discussions based on the current knowledge.     

Design and synthesis of tryptophan containing peptides as potential analgesic and anti‐inflammatory agents

A new series of smaller peptides with tryptophan at C‐terminal and varying N‐protected amino acids/peptides were designed, synthesized and characterized by analytical and spectroscopic techniques. Analgesic and anti‐inflammatory properties of these peptides were carried out in vivo using tail‐flick method and carrageenan‐induced paw edema method, respectively, at different doses and different time intervals. Most of the peptides synthesized displayed enhanced activity, and particularly tetra and hexapeptides 29–31 were found to be even more potent than the reference standards used. Moreover, some peptides have exhibited promising activity even after 24 h of administration, whereas the reference standards were active only up to 3 h. Further, the compounds did not present any ulcerogenic liability. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

DNA damage is a late event in resveratrol-mediated inhibition of Escherichia coli

Resveratrol is an important phytoalexin notable for a wide variety of beneficial activities. Resveratrol has been reported to be active against various pathogenic bacteria. However, it is not clear at the molecular level how this important activity is manifested. Resveratrol has been reported to bind to cupric ions and reduce it. In the process, it generates copper-peroxide complex and reactive oxygen species (ROS). Due to this ability, resveratrol has been shown to cleave plasmid DNA in several studies. To this end, we envisaged DNA damage to play a role in resveratrol mediated inhibition in Escherichia coli. We employed DNA damage repair deficient mutants from keio collection to demonstrate the hypersensitive phenotype upon resveratrol treatment. Analysis of integrity and PCR efficiency of plasmid DNA from resveratrol-treated cells revealed significant DNA damage after 6?h or more compared to DNA from vehicle-treated cells. RAPD-PCR was performed to demonstrate the damage in genomic DNA from resveratrol-treated cells. In addition, in situ DNA damage was observed under fluorescence microscopy after resveratrol treatment. Further resveratrol treatment resulted in cell cycle arrest of significant fraction of population revealed by flow cytometry. However, a robust induction was not observed in phage induction assay and induction of DNA damage response genes quantified by promoter fused fluorescent tracker protein. These observations along with our previous observation that resveratrol induces membrane damage in E. coli at early time point reveal, DNA damage is a late event, occurring after a few hours of treatment.     

Short AntiMicrobial Peptides (SAMPs) as a class of extraordinary promising therapeutic agents

The emergence of multidrug resistant bacteria has a direct impact on global public health because of the reduced potency of existing antibiotics against pathogens. Hence, there is a pressing need for new drugs with different modes of action that can kill microorganisms. Antimicrobial peptides (AMPs) can be regarded as an alternative tool for this purpose because they are proven to have therapeutic effects with broad‐spectrum activities. There are some hurdles in using AMPs as clinical candidates such as toxicity, lack of stability and high budgets required for manufacturing. This can be overcome by developing shorter and more easily accessible AMPs, the so‐called S hort A nti M icrobial P eptides (SAMPs) that contain between two and ten amino acid residues. These are emerging as an attractive class of therapeutic agents with high potential for clinical use and possessing multifunctional activities. In this review we attempted to compile those SAMPs that have exhibited biological properties which are believed to hold promise for the future. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Exquisite specificity of mitogenic lectin from <Emphasis Type="Italic">Cephalosporium curvulum</Emphasis> to core fucosylated N-glycans

Lectins are carbohydrate binding proteins that are gaining attention as important tools for the identification of specific glycan markers expressed during different stages of the cancer. We earlier reported the purification of a mitogenic lectin from human pathogenic fungus Cephalosporium curvulum (CSL) that has complex sugar specificity when analysed by hapten inhibition assay. In the present study, we report the fine sugar specificity of CSL as determined by glycan array analysis. The results revealed that CSL has exquisite specificity towards core fucosylated N-glycans. Fucosylated trimannosyl core is the basic structure required for the binding of CSL. The presence of fucose in the side chain further enhances the avidity of CSL towards such glycans. The affinity of CSL is drastically reduced towards the non-core fucosylated glycans, in spite of their side chain fucosylation. CSL showed no binding to the tested O-glycans and monosaccharides. These observations suggest the unique specificity of CSL towards core fucosylated N-glycans, which was further validated by binding of CSL to human colon cancer epithelial and hepatocarcinoma cell lines namely HT29 and HepG2, respectively, that are known to express core fucosylated N-glycans, using AOL and LCA as positive controls. LCA and AOL are fucose specific lectins that are currently being used clinically for the diagnosis of hepatocellular carcinomas. Most of the gastrointestinal markers express core fucosylated N-glycans. The high affinity and exclusive specificity of CSL towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins, makes it a promising molecule that needs to be further explored for its application in the diagnosis of gastrointestinal cancer.     

Mitogenic lectins from Cephalosporium curvulum (CSL) and Aspergillus oryzae (AOL) mediate host–pathogen interactions leading to mycotic keratitis

A core-fucose-specific lectin, CSL from Cephalosporium curvulum, has been reported earlier. Here we assign the role for CSL and another lectin AOL, from pathogenic fungus Aspergillus oryzae, in causing mycotic keratitis. CSL and AOL show strong binding to immortalized and primary human corneal epithelial cells (HCECs) which are inhibited by asialofetuin, confirming their glycan-mediated binding. CSL and AOL showed increase in viability at lower concentrations (0.07 µg/ml) whereas at higher concentrations (0.15 µg/ml and 0.30 µg/ml), have inhibitory effect on immortalized HCECs. Lectin-mediated effect was comparable with the effect induced by the Colony Forming Units (CFUs) of C. curvulum and A. oryzae. CFUs induced more than 1.5-fold increase in HCECs proliferation. Both lectins and fungal CFUs induce secretion of proinflammatory cytokines IL6 and IL8 implicated in ocular diseases. This was supported by upregulation of TLR2 and 4 by lectins as revealed by flow cytometry and RT-PCR. CSL and AOL mediate host–pathogen interactions leading to mycotic keratitis. The mechanism of pathogenesis is possibly initiated through surface binding of mycelia through the lectins to TLR2/4 followed by upregulation of proinflammatory cytokines IL6, IL8 and TLR2 and 4. Understanding the mechanism of pathogenesis is of clinical significance in designing and developing therapeutic strategy to control the infection.