Identification of inhibitors of the E. coli chaperone SurA using in silico and in vitro techniques

SurA is a gram-negative, periplasmic chaperone protein involved in the proper folding of outer membrane porins (OMPs), which protect bacteria against toxins in the extracellular environment by selectively regulating the passage of nutrients into the cell. Previous studies demonstrated that deletion of SurA renders bacteria more sensitive to toxins that compromise the integrity of the outer membrane. Inhibitors of SurA will perturb the folding of OMPs, leading to disruption of the outer membrane barrier and making the cell more vulnerable to toxic insults. The discovery of novel SurA inhibitors is therefore of great importance for developing alternative strategies to overcome antibiotic resistance. Our laboratory has screened over 10,000,000 compounds in silico by computationally docking these compounds onto the crystal structure of SurA. Through this screen and a screen of fragment compounds (molecular weight?less than?250?g/mol), we found twelve commercially readily available candidate compounds that bind to the putative client binding site of SurA. We confirmed binding to SurA by developing and employing a competitive fluorescence anisotropy-based binding assay. Our results show that one of these compounds, Fmoc-β-(2-quinolyl)-d-alanine, binds the client binding site with high micromolar affinity. Using this compound as a lead, we also discovered that Fmoc-l-tryptophan and Fmoc-l-phenylalanine, but not Fmoc-l-tyrosine, bind SurA with similar micromolar affinity. To our knowledge, this is the first report of a competitive fluorescence anisotropy assay developed for the identification of inhibitors of the chaperone SurA, and the identification of three small molecules that bind SurA at its client binding site.     

Screening of anti-inflammatory phytocompounds from Crateva adansonii leaf extracts and its validation by in silico modeling

Anti-inflammatory phytocompounds from Crateva adansonii DC leaf extracts were identified by GCMS analysis and its anti-inflammatory potential was evaluated by in silico molecular docking study against inflammatory molecular targets. Three different (Aqueous, Methanol and Petroleum ether) dried leaf extracts of Crateva adansonii were obtained from soxhlet extraction method. Preliminary phytoconstituents analysis of three different leaf extracts of C. adansonii confirmed the presence of various major classes of bioactive phytoconstituents such as polyphenols (tannins and flavonoids), steroids, alkaloid, coumarin, carbohydrate and terpenoids. Among three leaf extracts, methanolic leaf extract possess highest total phenolic content of 77?±?1.65?µg gallic acid equivalent (GAE)/g of dry weight of leaf extract, subsequently methanolic leaf extract also shows maximal in vitro antioxidant activity (DPPH scavenging activity) of 71.22?±?1.32% among three different leaf extracts. GC–MS analysis of petroleum ether leaf extract revealed the presence of nine phytocompounds representing 95.43% peak area percentage, among nine identified phytocompounds three phytocompounds of C. adansonii possess anti-inflammatory property namely phytol, 1-Hexyl-2-Nitrohexane and 2-Isopropyl-5-Methylcyclohexyl 3-(1-(4-Chlorophenyl)-3-Oxobutyl)-Coumarin-4-Yl Carbonate were chosen for in silico molecular docking study against four inflammatory receptor targets (COX-2, TNFα, IL-1β and IL-6) and they shows less binding energy with highest docking score ranging from ?15.9500 to 5.0869. The present study substantially indicated and proven that anti-inflammatory potential of phytocompounds from C. adansonii leaf extracts which can be exploited for commercial designing of novel anti-inflammatory drug to treat various inflammatory disorders.     

Suppression of histone deacetylase 3 (HDAC3) enhances apoptosis induced by paclitaxel in human maxillary cancer cells in vitro and in vivo

Inclusion of chemotherapeutic drugs in treatment of patients with newly diagnosed head and neck cancer has improved response rates and prolonged median survival. Nevertheless, most patients with advanced head and neck cancer are destined to relapse and to develop resistance to initially used drugs such as paclitaxel. Consequently, it has been more important in cancer therapy to determine the molecular mechanisms that are related to cell-killing effects of anti-cancer agents or cancer resistance against them. Consequently, we examined whether abrogation of histone deacetylase 3 (HDAC3) expression by anti-sense oligonucleotides (ASOs) potentiates the efficacy of paclitaxel in human maxillary cancer IMC-3 cells. Here, we showed that paclitaxel-induced apoptosis was enhanced significantly by addition of ASOs for HDAC3 in cultured cells. Furthermore, paclitaxel-induced apoptosis in IMC-3 tumors transplanted in nude mice was enhanced significantly by administration of ASOs for HDAC3, thereby suppressing tumor growth. We provide new evidence that HDAC3 is a novel molecular target whose inactivation can potentiate the efficacy of anti-cancer drugs disrupting microtubules such as paclitaxel.     

ECRG2, a novel candidate of tumor suppressor gene in the esophageal carcinoma,interacts directly with metallothionein 2A and links to apoptosis

Esophageal cancer related gene 2 (ECRG2) is a novel candidate of the tumor suppressor gene identified from human esophagus. To study the biological role of the ECRG2 gene, we performed a GAL4-based yeast two-hybrid screening of a human fetal liver cDNA library. Using the ECRG2 cDNA as bait, we identified nine putative clones as associated proteins. The interaction of ECRG2 and metallothionein 2A (MT2A) was confirmed by glutathione S-transferase pull-down assays in vitro and co-immunoprecipitation experiments in vivo. ECRG2 co-localized with MT2A mostly to nuclei and slightly to cytoplasm, as shown by confocal microscopy. Transfection of ECRG2 gene inhibited cell proliferation and induced apoptosis in esophageal cancer cells. In the co-transfection of ECRG2 and MT2A assays, cell proliferation was inhibited and apoptosis was slightly induced compared with control groups. When we used antisense MT2A to interdict the effect of MT2A, the inhibition of cell proliferation and induction of apoptosis were significantly enhanced. When we used antisense ECRG2 to interdict the effect of ECRG2 in the group of Bel7402 cells co-transfected with ECRG2 and MT2A, the inhibition of cell proliferation and induction of apoptosis disappeared. The results provide evidence for ECRG2 in esophageal cancer cells acting as a bifunctional protein associated with the regulation of cell proliferation and induction of apoptosis. ECRG2 might reduce the function of MT2A on the regulation of cell proliferation and induction of apoptosis. The physical interaction of ECRG2 and MT2A may play an important role in the carcinogenesis of esophageal cancer.     

Design,synthesis, and structure-activity relationships of new benzofuro[3,2-b]pyridin-7-ols as DNA topoisomerase II inhibitors

Human DNA topoisomerases have become attractive targets for developing more effective anticancer drugs. In this study, a series of new benzofuro[3,2-b]pyridin-7-ols were designed and synthesized for the first time and screened for their topoisomerase I and II inhibitory and antiproliferative activity. Structure-activity relationships revealed the position of ortho- and para-hydroxyl group at 2-phenyl ring, and meta-hydroxyl group at 4-phenyl ring of benzofuro[3,2-b]pyridin-7-ol are important for potent and selective topo II inhibitory activity. Compound 11 showed the most selective and potent topo II inhibition (100% inhibition at 100?µM) and strongest antiproliferative activity (IC₅₀?=?0.86?µM) than all the positive controls in HeLa cell line.     

Anticancer activity in vitro of a fucoidan from the brown alga Fucus evanescens and its low-molecular fragments, structurally characterized by tandem mass-spectrometry

Fucoidan was isolated and purified from the brown algae Fucus evanescens and subjected to autohydrolysis to obtain low-molecular-weight fragments. MALDI-TOFMS analysis has shown that monosulfated fucose and more heavily sulfated (up to 5) fucooligosaccharides with polymerization degree (DP) 2, 4 and 6, including galactose-containing sulfated oligosaccharides were the products of autohydrolysis. The structural features of these fragments were elucidated by negative-ion potential lift tandem MALDI-TOF mass-spectrometry using arabinoosazone as a matrix, which allowed reducing the in-source fragmentation. Native fucoidan has been shown to exert anticancer activities in both human malignant melanoma cell lines SK-MEL-28 and SK-MEL-5. Low-molecular-weight fragments exhibited almost no action to cell proliferation in both cell lines and colony formation on SK-MEL-5 cells, but its inhibition activity to the colony formation of SK-MEL-28 cell lines was as high as was demonstrated by native fucoidan (70%). Probably, the inhibiting activity for SK-MEL-28 depended on the presence of sulfates and (1 → 4)-linked α-l-Fucp residues in the main chain of fucoidan/oligosaccharides.     

Synthesis, structure and characterization of novel Cd(II) and Zn(II) complexes with the condensation product of 2-formylpyridine and selenosemicarbazide: Antiproliferative activity of the synthesized complexes and related selenosemicarbazone complexes

Two novel Cd(II) and Zn(II) complexes with the condensation product of 2-formylpyridine and selenosemicarbazide were synthesized. The structure of Cd(II) complex was determined by X-ray crystallography. The ligand is coordinated in a neutral form via pyridine and azomethine nitrogen atoms and the selenium donor. The cadmium ion completes its five-coordination by two chloride ligands, forming a square-pyramidal geometry. The structure of Zn(II) complex was established by analysis of spectroscopic data, which indicated coordination of the ligand as a bidentate via the selenium and the azomethine nitrogen atoms. The cytotoxic activity of the newly synthesized complexes, as well as if five structurally related complexes and the ligand evaluated against eight tumor cell lines. The new Cd(II) complex showed the highest activity similar to cisplatin with IC50 less than 10 μM for all cell lines. Cell cycle distribution and apoptosis study showed that Cd(II) complex and cisplatin might have some similarity in anticancer activity, which was not the case for cisplatin and other studied complexes. Effects of the complexes on matrix metalloproteinases (MMPs) MMP-9 and MMP-2 was also studied. Cd(II) and Zn(II) complexes and cisplatin increased MMP-2 activity in supernatants of tested cells, while Ni(II) complex with the same ligand decreased the activity, implying a possible activity in preventing tumor invasion and metastasis processes.     

The proteins encoded by the Drosophila Planar Polarity Effector genes inturned, fuzzy and fritz interact physically and can re-pattern the accumulation of “upstream” Planar Cell Polarity proteins

The frizzled/starry night pathway regulates planar cell polarity in a wide variety of tissues in many types of animals. It was discovered and has been most intensively studied in the Drosophila wing where it controls the formation of the array of distally pointing hairs that cover the wing. The pathway does this by restricting the activation of the cytoskeleton to the distal edge of wing cells. This results in hairs initiating at the distal edge and growing in the distal direction. All of the proteins encoded by genes in the pathway accumulate asymmetrically in wing cells. The pathway is a hierarchy with the Planar Cell Polarity (PCP) genes (aka the core genes) functioning as a group upstream of the Planar Polarity Effector (PPE) genes which in turn function as a group upstream of multiple wing hairs. Upstream proteins, such as Frizzled accumulate on either the distal and/or proximal edges of wing cells. Downstream PPE proteins accumulate on the proximal edge under the instruction of the upstream proteins. A variety of types of data support this hierarchy, however, we have found that when over expressed the PPE proteins can alter both the subcellular location and level of accumulation of the upstream proteins. Thus, the epistatic relationship is context dependent. We further show that the PPE proteins interact physically and can modulate the accumulation of each other in wing cells. We also find that over expression of Frtz results in a marked delay in hair initiation suggesting that it has a separate role/activity in regulating the cytoskeleton that is not shared by other members of the group.     

Simultaneous study of particle reworking, irrigation transport and reaction rates in sediment bioturbated by the polychaetes Heteromastus and Marenzelleria

The present study employed simultaneously methods to investigate particle and solute transport and reaction rates in sandy sediments inhabited by two worms (2500 m^− 2) with different feeding modes. Heteromastus filiformis is a head-down deposit-feeder and the main activities exerted by this worm are transport of particles as faecal pellets from subsurface to surface sediments and burrow ventilation. Marenzelleria viridis is a surface deposit-feeder that actively searches for food by burrowing near the sediment surface, producing a network of ventilated galleries in this zone. M. viridis exhibited 1.5 to 2.2 times higher particle mixing rates (D_b = 3.3 to 4 × 10^− 3 cm^− 2 d^− 1) compared to H. filiformis. In M. viridis treatments, continuous advection (eddy diffusion) was the major factor influencing solute transport resulting in apparent diffusion rates (D_a = 2.2 cm^− 2 d^− 1), which were 3 times higher than molecular diffusion within the sediment. In H. filiformis inhabited sediments, the transport of solutes was discontinuous and driven by a surprisingly high nonlocal exchange (α = 1.1-1.3 d^− 1), emphasizing its strong irrigation effects. Accordingly, the enhancement of solute fluxes was more pronounced for H. filiformis compared to M. viridis. Depth integrated TCO₂ production derived from diagenetic modelling, which takes into account three reaction zones, is in good agreement with rates obtained from measured fluxes, indicating the applicability of both approaches to get reliable rates. However, the reaction rates showed that the presence of animals had a modest effect on microbial carbon oxidation. The results proved that transport conditions are deeply related to feeding modes. Exchange of solutes was the most important transport process by H. filiformis, while M. viridis affected both mixing and solute transport.     

NADPH fluorescence in the cyanobacterium Synechocystis sp. PCC 6803: A versatile probe for in vivo measurements of rates,yields and pools

We measured the kinetics of light-induced NADPH formation and subsequent dark consumption by monitoring in vivo its fluorescence in the cyanobacterium Synechocystis PCC 6803. Spectral data allowed the signal changes to be attributed to NAD(P)H and signal linearity vs the chlorophyll concentration was shown to be recoverable after appropriate correction. Parameters associated to reduction of NADP⁺ to NADPH by ferredoxin–NADP⁺-oxidoreductase were determined: After single excitation of photosystem I, half of the signal rise is observed in 8 ms; Evidence for a kinetic limitation which is attributed to an enzyme bottleneck is provided; After two closely separated saturating flashes eliciting two photosystem I turnovers in less than 2 ms, more than 50% of the cytoplasmic photoreductants (reduced ferredoxin and photosystem I acceptors) are diverted from NADPH formation by competing processes. Signal quantitation in absolute NADPH concentrations was performed by adding exogenous NADPH to the cell suspensions and by estimating the enhancement factor of in vivo fluorescence (between 2 and 4). The size of the visible (light-dependent) NADP (NADP⁺ + NADPH) pool was measured to be between 1.4 and 4 times the photosystem I concentration. A quantitative discrepancy is found between net oxygen evolution and NADPH consumption by the light-activated Calvin–Benson cycle. The present study shows that NADPH fluorescence is an efficient probe for studying in vivo the energetic metabolism of cyanobacteria which can be used for assessing multiple phenomena occurring over different time scales.     

Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells

Background

The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNA-related lectins induce cell death are still only rudimentarily understood.

Methods

In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)-induced L929 cell apoptosis.

Results

Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis.

General significance

These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations.     

Ultrastructural changes in the cuticle of the sheep blowfly, Lucilia, induced by certain insecticides and biological inhibitors

The ultrastructural effects on larval cuticle of Lucilia cuprina of two inhibitors of chitin synthesis, diflubenzuron and polyoxin D and an inhibitor of dihydrofolate reductase, aminopterin, are compared with those of the insecticide, cyromazine. Diflubenzuron and polyoxin D both prevent the formation of a normal lamellate appearance in procuticle and interfere with deposition of epicuticle. Aminopterin and cyromazine cause necrotic lesions in the cuticle which, in the case of cyromazine, are contiguous with invasive processes of epidermal cells. There is an accumulation of electron-dense granules in some epidermal cells in larvae poisoned with aminopterin or cyromazine. Aminopterin has a more drastic cytotoxic effect than cyromazine and it also interferes with the formation of epicuticle. The lesions produced by cyromazine treatment are not mimicked precisely by any of the other chemicals. However, there is closer accord between the effects of cyromazine and aminopterin than between cyromazine and the inhibitors of chitin formation.     

Tarsal morphology and attachment ability of the codling moth Cydia pomonella L. (Lepidoptera, Tortricidae) to smooth surfaces

Despite several studies on the attachment ability of different insect taxa, little is known about this phenomenon in adult Lepidoptera. In this study we combined morphological and experimental analyses of tarsal adhesive devices and the attachment ability of the codling moth Cydia pomonella (L.) (Lepidoptera, Tortricidae) to smooth surfaces. Pretarsi of C. pomonella attach to smooth substrates by means of their smooth, flexible and well developed arolia. Using the centrifugal force measurement technique, friction forces of males and females were assessed on hydrophobic and hydrophilic glass surfaces. Adults of both sexes generated similar forces in spite of the noticeable difference in their body masses. That is why males showed significantly higher safety factors (attachment force divided by body weight) compared to those of females. Hydrophobicity of the substrate had no considerable effect on friction forces. For females, friction forces (sliding parallel to the substrate plane) were compared with adhesive forces (pulling off perpendicularly from the substrate plane) measured on Plexiglas surfaces. It can be concluded that the attachment system of C. pomonella is rather robust against physico-chemical properties of the substrate and is able to achieve a very good attachment on vertical and horizontal substrata.