Structural investigations into the binding mode of novel neolignans Cmp10 and Cmp19 microtubule stabilizers by in silico molecular docking,molecular dynamics,and binding free energy calculations

Microtubule stabilizers provide an important mode of treatment via mitotic cell arrest of cancer cells. Recently, we reported two novel neolignans derivatives Cmp10 and Cmp19 showing anticancer activity and working as microtubule stabilizers at micromolar concentrations. In this study, we have explored the binding site, mode of binding, and stabilization by two novel microtubule stabilizers Cmp10 and Cmp19 using in silico molecular docking, molecular dynamics (MD) simulation, and binding free energy calculations. Molecular docking studies were performed to explore the β-tubulin binding site of Cmp10 and Cmp19. Further, MD simulations were used to probe the β-tubulin stabilization mechanism by Cmp10 and Cmp19. Binding affinity was also compared for Cmp10 and Cmp19 using binding free energy calculations. Our docking results revealed that both the compounds bind at Ptxl binding site in β-tubulin. MD simulation studies showed that Cmp10 and Cmp19 binding stabilizes M-loop (Phe272-Val288) residues of β-tubulin and prevent its dynamics, leading to a better packing between α and β subunits from adjacent tubulin dimers. In addition, His229, Ser280 and Gln281, and Arg278, Thr276, and Ser232 were found to be the key amino acid residues forming H-bonds with Cmp10 and Cmp19, respectively. Consequently, binding free energy calculations indicated that Cmp10 (?113.655 kJ/mol) had better binding compared to Cmp19 (?95.216 kJ/mol). This study provides useful insight for better understanding of the binding mechanism of Cmp10 and Cmp19 and will be helpful in designing novel microtubule stabilizers.     

Restriction endonuclease resistant chromatin in human chromosomes

Summary The recent addition of restriction endonucleases in obtaining selective bands in the human genome has added a new dimension to molecular genetics. However, a considerable discrepancy exists in banding patterns produced by AluI in chromosomes 19 and 20, by MboI in chromosomes 4, 5, 8, 21 and 22 and by RsaI in chromosomes 12, 21 and 22. The principal causes of these differences are highlighted.     

Enhanced degradation of gamma-irradiated lignocelluloses by a new xylanolytic Flavobacterium sp. isolated from soil

An extracellular chitosanase produced by Rhodotorula gracilis CFR-1 that catalyses a limited degradation of chitosan with no detectable generation of glucosamine or reducing groups was identified. Ultracentrifugation, polyacrylamide gel electrophoresis and gel permeation studies suggest that chitosan of average molecular mass 36000 Da was reduced by the enzymic catalysis to nearly one-fourth this size without further hydrolysis of the products. The enzyme, produced constitutively by this yeast, was partially purified and some of its properties were studied.     

Biomass, Productivity and Energetics in Himalayan Alder Plantations

Biomass, net primary production and energy fixation in an agesequence of Himalayan alder (Alnus nepalensis D. Don) plantationswere estimated in the Kalimpong forest division of the easternHimalayas. Biomass in the plantations ranged from 106 t ha^–1(7-year stand) to 606 t ha^–1 (56-year stand) demonstratingthe potential of the alder for accumulating large biomass. Netprimary production and net energy fixation rates of the plantationswere reduced by nearly half in the 7-year stand (25 t ha^–1year^–1; 421 x 10⁶ kJ ha^–1 year^–1) comparedwith the 56-year stand (13 t ha^–1 year^–1; 215 x10⁶ kJ ha^–1 year^–1). Compartmental models of energystorage and flow rates were developed for the 7-year and 56-yearstands. The production efficiency, energy conversion efficiencyand energy efficiency in N₂ fixation have inverse relationshipswith plantation age. These efficiencies, when treated with eachother, showed significant exponential functions. Alnus nepalensis D. Don, Himalayan alder, plantation age, biomass, net primary production, energy flow, efficiencies     

Suppression of fungal β-glucan-induced plant defence in soybean (Glycine max L.) by cyclic 1,3-1,6-β-glucans from the symbiont Bradyrhizobium japonicum

The production of antimicrobial phytoalexins is one of the best-known inducible defence responses following microbial infection of plants or treatment with elicitors. In the legume soybean (Glycine max L.), 1,3-1,6--glucans derived from the fungal pathogen Phytophthora sojae have been identified as potent elicitors of the synthesis of the phytoalexin, glyceollin. Recently it has been reported that during symbiotic interaction between soybean and the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110 the bacteria synthesize cyclic 1,3-1,6--glucans. Here we demonstrate that both the fungal and the bacterial -glucans are ligands of -glucan-binding sites which are putative receptors for the elicitor signal compounds in soybean roots. Whereas the fungal -glucans stimulate phytoalexin synthesis at low concentrations, the bacterial cyclic 1,3-1,6--glucans appear to be inactive even at relatively high concentrations. Competition studies indicate that increasing concentrations of the bacterial 1,3-1,6--glucans progressively inhibit stimulation of phytoalexin synthesis in a bioassay induced by the fungal 1,3-1,6--glucans. Another type of cyclic -glucan, a 1,2--glucan from Rhizobium meliloti, that does not nodulate on soybean, seems to be inactive as elicitor and as ligand of the -glucan-binding sites. These results may indicate a novel mechanism for a successful plant-symbiont interaction by suppressing the plant's defence response.Abbreviations HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-l-[hexaglucosyl]deoxyglucitol - HG-AzPEA l-[2-(4-azidophenyl)-ethyl]amino-l-[hexaglucosyl]deoxyglucitol - IC₅₀ concentration for half-maximal displacement We thank Ines Arlt for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 369), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Fonds der Chemischen Industrie (J.E.), and USDA CSRS NRI Competitive Research grant 93373059233 (A.A.B.).     

DETECTION OF LISTERIA MONOCYTOGENES BY ADSORPTION AND ELUTION FROM HYDROPHOBIC MATRICES

The binding of L. monocytogenes Scott A strain to three hydrophobic matrices, octyl, phenyl and butyl Sepharose, was investigated. Optimal adsorption of L. monocytogenes to octyl Sepharose was obtained at pH 3.5 and 4 M NaCl. However, it was difficult to elute the bacteria from octyl Sepharose, even after changing the pH and lowering the salt concentration. Good adsorption of L. monocytogenes to phenyl Sepharose at pH 3.5 and 4 M NaCl was also observed. L. monocytogenes was found to adsorb weakly to butyl Sepharose, which is less hydrophobic than phenyl Sepharose. Bacteria were eluted under various conditions. The best elution was obtained with 10 mM sodium phosphate, followed by an increasing gradient of ethylene glycol. To test the potential application of hydrophobic chromatography for separating L. monocytogenes from food matrices, milk was inoculated with L. monocytogenes and then passed through a column of phenyl Sepharose at pH 3.5 and 4 M NaCl. Nearly all L. monocytogenes were bound to the hydrophobic gel and were eluted in a pure and viable form by changing the pH and lowering the salt concentration, and by using a polar reducing agent, ethylene glycol. This study shows that hydrophobic interaction chromatography can be used to separate L. monocytogenes from milk and may be applicable to other food suspensions. It is a gentle method that makes use of the hydrophobic surface properties of Listeria for attachment to hydrophobic gels, as well as using mild elution conditions to avoid inactivation of the organism.     

A greenhouse technique to screen pigeonpea for resistance to Heterodera cajani*

Heterodera cajani is an important nematode pest of pigeonpea in India and a simple and reliable greenhouse procedure has been developed to screen pigeonpea genotypes for resistance to it. In pot experiments, white cysts of H. cajani were counted on the roots of the susceptible genotype ICPL 87 at 15, 30 and 45 days after seedling emergence in soils infested with different levels of H. cajani. The seedlings were rated for the number of white cysts per root system on a one (highly resistant, no cysts) to nine (highly susceptible, more than 30 cysts) scale. White cysts were not easy to see on wet roots but were clearly visible on slightly dried roots. Cyst counts and ratings were more uniform when roots of 30 day old seedlings were evaluated than when 15 or 45 day old seedlings were examined. Effects of different H. cajani infestation levels on the ratings were not significant although the use of higher inoculum densities (16 to 27 eggs and juveniles/cm³ soil) was effective in reducing variability. This procedure was used to screen 60 pigeonpea genotypes and all of them were rated seven or nine. Ten accessions of Atylosia spp. and Rhynchosia spp. were rated three.     

Factors influencing oviposition behaviour of the sorghum midge, Contarinia sorghicola Coq.

Visual stimuli influence the orientation behaviour of the sorghum midge, Contarinia sorghicola Coq. (Diptera: Cecidomyiidae). Yellow, red and white colours are attractive to the midge while blue and black are least attractive. Sorghum panicles covered with blue- or black-coloured bags in a headcage showed maximum midge damage, while the reverse was true for panicles covered with yellow, red, and white coloured bags.
Panicles at half-anthesis with viable pollen and receptive stigmata suffered higher damage than those at the pre- and post-anthesis. Physical removal of anthers and stigmata significantly reduced the oviposition by the sorghum midge. Reduced oviposition/adult emergence was also recorded in male sterile sorghum lines (2219A and 296A) or through chemically- (Ethrel) (2-Chloro ethyl-phosphonic acid) induced male sterility in panicles of the sorghum cultivar, Swarna. Chemical stimuli from viable pollen and receptive stigmata and to a limited extent physical stimuli, govern the oviposition behaviour of the sorghum midge.
Sorghum cultivars IS 12573C, S-GIRL-MR1 and IS 2816C showed antixenosis to adult midges. However, these cultivars became susceptible under no-choice conditions in the headcage. DJ 6514 and IS 12666C were attractive to the adult midges, but showed antixenosis to oviposition under natural and no-choice conditions. Genotypes with short florets showed antixenosis for oviposition. Ovary and anther breadth and tannin content of grain showed negative associations with oviposition. Cultivar antixenosis to adult midges and oviposition is an important component of resistance to the sorghum midge.     

Human Epidermal Melanocyte and Keratinocyte Melanocortin Receptors: Visualization by Melanotropic Peptide Conjugated Microspheres (Latex Beads)

The objectives of this research were to determine whether melanocortin receptors are characteristic (constant) membrane markers of human epidermal melanocytes. Methodologies were developed to visualize melanotropin receptors by scanning electron microscopy (SEM). Multiple copies (up to a hundred) of [Nle⁴,D-Phe⁷]α-MSH, a superpotent analog of α-melanocyte stimulating hormone (α-MSH), were conjugated to a macromo-lecular carrier (latex beads: microspheres). Incubation in the presence of the melanotropin-conjugated microspheres resulted in binding of human normal epidermal melanocytes to the beads. Almost every (possibly all) melanocyte possesses melanocortin receptors as visualized by SEM. Specificity of binding of the macromolecular conjugate was demonstrated by several studies: 1) Binding of melanocytes to the microspheres was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; 2) microspheres lacking bound ligand did not bind to the melanocytes; 3) micro-spheres that were first treated with reducing agents (e.g., dithiothreitol) did not subsequently bind to melanocytes; 4) another peptide hormone ligand (e.g., a substance-P analog) attached to the latex beads failed to bind to the cells; 5) B16/F10 mouse melanoma cells known to express melanocortin receptors bound to the microspheres; and 6) cells of nonmelanocyte origin (e.g., mammary cancer cells, small-cell lung cancer cells, fibroblasts) did not bind to the macromolecular conjugate. One exception was that human epidermal keratinocytes also expressed melanocortin receptors as determined by all the criteria established above for epidermal melanocytes. Thus, cell specific melanocortin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.