Florigenic Acid from fungal culture

The extraction procedures which have been successfully employed in the preparation of a florigenic principle from the tissues of Xanthium, are applicable to the derivation of an entity of similar activity from Calonectria culture. The Xanthium principle is acedic, with pK_a values characteristic of a carboxylic acid (6). Although definitive chemical comparisons have not been completed, the extraction and solvent partition procedures that have been applicable to the extraction of the active entity from higher plant tissues have yielded florigenic preparations from fungal culture. The chemical principle from higher plant tissue may be the same or similar to the florigenic agent of Calonectria.

The many responses of higher plants to growth regulators produced by micro-organisms are well known. The presence of a flower-producing principle from Calonectria (Fusarium) rigidiuscula parallels closely the pattern exhibited by those fungal species capable of the production of auxin and gibberellins.

     

Chemical studies of a factor which elicits feeding behaviour in Dugesia dorotocephala

Extracts of brine shrimp have been studied and chemically fractionated in order to clarify the nature of the compounds which elicit feeding behaviour in Dugesia dorotocephala. The active principle is heat stable and has a molecular weight between 700 and 2000. Neither oxidation with hydrogen peroxide nor digestion with proteolytic enzymes destroyed the factor, although activity was lost after hydrolysis with acid. The factor has been partially purified. The available data suggest that the factor active with D. dorotocephala is larger in molecular weight, and probably chemically more complex, than those active in the coelenterates. At all stages of purification, the factor elicits the entire feeding behaviour of D. dorotocephala. No evidence was obtained for the existence of more than a single factor.     

Effects of a bacteriocin produced by Streptococcus faecalis var. zymogenes (E-1) on susceptible microorganisms

The bacteriocin produced by Streptococcus faecalis var. zymogenes (E-1) is most active against Diplococcus pneumoniae and least against other strains of S. faecalis. Clostridium perfringens showed an intermediate susceptibility to the active principle. By utilizing the gas production of C. perfringens as an indicator of metabolic activity, a decrease in sensitivity to bacteriocin was demonstrated with aging of the culture. Non-viable pinpoint clostridial colonies frequently developed by exposure of C. perfringens to a 2 or 3 hr old E-1 broth culture. The action of E-1 as studied on C. perfringens appears to be bactericidal and only partially bacteriolytic. The extent of E-1 bactericidal activity on susceptible D. pneumoniae, C. perfringens, and S. faecalis was shown to be dependent upon bacteriocin concentration.     

Genkwadaphnin,a potent antileukemic diterpene from Daphne genkwa

In vivo P-388 assay-directed fractionation of an active extract of Daphne genkwa (Yuán Huā) has led to the isolation and characterization of a new antileukemic principle. genkwadaphnin.     

Bioautography of cell attachment proteins.

A simple method, termed bioautography, is described which permits the visualization of bands of biologically active collagen-dependent cell attachment protein (c-CAP) after gel electrophoresis. The principle of bioautography depends on the “staining” of electrophoretically separated c-CAP's by live mammalian cells. Bioautography demonstrates (a) two bands of cell attachment protein (c-CAP) in serum; and (b) electrophoretic mobility differences between c-CAP's derived from the sera of various mammalian species.     

The synthesis of immunomodulating peptide alloferon, the active principle of antiviral drug allokine-alpha

Two variants of the synthesis of tridecapeptide alloferon, the active principle of antiviral preparation allokine-alpha, were developed on the basis of fragment condensation in solution or on the Merrifield resin. The solid phase variant of the synthesis was shown to be more technological; it allows the preparation of the product at a higher total yield (40% vs. 17% for conventional synthesis in solution from the starting derivatives of the C-terminal dipeptide). The by-products formed during the synthesis of alloferon were identified.     

The effect of fast and slow motor unit activation on whole-muscle mechanical performance: the size principle may not pose a mechanical paradox

The output of skeletal muscle can be varied by selectively recruiting different motor units. However, our knowledge of muscle function is largely derived from muscle in which all motor units are activated. This discrepancy may limit our understanding of in vivo muscle function. Hence, this study aimed to characterize the mechanical properties of muscle with different motor unit activation. We determined the isometric properties and isotonic force–velocity relationship of rat plantaris muscles in situ with all of the muscle active, 30% of the muscle containing predominately slower motor units active or 20% of the muscle containing predominately faster motor units active. There was a significant effect of active motor unit type on isometric force rise time (p < 0.001) and the force–velocity relationship (p < 0.001). Surprisingly, force rise time was longer and maximum shortening velocity higher when all motor units were active than when either fast or slow motor units were selectively activated. We propose this is due to the greater relative effects of factors such as series compliance and muscle resistance to shortening during sub-maximal contractions. The findings presented here suggest that recruitment according to the size principle, where slow motor units are activated first and faster ones recruited as demand increases, may not pose a mechanical paradox, as has been previously suggested.     

Purification of GidA protein,a novel topoisomerase II inhibitor produced by Streptomyces flavoviridis

The presence of topoisomerase II inhibition activities in the intracellular extract of Streptomyces flavoviridis was investigated. One active compound inhibiting relaxation activity of topoisomerase II was determined to be a protein. This active principle was purified to homogeneity by gel filtration followed by ion exchange chromatography. The apparent molecular mass was 42 kDa as determined by SDS-PAGE. MALDI TOF peptide mass fingerprinting analysis confirmed this topoisomerase II inhibitor, as glucose-inhibited division protein A (GidA) by MOWSE score of 72. The effects of purified GidA protein on DNA relaxation and decatenation by topoisomerase II were investigated. It inhibited topoisomerase II activity and acted as a topoisomerase poison that significantly stabilized the covalent DNA-topoisomerase II reaction intermediate “cleavable complex”, as observed with etoposide. Collectively, these findings indicate that GidA is a potent inhibitor of topoisomerase II enzyme, which can be exploited for rational drug design in human carcinomas.     

Co-Factor Binding Confers Substrate Specificity to Xylose Reductase from Debaryomyces hansenii

Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards “non-substrate” sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K_d∼5.0–10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4–5 fold molar excess. Comparison of K_d values with K_m values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.     

Métabolisme du chloro-2 (p-chlorophényl)-3 propionate de méthyle et problème de sélectivité

The product of hydrolysis of the active principle of the herbicide Bidisine® is degraded in at least two different ways. One way gives p-chlorobenzoic acid. The other, more important one, gives a conjugate with l-cysteine, which is further oxidized. The ability to oxidize the conjugate provided a means of distinguishing between susceptible and resistant plant species.     

Comparative phytochemical analysis of wild and in vitro-derived greenhouse-grown tubers, in vitro shoots and callus-like basal tissues of Harpagophytum procumbens

Comparative phytochemical analysis of wild and in vitro-derived greenhouse-grown tubers, in vitro shoots and callus-like basal tissues of Harpagophytum procumbens was done. Dried samples were ground to fine powders and their total iridoid (colorimetric method), phenolic [Folin-Ciocalteu (Folin C) method] and gallotannin (Rhodanine assay) contents as well as anti-inflammatory activity [cyclooxygenase assays (COX-1 and COX-2)] were determined. The tissue culture-derived tubers had the highest total iridoid content which was significantly higher than that of the tubers collected from the wild and other tissue cultured materials evaluated. This suggests that cultivated plants can be a viable alternative source of the active principle(s). The total phenolic and gallotannin contents of the wild tubers were significantly higher than the tissue culture-derived tubers and other in vitro-derived plant materials. The presence of phenolic compounds including gallotannins in the tissue cultured materials is of interest from a pharmacological point of view given the pharmacological role of phenolics. In general, extracts from wild tubers demonstrated better inhibitory activities in both COX-1 and COX-2 assays when compared to the tissue culture-derived tubers. All the petroleum ether (PE) and dichloromethane (DCM) extracts showed moderate (50-70%) to good (> 70%) inhibitory activities whereas the ethanol (EtOH) extracts showed poor or no inhibition in both assays. Based on previous reports indicating weak inhibition of COX-2 enzyme by harpagoside, the inhibitory activities of both COX enzymes exhibited by PE and DCM extracts in the current study could be due to the presence of other constituents in the extracts. This points towards the need to identify other active constituents and evaluate their role and mode of action in relation to harpagoside — the main active principle.     

Bioassay-guided isolation of a sesquiterpene lactone of deoxyelephantopin from Elephantopus scaber Linn. active on Trypanosome brucei rhodesience

Methanolic extracts of 70 Malaysia plants were screened for their in vitro antitrypanosomal activity using Trypanosome brucei rhodesience, strain STIB 900 and mouse skeletal cell (L-6) in cytotoxicity activity assay. Results indicated that methanol extract from Elephantopus scaber Linn. (E. scaber) possessed the highest value of antitrypanosomal activity with good selectivity index (antitrypanosomal IC₅₀ of 0.22 ± 0.02 μg/ml, SI value of 204.55). Based on these results, E. scaber was chosen for further study by applying bioassay guided fractionation to isolate its antiprotozoal principle. The antiprotozoal principle was isolated from the ethyl acetate partition through solvent fractionation and crystallization process. The isolated active compound 1 was identified as deoxyelephantopin on the basis of its spectral analysis (FTIR, MS, 1D and 2D NMR).     

In vitro anti-influenza virus activity of a cardiotonic glycoside from Adenium obesum (Forssk.)

Methanolic extracts of six Saudi plants were screened for their in vitro antiviral activity using influenza virus A/PR/8/34 (H1N1) and MDCK cells in an MTT assay. The results indicated that the extracts of Adeniumobesum and Tephorosianubica possessed antiviral activity (99.3 and 93.3% inhibition at the concentration of 10 μg/ml, respectively). Based on these results A. obesum was selected for further study by applying bioactivity-guided fractionation to isolate its antiviral principle. The antiviral principle was isolated from the chloroform fraction through solvent fractionation, combined open liquid chromatography and HPLC. The isolated active compound A was identified as oleandrigenin-β-d-glucosyl (1 → 4)-β-d-digitalose, on the basis of its spectral analysis (MS, 1D and 2D NMR). The isolated glycoside showed reduction of virus titre by 69.3% inhibition at concentration of 1 μg/ml (IC₅₀ = 0.86 μg/ml).     

Inhibitor for FcεRI expression on mast cell from Verbasucum thapsus L.

We found out 2′,3′-dihydroxypuberulin from South American medicinal plant, V. thapsus L., as a candidate of an anti-allergic lead which inhibits the expression of high-affinity receptor of IgE (FcεRI) on the surface of mast cells. Furthermore, the analysis of structure-activity relationship by using synthesized 2′,3′-dihydroxypuberulin analogs revealed that both hydroxy groups in the side chain and both of methyl moieties on phenolic hydroxy groups were crucial for potent activity, but absolute configuration of C-3′ position wasn’t. The active principle, 2′,3′-dihydroxypuberulin, was disclosed to down-regulate the mRNA level of β-chain of FcεRI, different from previous reported active natural product reducing γ-chain level.     

Effect of garlic oil on incorporation of amino acids into proteins of Culex pipiens quinquefasciatus Say larvae

Incorporation of [¹⁴C]leucine into proteins of 3rd instar Culex pipiens quinquefasciatus Say larvae increases linearly with time between 1 and 4 h. Garlic oil as well as the active larvacidal principle from it, viz. diallyl disulphide, inhibits significantly synthesis of the larval proteins. The maximum reduction in incorporation is observed during the first hour of treatment. The incorporation of [¹⁴C]phenylalanine is also inhibited by garlic oil and the effect is irreversible. Garlic oil does not seem to have any effect on proteins already labelled and it does not suppress substantially oxygen uptake by the larvae.     

A Repurposing Strategy for Hsp90 Inhibitors Demonstrates Their Potency against Filarial Nematodes

Novel drugs are required for the elimination of infections caused by filarial worms, as most commonly used drugs largely target the microfilariae or first stage larvae of these infections. Previous studies, conducted in vitro, have shown that inhibition of Hsp90 kills adult Brugia pahangi. As numerous small molecule inhibitors of Hsp90 have been developed for use in cancer chemotherapy, we tested the activity of several novel Hsp90 inhibitors in a fluorescence polarization assay and against microfilariae and adult worms of Brugia in vitro. The results from all three assays correlated reasonably well and one particular compound, NVP-AUY922, was shown to be particularly active, inhibiting Mf output from female worms at concentrations as low as 5.0 nanomolar after 6 days exposure to drug. NVP-AUY922 was also active on adult worms after a short 24 h exposure to drug. Based on these in vitro data, NVP-AUY922 was tested in vivo in a mouse model and was shown to significantly reduce the recovery of both adult worms and microfilariae. These studies provide proof of principle that the repurposing of currently available Hsp90 inhibitors may have potential for the development of novel agents with macrofilaricidal properties.     

Effect of nutritional factors on cryptotanshinone and ferruginol production by cell suspension cultures of Salvia miltiorrhiza

The effects of the components of Murashige-Skoog (MS) medium on the production of the diterpenes cryptotanshinone and ferruginol in cell suspension cultures of Salvia miltiorrhiza were examined. Sucrose, a nitrogen source and thiamine were needed for the production of these compounds, and phosphate, MnSO₄ and kinetin showed slight beneficial effects. All the other components of MS medium were found to be either unnecessary or inhibitory for the production of these compounds, when tested separately. A revised medium for the production of cryptotanshinone, a clinically active principle in this plant, was designed.     

Effect of Liquid Nutrient Culture,Vacuum Distillation and Dialysis on Hatching Activity of Sugar Beet Root Diffusate for Heterodera schachtii

Roots of sugar beets grown in liquid culture excrete substances that stimulate egg hatch and emergence of larvae from cysts of Heterodera schachtii. Their hatching effect is comparable to that of sugar beet root diffusate leached from soil-grown sugar beet plants. Consequently, liquid culture provides a way of obtaining H. schachtii hatch-stimulant free of contaminants from soil. Root diffusate, concentrated 50-fold or dried by vacuum distillation, retained hatching activity. The active principle of diffusate is dialyzable with a diffusion rate between those of inorganic salts and compounds with molecular weights greater than 15,000.     

Antifeedant activity of ethanolic extract from Flourensia oolepis and isolation of pinocembrin as its active principle compound

The ethanolic extract from Flourensia oolepis aerial parts showed strong antifeedant activity against the pest larvae, Epilachna paenulata, with an antifeedant index (AI%) of 99.1% at 100 μg/cm². Based on chromatographic fractionation of the extract, guided by bioassays on E. paenulata, the flavanone pinocembrin (1) was isolated as the most active principle. In a choice assay, compound 1 showed strong antifeedant activity against E. paenulata, Xanthogaleruca luteola and Spodoptera frugiperda with an AI% of 90, 94 and 91% (p < 0.01) respectively, at 50 μg/cm². The dosages necessary for 50% feeding inhibition of the insects (ED₅₀) were 7.98, 6.13 and 8.86 μg/cm², respectively. The feeding inhibitory activity of 1 against E. paenulata was compared with the activity of other structurally related flavonoids like naringenin, which was inactive up to 100 μg/cm², catechin which was nearly 6 times less active than 1, and quercetin which was equally active as 1. The effect of these on the feeding behavior of E. paenulata was also studied.     

Physiological changes in central neuronal pathways contributing to the generation of a reflex reversal

1. In the stick insect Carausius morosus the properties of the neuronal network governing the femur-tibia joint depend on the behavioral state of the animal. In the inactive animal flexion of the femur-tibia joint results in the generation of a resistance reflex, while in the active animal the same stimulus induces the so-called active reaction, the first part of which is a reflex reversal.
2. Recordings from motoneurons innervating the extensor tibiae muscle indicated that their time course of activity during the active reaction is due to inputs from intercalated pathways. We therefore investigated the role of identified nonspiking interneurons that transmit sensory information from the chordotonal organ onto the extensor motoneurons in the inactive animal. We can show that (i) the nonspiking interneurons received altered inputs whereas (ii) they provided qualitatively the same synaptic drive onto leg motoneurons.
3. From our results it is clear that (i) neuronal pathways contributing to the generation of the resistance reflex are also involved in the generation of the reflex reversal in the same control loop, (ii) thereby adopting the same principle of information processing (parliamentary principle), because both, supporting and opposing pathways contribute to the generation of the motor output.