Inhibition of Site I mitochondrial electron transport by an extract of the seeds of Millettia thonningii: a potential mechanism for the plant's molluscicidal and schistosome larvicidal activity

A dichloromethane extract of the seeds of Millettia thonningii (Leguminosae) which contains a mixture of isoflavonoids (predominantely robustic acid, alpinumisoflavone and dimethylalpinumisoflavone) is known to have larvicidal activity towards the miracidia and cercariae of schistosomes and to possess significant molluscicidal activity. The present investigation has assayed the effects of this extract on the electron transport systems of isolated rat liver mitochondria. The extract was found to inhibit mitochondrial electron transport at Site I (NADH dehydrogenase) at concentrations of 30-159 mg x l(-1). Although the extract is not as potent an inhibitor at Site I as rotenone, a known inhibitor of NADH dehydrogenase, such observations could explain the molluscicidal and schistosomicidal activity of dichloromethane extracts of the seeds of M. thonningii.     

A pyrano-isoflavone from seeds of Milletia thonningii

Extraction of the seeds of Milletia thonningii gave alpinumisoflavone, its monomethyl and dimethyl derivatives, robustic acid and a new pyrano-isoflavone. The structure of the latter was established by chemical transformation and spectroscopic means.     

Anti-phospholipase A2 and anti-inflammatory activity of Santolina chamaecyparissus

The activity of the Santolina chamaecyparissus methanol extract was tested against the phospholipase A2 (PLA2)-induced mouse paw edema and in vitro inhibition of PLA2 activity. After fractionation, only the dichloromethane extract was active against the PLA2 in vitro test. In addition, it reduced the edema induced by arachidonic acid, and by 12-O-tetradecanoylphorbol-13-acetate in a multidose test. After chromatography on silicagel and gel filtration on Sephadex, and using an in vitro anti-PLA2 assay-guided process, we have isolated and identified from the dichloromethane extract the flavone nepetin and four sesquiterpenes.     

Update on paramyosin in parasitic worms

Paramyosin was first identified as a structural component of invertebrate muscle. Analysis of crude, native, adult schistosome worm preparations identified a highly immunogenic protein which was later identified as paramyosin. Early vaccination/challenge studies with native paramyosin produced encouraging levels of protective efficacy against schistosomes, which led to the question as to how a sub-tegumental (muscular) protein could provide a target for vaccine-mediated immunological attack. Immunolocalisation studies of schistosomes confirmed the presence of paramyosin within the post-acetabular glands of cercariae and on the tegumental surface of lung schistosomula. Here we present an update on the more recent research on paramyosin in parasitic worms that has focused primarily in two directions: (i) further testing of the vaccine potency of paramyosin against schistosomes and other parasitic worms; and (ii) characterisation of the protein at the molecular and biochemical levels.     

Antimicrobial activity and main chemical composition of two smoke condensates from Peganum harmala seeds

The smoke of Peganum harmala seeds is traditionally used in Iran as a disinfectant agent. The aim of this study was to determine the antimicrobial activity of two smoke condensates from Peganum harmala seeds. Furthermore the composition of smoke preparations was studied using gas chromatography and mass spectroscopy analysis. The most prevalent compound detected in a dichloromethane extract was harmine. Standard harmine as well as the dichloromethane extract showed antimicrobial activity against all test strains. Harmine was not detected in an n-hexane extract and we did not observe antimicrobial activity from this smoke preparation at the tested concentrations.     

Cytotoxic isoflavones from Erythrina indica.

Bioassay-directed fractionation of the CH(2)Cl(2)-MeOH (1:1) extract of the stem bark of Erythrina indica, has resulted in the isolation of two new isoflavone derivatives named indicanines D and E together with 11 known compounds including: six isoflavones (genistein, wighteone, alpinumisoflavone, dimethylalpinumisoflavone, 8-prenyl erythrinin C, and erysenegalensein E), one cinnamate (erythrinassinate B), two pentacyclic triterpenes (oleanolic acid and erythrodiol), and two phytosterols (stigmasterol and its 3-O-beta-D-glucopyranoside). The structures of the new compounds were elucidated by means of spectroscopic analysis. The in vitro cytocidal activity against KB cells of some of the isolated compounds is also reported.     

Schistosoma mansoni: topochemical features of cercariae, schistosomula, and adults

The teguments of developing and mature cercariae, recently transformed, and 1-wk-old schistosomula and adult worms were examined for the ultrastructural location of macromolecular carbohydrates and polyelectrolytes. The surface of mature cercariae within sporocysts and cercariae released from the snail is covered by a filamentous coat which reacts with cytochemical reagents for the demonstration of vicinal glycols, but neither the coat nor the surface of the tegument plasmalemma binds cationic colloidal iron at low pH.Upon penetrating mammalian skin, the cercaria sheds its surface coat; the tegument surface of newly transformed schistosomula, older schistosomula and adult worms stains en bloc with acidic colloidal iron, as does the tegument plasmalemma of mature cercariae if the overlying filamentous coat is first removed by physicochemical means. The cercarial coat thus serves to mask anionic groups at the surface of the tegument plasmalemma which become functionally exposed after penetration of the mammalian host. The distribution of colloidal iron binding sites coincides with those for the carbohydrate-complexing phytohemmagglutnin, concanavalin A, which suggests that these membrane-fixed anions are acid mucopolysaccharides, glycoproteins or glycolipids. Carbohydrate-containing material was also localized within membrane-bound vesicles of the tegument matrix and perikarya of developing cercariae and postcercarial schistosomes, suggesting that surface mucosubstances contributing to the tegument glycocalyx of these worms are elaborated, at least in part, by the tegument itself.     

Sativa seeds against Schistosoma mansoni different stages

The schistosomicidal properties of Nigella sativa seeds were tested in vitro against Schistosoma mansoni miracidia, cercariae, and adult worms. Results indicate its strong biocidal effects against all stages of the parasite and also showed an inhibitory effect on egg-laying of adult female worms. In the present work we also studied the effects of crushed seeds on some antioxidant enzymes; which have a role in protection of adult worms against host oxidant killing; as well as some enzymes of glucose metabolism; which have a crucial role in the survival of adult worms inside their hosts. The data revealed that the used drug induce an oxidative stress against adult worms which indicated by a decrease in the activities of both antioxidant enzymes, superoxide dismutase, glutathione peroxidase, and glutathione reductase and enzymes of glucose metabolism, hexokinase and glucose-6-phosphate dehydrogenase. Disturbing of such enzymes of adult worms using N. sativa seeds could in turn render the parasite vulnerable to damage by the host and may play a role in the antischistosomal potency of the used drug.     

Schistosoma mansoni-New Zealand rabbit model: resistance induced by infection followed by active immunization with protective antigens

In previous studies, rabbits immunized with adult worm antigens released from fresh adult schistosomes incubated in saline media showed a significant level of protection against challenge parasites. Focusing on the rabbit-Schistosoma mansoni model, concomitant immunity was investigated. A peculiar form of response to cercarial infection was observed: rabbits subjected to percutaneous infection and similar reinfections at different times after primary infection killed schistosomula from the challenge infection as well as established parasites from the primary infection. In this study the challenge infection stimulus was replaced by active immunization with an adult worm-derived protective antigenic mixture. The results show that immunization of New Zealand rabbits with an adult worm antigenic extract is capable of inducing a response that results in a significant reduction of the mean worm burden of the primary infection earlier than did homologous infection, as compared to worm reduction due to a second infection.     

Schistosoma mansoni dermaseptin-like peptide: structural and functional characterization

Analysis of the Schistosoma mansoni peptidome for immunomodulatory molecules by solvent extraction and reverse-phase HPLC revealed a 27-amino-acid residue peptide from an extract of cercariae. Using matrix-assisted, laser desorption-ionization, time-of-flight mass spectrometry, the peptide yielded a protonated molecular ion [M + H]+ of m/z 2789. The unequivocal sequence was deduced by automated Edman degradation as: DLWNSIKDMAAAAGRAALNAVTGMVNQ. The peptide exhibited an 80.76% identity with dermaseptin 3.1 from the leaf frog Agalychnis annae, and was therefore named Schistosoma mansoni dermaseptin-like peptide (SmDLP). Immunocytochemical staining using a primary antidermaseptin B2 antibody located SmDLP in acetabular glands of cercariae, in and around schistosomula, and in adult worms and their eggs. Dot-blotting confirmed its presence in extracts (cercariae and worms) and excretion/secretion (E/S) products (transforming cercariae and eggs). This was corroborated by use of a MALDI-ToF spectra database of E/S products from cercariae. Functional characterization of the peptide indicated that SmDLP had typical amphipathic antimicrobial peptide properties, i.e., the ability to lyse human erythrocytes causing a decrease in the levels of nitric oxide produced by monocytic cells. This last function strongly suggests that SmDLP plays a vital role in the parasite's immunoevasion strategy. The possibility that schistosomes acquired this gene from amphibians has been discussed by constructing a phylogenetic tree.     

Uncovering Biological Application of Brazilian Green Propolis: A Phenotypic Screening against Schistosoma mansoni

The chemotherapy of schistosomiasis remains centered in the use of praziquantel, however, there has been growing resistant parasites to this drug. Thus, the aim of this work was to evaluate in vitro schistosomicidal activity of the hexanes/dichloromethane 1 : 1 extract of Brazilian green propolis (Pex), as well as its major isolated compounds artepillin C, caffeic acid, coumaric acid and drupanin against Schistosoma mansoni. The Pex was active by displaying an IC₅₀ value of 36.60 (26.26–51.13) μg mL^?1 at 72 h against adult worms of S. mansoni. The major isolated compounds were inactive with IC₅₀ values >100 μM, however, the combination of the isolated compounds (CM) in the same range found in the extract was active with an IC₅₀ value of 41.17 (39.89–42.46) μg mL^?1 at 72 h. Pex and CM induced alteration in the tegument of S. mansoni, and caffeic acid caused alteration in egg's maturation. Pex displayed in vitro activity against adult worms’ and eggs’ viability of S. mansoni, which opens new perspectives to better understand the synergistic and/or additive effects promoted by both Pex extract and CM against schistosomiasis features.     

Two isoforms of a divalent metal transporter (DMT1) in Schistosoma mansoni suggest a surface-associated pathway for iron absorption in schistosomes

We describe two homologues of the mammalian divalent metal transporter (DMT1) for Schistosoma mansoni, a pathogenic intravascular parasite of humans. Schistosomes have a high nutritional and metabolic demand for iron. Nucleotide sequences of the parasite homologues, designated SmDMT1A and -B, are identical in all but the 5'-regions. The predicted amino acid sequences share at least 60% identity with DMT1 (=Nramp2) of humans, mice, and rats, and at least 55% identity with Nramp1 from mice, humans and Caenorhabditis elegans. SmDMT1A is expressed in differentiating eggs, miracidia, cercariae, schistosomula, and adults, whereas SmDMT1B is expressed in all but the miracidium and occurs at lower levels than SmDMT1A in differentiating eggs and cercariae. An iron-responsive element, present at the 3'-untranslated region of many DMT1 molecules, is not present in schistosome mRNAs studied here. A Western blot analysis of adult worm preparations using a homologous rabbit serum raised against a schistosome DMT1 peptide and a heterologous serum raised against mammalian DMT1, revealed a band approximating 115 kDa. By immunofluorescence microscopy, the schistosome DMT1s localize primarily to the tegument. Iron uptake assays demonstrated that SmDMT1s were able to rescue yeast growth in ferrous iron-transport deficient yeast (fet3fet4). The results suggest that schistosomes express molecules for ferrous iron transport in their tegument, suggesting trans-tegumental transport as one means of iron acquisition for these parasites.     

Anti-staphylococcal acylphloroglucinols from Hypericum beanii

As part of an ongoing project to investigate the anti-staphylococcal properties of the Hypericum genus, an acylphloroglucinol, 1,5-dihydroxy-2-(2'-methylpropionyl)-3-methoxy-6-methylbenzene (1), was isolated from the dichloromethane extract of the aerial parts of H. beanii (Guttiferae), together with a minor related acylphloroglucinol 1,5-dihydroxy-2-(2'-methylbutanoyl)-3-methoxy-6-methylbenzene (2) as a mixture in a 5:2 ratio. The known compounds 1,7-dihydroxyxanthone (3), stigmasterol, catechin and shikimic acid were also isolated from this plant. The structures of the compounds were characterized by extensive 1- and 2D NMR spectroscopy and mass spectrometry. The minimum inhibitory concentration (MIC) values the acylphloroglucinol mixture and (3) against a panel of multidrug-resistant strains of Staphylococcus aureus ranged from 16-32 microg/ml to 128-256 microg/ml, respectively.     

Biological effects of active fraction isolated from Hydnocarpus pentandra (Bunch. –Ham.) Oken seeds against Helicoverpa armigera (Hub.) (Lepidoptera: Noctuidae)

Hexane, chloroform, ethyl acetate and methanol extracts of Hydnocarpus pentandra (Flacourtiaceae) seeds were tested for antifeedant, larvicidal, pupal mortality and adult deformations activities against Helicoverpa armigera. Crude extracts were screened at 0.5, 1.0, 1.5 and 2.0% concentrations. Bioassay-guided fractionation method was followed to isolate the active fraction from the crude extract. Active fraction was analysed by FT-IR, ¹H NMR, ¹³C NMR and GC-MS. Hexane extract presented the highest antifeedant (87.89%), pupal mortality (41.67%) and adult malformation activities at 2% concentration. Seven different fractions were isolated from hexane extract, among which fraction-2 showed the highest antifeedant (81.43%) activity and recorded the lowest LC₅₀ of 792.07 ppm. The fraction-2 contained two cyclopentenyl carboxylic acids, such as hydnocarpic acid (1) and chaulmoogric acid (2) in the ratio of 2:1. These compounds were major constituents in the active fraction of hexane extract of H. pentandra seeds. Fraction-2 can be used for agricultural pest management.     

Studies on the Constituents of Chinese Traditional Drug Suan Zau Ren

Suan Zau Ren (the seeds of Zizyphus spinosus Hu) is a widely used chinese traditional drag for treatment of insomnia, neurasthenia and sometimes for sleepiness caused by physical emaciation in chinese traditional medicine. Its dichloromethane extract afforded four pentacyclic triterpenes of the lup-20(29)-en group, namely betulin (Ⅱ), betulinic acid(Ⅰ), ceanothic acid(Ⅲ), and alphitolic acid (Ⅳ), and one sterol, daucosterol (Ⅴ). Their structures were identified by the spectroscopic analysis. The later three compounds were isolated from Suan Zau Ren for the first time.     

Fasciola hepatica and Schistosoma mansoni: immunofluorescent antigen localization and cross-reactivity

In an attempt to identify the tissue sources of biochemically purified antigenic fractions of Fasciola hepatica and Schistosoma mansoni, antisera were tested against plastic-embedded sections of worms of various ages by an indirect fluorescent-antibody-labeling technique. Antibodies prepared against antigens purified by chromatography of F. hepatica whole worm extract through concanavalin A-Sepharose 4B labeled the parenchyma and tegument of adult F. hepatica strongly while antibodies developed against antigens purified by antibody-affinity chromatography against antibodies of S. mansoni labeled only the parenchyma. Antigens common to these two groups clearly originated from F. hepatica parenchyma. Certain of these common antigens are known to provide significant protection in mice to challenge with S. mansoni cercariae, and in the present study antisera against F. hepatica extracts cross-labeled S. mansoni adult male parenchyma. Reciprocal cross-reactions between antisera against S. mansoni and the parenchyma of adult F. hepatica were also noted. FhFIIb, an extract of F. hepatica which Tailliez described as not cross-reacting with S. mansoni, was found to contain no F. hepatica parenchymal antigens. Antigenic fractions of F. hepatica and S. mansoni collected from the surface of worms after incubation in nonionic detergent were unexpectedly found to contain much parenchymal antigen, suggesting leakage of internal components into the supernatant during preparation. Antisera to F. hepatica developed during a natural infection in rabbits labeled tegumental components and gut strongly but did not react with parenchymal tissue. Antisera against extracts of adult schistosomes labeled the parenchyma of male worms and the glycocalyx of the cercarial tegument, indicating the presence of common antigens in the adult and the cercarial stage. Reciprocal reactions between anticercarial sera and adult sections provided further evidence of shared antigenicity. Antisera against S. mansoni egg antigens strongly labeled sections of eggs in liver tissue and cross-reacted with cercarial glycocalyx, indicating the existence of common antigens between these two stages. The antisera also cross-reacted with what appeared to be non-membrane-bound protein in the tegument of F. hepatica. The soluble egg antigen extract shared antigenicity with the parenchyma of both S. mansoni and F. hepatica but circumoval precipitin had no cross-reactivity with this tissue. Thus S. mansoni eggs contain nondiffusable components sharing antigenic specificity with adult parenchymal tissue.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pattern of cercarial emergence of Schistosoma curassoni from Niger and comparison with three sympatric species of schistosomes.

The emergence pattern of Schistosoma curassoni cercariae from Bulinus umbilicatus, whose adult worms parasitize bovine, caprine, and ovine ungulates in Niger, is of a circadian type with a mean emission time at 0855 hr +/- 1 hr 6 min, characteristic of the schistosome species parasitizing domestic or wild cattle. The comparison of this cercarial emergence pattern with those of the other 3 sympatric species of schistosomes (Schistosoma haematobium, Schistosoma bovis, and Schistosoma mansoni) shows a significant difference between the chronobiology of the cercariae infective for human and those infective for bovine hosts. This difference may improve epidemiological surveys based on snail prevalences by allowing the distinction between bulinids infected with human and bovine parasites.     

Secondary metabolites of ponderosa lemon (Citrus pyriformis) and their antioxidant, anti-inflammatory, and cytotoxic activities

Column chromatography of the dichloromethane fraction from an aqueous methanolic extract of fruit peel of Citrus pyriformis Hassk. (Rutaceae) resulted in the isolation of seven compounds including one coumarin (citropten), two limonoids (limonin and deacetylnomilin), and four sterols (stigmasterol, ergosterol, sitosteryl-3-beta-D-glucoside, and sitosteryl-6'-O-acyl-3-beta-D-glucoside). From the ethyl acetate fraction naringin, hesperidin, and neohesperidin were isolated. The dichloromethane extract of the defatted seeds contained three additional compounds, nomilin, ichangin, and cholesterol. The isolated compounds were identified by MS (EI, CI, and ESI), 1H, 13C, and 2D-NMR spectral data. The limonoids were determined qualitatively by LC-ESI/MS resulting in the identification of 11 limonoid aglycones. The total methanolic extract of the peel and the petroleum ether, dichloromethane, and ethyl acetate fractions were screened for their antioxidant and anti-inflammatory activities. The ethyl acetate fraction exhibited a significant scavenging activity for DPPH free radicals (IC50 = 132.3 microg/mL). The petroleum ether fraction inhibited 5-lipoxygenase with IC50 = 30.6 microg/mL indicating potential anti-inflammatory properties. Limonin has a potent cytotoxic effect against COS7 cells [IC50 = (35.0 +/- 6.1) microM] compared with acteoside as a positive control [IC50 = (144.5 +/- 10.96) microM].     

Schistosoma mansoni: surface membrane stability in vitro and in vivo

The human complement component C3b is known to bind in vitro to the surfaces of all developmental stages of schistosomes as a consequence of complement activation by the alternative pathway. C3b bound to Schistosoma mansoni parasites has now been used in combination with fluorescent labeled antibodies against C3b to label the surfaces of living schistosomes. Binding of complement components and labeled antibodies to adult schistosomes rendered their surface membrane homogeneously fluorescent. At the ultrastructural level, the label was seen as a dense deposit lying on the tegumental membrane. Surface damage was not observed in labeled adults by electron microscopy. Fluorescent schistosomes were cultured in vitro for periods of up to 2 weeks, during which time the parasites remained fully viable and their surface membrane was still fluorescent. The electron dense deposit persisted, and tegumental damage at the electron microscope level was minimal or absent. Consequently, adult schistosomes would seem able to survive in vitro in the absence of rapid and general turnover of their surface membrane. Loss of fluorescence was observed consistently only at the anterior end of the parasite, including the suckers, a finding which indicates that membrane turnover may occur at different rates on different parts of the body. Fluorescent 3-week-old juveniles and 6-day-old lung stage parasites were cultured under the same conditions with similar results: they remained viable and fluorescent for at least 2 weeks. Results with skin schistosomula were different in the sense that many worms died during culture, and those which survived lost large parts of their fluorescent surface. A few of the surviving and fluorescent schistosomula developed the elongate shape typical of lung stage parasites. Fluorescent viable skin schistosomula were injected intravenously into mice and subsequently recovered from the lungs after varying periods. Fluorescence was lost in a patchy way within a few minutes from some individuals and within several hours from most of the worms. These data permit the following conclusions: C3b is a suitable tracer for membrane renewal in all developmental stages of schistosomes. Very slow membrane renewal in vitro and very rapid renewal in vivo are both compatible with parasite survival.     

Various stages of schistosoma express Lewis(x), LacdiNAc, GalNAcbeta1-4 (Fucalpha1-3)GlcNAc and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc carbohydrate epitopes: detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins

We report here that fucosylated epitopes such as Lewis(x), LacdiNAc, fucosylated LacdiNAc (LDN-F) and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) are expressed by schistosomes throughout their life cycle. These four epitopes were enzymatically synthesized and coupled to bovine serum albumin to yield neoglycoproteins. Subsequently these neoglycoproteins were used to probe a panel of 188 monoclonal antibodies obtained from infected or immunized mice, in ELISA and surface plasmon resonance analysis. Of these antibodies, 25 recognized one of the fucosylated structures synthesized, indicating that these structures are immunogenic during infection. The MAbs identified could be subdivided in four different groups based on the recognition of either the Lewis(x)-, the LacdiNAc-, the LDN-DF-, or both the LDN-F- and LDN-DF epitope. These monoclonal antibodies were then used to investigate the localization of the fucosylated epitopes in various stages of Schistosoma mansoni using indirect immunofluorescence. Lewis(x)epitopes were mainly found in the gut and on the tegument of adult worms, on egg shells, and on the oral sucker of cercariae. The LacdiNAc epitope was expressed on the tegument of adult worms, on miracidia, and on the oral sucker of cercariae. In contrast, LDN-DF epitopes were mainly present in the excretory system of adult worms, on miracidia and on whole cercariae. These also stained positive with the LDN-F/LDN-DF epitope antibodies, while whole parenchyma reacted characteristically only with the latter antibodies. The identification of different carbohydrate structures in various stages of schistosomes may lead to a better understanding of the function of glycans in the immune response during infection.