Therapeutic application of natural inhibitors against snake venom phospholipase A(2)

Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed.     

Molecular docking of glucosamine-6-phosphate synthase in Rhizopus oryzae

Recent expansion of immunocompromised population has led to significant rise in zygomycosis caused by filamentous fungus Rhizopus oryzae. Due to emergence of fungal resistance and side-effects of antifungal drugs, there is increased demand for novel drug targets. The current study elucidates molecular interactions of peptide drugs with G-6-P synthase (catalyzing the rate-limiting step of fungal cell wall biosynthetic pathway) of R.oryzae by molecular docking studies. The PDB structures of enzyme in R.oryzae are not known which were predicted using I-TASSER server and validated with PROCHECK. Peptide inhibitors, FMDP and ADGP previously used against enzyme of E.coli (PDBid: 1XFF), were used for docking studies of enzyme in R.oryzae by SchrödingerMaestro v9.1. To investigate binding between enzyme and inhibitors, Glide and Induced Fit docking were performed. IFD results of 1XFF with FMDP yielded C1, R73, W74, T76, G99 and D123 as the binding sites. C379 and Q427 appear to be vital for binding of R.oryzae enzymes to inhibitors. The comparison results of IFD scores of enzyme in R.oryzae and E.coli (PDBid: 2BPL) yield appreciable score, hinting at the probable effectiveness of inhibitors FMDP and ADGP against R.oryzae, with ADGP showing an improved enzyme affinity. Moreover, the two copies of gene G-6-P synthase due to extensive fungal gene duplication, in R. oryzae eliminating the problem of drug ineffectiveness could act as a potential antifungal drug target in R. oryzae with the application of peptide ligands.     

p-Hydroxyphenylpyruvate dioxygenase is a herbicidal target site for beta-triketones from Leptospermum scoparium

p-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism and is the molecular target site of β-triketone pharmacophores used to treat hypertyrosinemia in humans. In plants, HPPD is involved in the biosynthesis of prenyl quinones and tocopherols, and is the target site of β-triketone herbicides. The β-triketone-rich essential oil of manuka (Leptospermum scoparium), and its components leptospermone, grandiflorone and flavesone were tested for their activity in whole-plant bioassays and for their potency against HPPD. The achlorophyllous phenotype of developing plants exposed to manuka oil or its purified β-triketone components was similar to that of plants exposed to the synthetic HPPD inhibitor sulcotrione. The triketone-rich fraction and leptospermone were approximatively 10 times more active than that of the crude manuka oil, with I₅₀ values of 1.45, 0.96 and 11.5 μg mL⁻¹, respectively. The effect of these samples on carotenoid levels was similar. Unlike their synthetic counterpart, steady-state O₂ consumption experiments revealed that the natural triketones were competitive reversible inhibitors of HPPD. Dose-response curves against the enzyme activity of HPPD provided apparent I₅₀ values 15.0, 4.02, 3.14, 0.22 μg mL⁻¹ for manuka oil, triketone-rich fraction, leptospermone and grandiflorone, respectively. Flavesone was not active. Structure-activity relationships indicate that the size and lipophilicity of the side-chain affected the potency of the compounds. Computational analysis of the catalytic domain of HPPD indicates that a lipophilic domain proximate from the Fe²⁺ favors the binding of ligands with lipophilic moieties.     

Novel properties of antimicrobial peptide anoplin

Antimicrobial decapeptide anoplin was tested for its antifungal activity against plant pathogen Leptosphaeria maculans and protection of Brassica napus plants from disease. To reveal the mode of action of the peptide, a natural form of anoplin amidated on C-terminus (ANP-NH₂), and its carboxylated analog (ANP-OH) were used in the study. We demonstrated strong antifungal activity of anoplin in vitro regardless C-terminus modification. In addition we show that both ANP-NH₂ and ANP-OH induce expression of defence genes in B. napus and protects plants from L. maculans infection. The results indicate that the amidation of anoplin is not essential for its antifungal and plant defence stimulating activities.     

Chemical and biological explorations of the electrophilic reactivity of the bioactive marine natural product halenaquinone with biomimetic nucleophiles

The electrophilic reactivity of the bioactive marine sponge natural product halenaquinone has been investigated by reaction with the biomimetic nucleophiles N-acetyl-l-cysteine and N_α-acetyl-l-lysine. While cysteine reacted at the vacant quinone positions C-14 and C-15, lysine was found to react preferentially at the keto-furan position C-1. A small library of analogues was prepared by reaction of halenaquinone with primary amines, and evaluated against a range of biological targets including phospholipase A₂, farnesyltransferases (FTases) and Plasmodium falciparum. Geranylamine analogue 11 exhibited the most potent activity towards FTases (IC₅₀ 0.017-0.031 μM) and malaria (IC₅₀ 0.53-0.62 μM).     

Lysophosphatidylcholine as an effector of fatty acid-induced insulin resistance

The mechanism of FFA-induced insulin resistance is not fully understood. We have searched for effector molecules(s) in FFA-induced insulin resistance. Palmitic acid (PA) but not oleic acid (OA) induced insulin resistance in L6 myotubes through C-Jun N-terminal kinase (JNK) and insulin receptor substrate 1 (IRS-1) Ser307 phosphorylation. Inhibitors of ceramide synthesis did not block insulin resistance by PA. However, inhibition of the conversion of PA to lysophosphatidylcholine (LPC) by calcium-independent phospholipase A₂ (iPLA₂) inhibitors, such as bromoenol lactone (BEL) or palmitoyl trifluoromethyl ketone (PACOCF₃), prevented insulin resistance by PA. iPLA₂ inhibitors or iPLA₂ small interfering RNA (siRNA) attenuated JNK or IRS-1 Ser307 phosphorylation by PA. PA treatment increased LPC content, which was reversed by iPLA₂ inhibitors or iPLA₂ siRNA. The intracellular DAG level was increased by iPLA₂ inhibitors, despite ameliorated insulin resistance. Pertussis toxin (PTX), which inhibits LPC action through the G-protein coupled receptor (GPCR)/Gα_i, reversed insulin resistance by PA. BEL administration ameliorated insulin resistance and diabetes in db/db mice. JNK and IRS-1Ser307 phosphorylation in the liver and muscle of db/db mice was attenuated by BEL. LPC content was increased in the liver and muscle of db/db mice, which was suppressed by BEL. These findings implicate LPC as an important lipid intermediate that links saturated fatty acids to insulin resistance.     

Antifouling activity of synthetic γ-hydroxybutenolides

The desire to provide greener antifouling (AF) biocides for incorporation into marine paints has led to the discovery of many AF natural products. However, economical and sustainable supply of these natural products is often lacking. Twenty one structurally diverse γ-hydroxybutenolides – inspired by two natural AF sesterterpene γ-hydroxybutenolides (cavernosolide and lintenolide A) – were synthesized and tested for in vitro AF efficacy against the marine bryozoan larvae (Bugula neritina) and algae (Isochrysis galbana). The most potent analogues (EC₅₀ ca. 2 μg/mL) had AF activity comparable to the natural products (EC₅₀ ca. 0.6 μg/mL). Sites of attachment of side chains to the γ-hydroxybutenolide core structure had little effect on AF activity, however a clear relationship was established between lipophilicity and AF activity. Four analogues were chosen for field trials, incorporated into paints and deployed on panels in the marine environment. These analogues were chosen to reflect logistics of large scale synthesis and to provide representation of diverse structural motifs. Without any primary or co-biocides (except ZnO as a paint pigment), two analogues demonstrated a pronounced AF effect by resisting biofouling for over 18 weeks, compared to the ZnO control of between 7 and 10 weeks. Fine tuning of the structural motifs and/or the inclusion of co-biocides in paint formulations provide the potential to increase AF efficacy further.     

Roles of H2S in adaptation of alpine plants Lamiophlomis rotata to altitude gradients

Hydrogen sulfide (H₂S) is an important gaseous transmitter in organisims. It widespreads in the organs and tissues of animals and participates in the biological process of cardiovascular relaxation, cell apoptosis and protection, inflammation and neuromodulation. H₂S also can be synthesized in plants system and is involved in stress responses and the biological process of growth and development. This review describes the synthesis and biological function of H₂S in plants. Based on our research for the adaptation of Lamiophlomis rotata to different altitude gradients, we firstly proposed H₂S plays an important role in the adaptation of Lamiophlomis rotata to alpine environment.     

Synthesis,in vitro urease inhibition and molecular docking studies of some novel quinazolin-4(3H)-one derivatives containing triazole,thiadiazole and thiosemicarbazide functionalities

A new series of quinazolinone derivatives containing triazole, thiadiazole, thiosemicarbazide functionalities was synthesized and then screened for their in vitro urease inhibition properties. Most of the compounds showed excellent activity with IC₅₀ values ranging between 1.88 ± 0.17 and 6.42 ± 0.23 µg/mL, compared to that of thiourea (IC₅₀ = 15.06 ± 0.68) and acetohydroxamic acid (IC₅₀ = 21.03 ± 0.94), as reference inhibitors. Among the synthesized molecules, compounds 5c, 5e and 5a showed the best inhibitory effect against urease enzyme with IC₅₀ values of 1.88 ± 0.17 µg/mL, 1.90 ± 0.10 and 1.96 ± 0.07 µg/mL, respectively. Moreover in order to give better understanding of the inhibitory activity of synthesized compounds, molecular docking studies were applied at the target sites of jack bean urease enzyme (JBU). Their binding poses and energy calculations were analyzed using induced fit docking (IFD) and prime-MMGBSA tool. Binding poses of studied compounds were determined using induced fit docking (IFD) algorithms.     

Comparative Study of Antibiofilm Activity of Copper Oxide and Iron Oxide Nanoparticles Against Multidrug Resistant Biofilm Forming Uropathogens

The effectiveness of the metal oxide nanoparticles viz. CuO and Fe₂O₃ as antibacterial agents against multidrug resistant biofilm forming bacteria was evaluated. CuO nanoparticles were also experimented for antibiofilm and time kill assay. The CuO displayed maximum antibacterial activity with zone of inhibition of (22 ± 1) mm against methicillin resistant Staphylococcus aureus (MRSA) followed by Escherichia coli (18 ± 1) mm. The Fe₂O₃ showed the zone of inhibition against MRSA of (14 ± 1) mm followed by E. coli (12 ± 1) mm. CuO proved to be more toxic than Fe₂O₃ nanoparticles showing significantly high antibacterial activity and found to possess dose dependent antibiofilm properties.     

Paranoid potato: Phytophthora-resistant genotype shows constitutively activated defense

Phytophthora is the most devastating pathogen of dicot plants. There is a need for resistance sources with different modes of action to counteract the fast evolution of this pathogen. In order to better understand mechanisms of defense against P. infestans, we analyzed several clones of potato. Two of the genotypes tested, Sarpo Mira and SW93-1015, exhibited strong resistance against P. infestans in field trials, whole plant assays and detached leaf assays. The resistant genotypes developed different sizes of hypersensitive response (HR)-related lesions. HR lesions in SW93-1015 were restricted to very small areas, whereas those in Sarpo Mira were similar to those in Solanum demissum, the main source of classical resistance genes. SW93-1015 can be characterized as a cpr (constitutive expressor of PR genes) genotype without spontaneous microscopic or macroscopic HR lesions. This is indicated by constitutive hydrogen peroxide (H₂O₂) production and PR1 (pathogenesis-related protein 1) secretion. SW93-1015 is one of the first plants identified as having classical protein-based induced defense expressed constitutively without any obvious metabolic costs or spontaneous cell death lesions.     

RELATIONSHIPS BETWEEN HEART RATE AND PHYSIOLOGICAL PARAMETERS OF PERFORMANCE IN TOP-LEVEL WATER POLO PLAYERS

The aim of this study was to measure the heart rate (HR) response of eight elite water polo players during the four 7-min quarters of the game and to check for relationships with the physiological parameters of performance (V.O₂max, Th1_vent, Th2_vent). Each athlete performed a V.O₂max treadmill test and played a water polo game wearing a heart rate monitor. The game fatigue index was calculated as the ratio of the fourth-quarter HR to the first-quarter HR: HR₄/HR₁. The results showed a slight decrease in fourth-quarter HR compared with the first quarter, with the mean four-quarter HR equal to 79.9±4.2% of HR_max. Stepwise multiple regression analysis showed V.O₂max to be the main explanatory factor of game intensity, i.e. game HR expressed in %HR_reserve (R=0.88, P<0.01). We observed that higher aerobic capacity resulted in higher game intensity. We also observed a decrease in the playing intensity in the fourth quarter compared with the first, likely due to very high game involvement. We concluded that high aerobic capacity seems necessary to ensure high game intensity in water polo. This suggests that coaches should encourage their athletes to reach a minimum level of V.O₂max and that HR monitoring could be of great interest in the control of water polo training sessions.     

Growth of Arabidopsis seedlings on high fungal doses of Piriformospora indica has little effect on plant performance,stress, and defense gene expression in spite of elevated jasmonic acid and jasmonic acid-isoleucine levels in the roots

The endophytic fungus Piriformospora indica colonizes the roots of many plant species including Arabidopsis and promotes their performance, biomass, and seed production as well as resistance against biotic and abiotic stress. Imbalances in the symbiotic interaction such as uncontrolled fungal growth result in the loss of benefits for the plants and activation of defense responses against the microbe. We exposed Arabidopsis seedlings to a dense hyphal lawn of P. indica. The seedlings continue to grow, accumulate normal amounts of chlorophyll, and the photosynthetic parameters demonstrate that they perform well. In spite of high fungal doses around the roots, the fungal material inside the roots was not significantly higher when compared with roots that live in a beneficial symbiosis with P. indica. Fifteen defense- and stress-related genes including PR2, PR3, PAL2, and ERF1 are only moderately upregulated in the roots on the fungal lawn, and the seedlings did not accumulate H₂O₂/radical oxygen species. However, accumulation of anthocyanin in P. indica-exposed seedlings indicates stress symptoms. Furthermore, the jasmonic acid (JA) and jasmonic acid-isoleucine (JA-Ile) levels were increased in the roots, and consequently PDF1.2 and a newly characterized gene for a 2-oxoglurate and Fe²⁺-dependent oxygenase were upregulated more than 7-fold on the dense fungal lawn, in a JAR1- and EIN3-dependent manner. We conclude that growth of A. thaliana seedlings on high fungal doses of P. indica has little effect on the overall performance of the plants although elevated JA and JA-Ile levels in the roots induce a mild stress or defense response.     

Plant Sources of Chinese Herbal Remedies: Laboratory Efficacy,Suppression of Meloidogyne javanica in Soil,and Phytotoxicity Assays

Extracts of Chinese herbal medicines from plants representing 13 families were tested for their ability to suppress plant-parasitic nematodes. Effective concentration (EC₅₀ and EC₉₀) levels for 18 of the extracts were determined in laboratory assays with Meloidogyne javanica juveniles and all stages of Pratylenchus vulnus. Efficacy of 17 extracts was tested against M. javanica in soil. Generally, EC₅₀ and EC₉₀ values determined in the laboratory were useful indicators for application rates in the soil. Extracts tested from plants in the Liliaceae reduced galling of tomato by M. javanica and were not phytotoxic. Similarly, isothiocyanate-yielding plants in the Brassicaceae suppressed root galling without phytotoxicity. Other plant extracts, including those from Azadirachta indica, Nerium oleander, and Hedera helix, suppressed root galling but were phytotoxic at the higher concentrations tested. Many of these plant sources have been tested elsewhere. Inconsistency in results across studies points to the need for identification of active components and for determination of concentration levels of these components when plant residues or extracts are applied to soil.     

Lipase inhibitory activity of ethyl acetate fraction from Ecklonia cava extracts

Obesity is a key contributing risk factor to cardiovascular disease, certain cancers, and diabetes. Much effort has being made to investigate potential inhibitors against lipase from natural products. The ethyl acetate (EA) extract of Ecklonia cava (EC) were tested for their ability to inhibit pancreatic lipase activity in vitro. The 22 sub-fractions from EA extract were separated using silica gel column chromatography. Among the sub-fractions, the EA6 sub-fraction exhibited the highest inhibitory activity. Dieckol compound was isolated from the EA6 sub-fraction, which inhibited the lipase activity in a concentrationdependent manner with IC₅₀ value at 0.26 mg/mL. These results suggest that EC has potential as a natural antiobesity agent.     

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of blaNDM-1

New Delhi metallo-β-lactamase-1 gene (bla_NDM-1) codes for New Delhi metallo-beta-lactamase-1 (NDM-1) enzyme that cleaves the amide bond of β-lactam ring, and provides resistance against major classes of β-lactam antibiotics. Dissemination of the plasmid borne bla_NDM-1 through horizontal gene transfer is a potential threat to the society. In this study, a rapid non-culture method for detecting NDM-1 positive bacteria was developed by Loop Mediated Isothermal Amplification (LAMP) of bla_NDM-1. Sensitivity of this method was found to be one femtogram of plasmid DNA, which translates into 2.6–25.8 copies depending on the size of the plasmid DNA. This method was applied to detect NDM-1 positive bacteria in 81 water samples that were collected from environmental and drinking water sources. NDM-1 positive bacteria were detected in three drinking water samples by LAMP but not by PCR. These three samples were collected from the water sources that were treated with chlorine for decontamination before public distribution. NDM-1 positive bacteria were not detected in lake water samples or in the samples that were collected from the water sources that were purified by reverse osmosis before public distribution. Detection of NDM-1 positive bacteria using LAMP was found to be safe, sensitive and rapid for screening large number of samples from diverse sources. This method could be developed as on-field detection kit by using fluorescent dyes to visualize the amplified bla_NDM-1 gene.     

Inherited phenotype instability of inflorescence and floral organ development in homeotic barley double mutants and its specific modification by auxin inhibitors and 2,4-D

Background and Aims Barley (Hordeum vulgare) double mutants Hv-Hd/tw₂, formed by hybridization, are characterized by inherited phenotypic instability and by several new features, such as bract/leaf-like structures, long naked gaps in the spike, and a wide spectrum of variations in the basic and ectopic flowers, which are absent in single mutants. Several of these features resemble those of mutations in auxin distribution, and thus the aim of this study was to determine whether auxin imbalances are related to phenotypic variations and instability. The effects of auxin inhibitors and 2,4-D (2,4-dichlorophenoxyacetic acid) on variation in basic and ectopic flowers were therefore examined, together with the effects of 2,4-D on spike structure.Methods The character of phenotypic instability and the effects of auxin inhibitors and 2,4-D were compared in callus cultures and intact plants of single homeotic Hv-tw₂ and Hv-Hooded/Kap (in the BKn3 gene) mutants and alternative double mutant lines: offspring from individual plants in distal hybrid generations (F₉–F₁₀) that all had the same BKn3 allele as determined by DNA sequencing. For intact plants, two auxin inhibitors, 9-hydroxyfluorene-9-carboxylic acid (HFCA) and p-chlorophenoxyisobutyric acid (PCIB), were used.Key Results Callus growth and flower/spike structures of the Hv-tw₂ mutant differed in their responses to HFCA and PCIB. An increase in normal basic flowers after exposure to auxin inhibitors and a decrease in their frequencies caused by 2,4-D were observed, and there were also modifications in the spectra of ectopic flowers, especially those with sexual organs, but the effects depended on the genotype. Exposure to 2,4-D decreased the frequency of short gaps and lodicule transformations in Hv-tw₂ and of long naked gaps in double mutants.Conclusions The effects of auxin inhibitors and 2,4-D suggest that ectopic auxin maxima or deficiencies arise in various regions of the inflorescence/flower primordia. Based on the phenotypic instability observed, definite trends in the development of ectopic flower structures may be detected, from insignificant outgrowths on awns to flowers with sterile organs. Phenotypically unstable barley double mutants provide a highly promising genetic system for the investigation of gene expression modules and trend orders.