Granulomatous hypersensitivity and antibody production in response to antigens of Capillaria hepatica eggs

Soluble egg antigens of Capillaria hepatica were fractionated by gel filtration chromatography into two major protein peaks of 300,000 and 14,000 mol.wt. The unfractionated antigen and both peaks were capable of sensitizing mice to produce quantitatively larger liver granulomas upon experimental innoculation of eggs; the increase in granuloma size was related to the amount of antigen used to sensitize. The antibody response in C. hepatica infected mice was primarily directed toward the high molecular weight components. Comparison of these findings with those reported for the Schistosoma mansoni egg granuloma suggests a diversity in the mechanisms of granuloma formation among helminth parasites.     

Real-time PCR detection of Holophagae (Acidobacteria) and Verrucomicrobia subdivision 1 groups in bulk and leek (Allium porrum) rhizosphere soils

In the light of the poor culturability of Acidobacteria and Verrucomicrobia species, group-specific real-time (qPCR) systems were developed based on the 16S rRNA gene sequences from culturable representatives of both groups. The number of DNA targets from three different groups, i.e. Holophagae (Acidobacteria group 8) and Luteolibacter/Prosthecobacter and unclassified Verrucomicrobiaceae subdivision 1, was determined in DNA extracts from different leek (Allium porrum) rhizosphere soil compartments and from bulk soil with the aim to determine the distribution of the three bacterial groups in the plant-soil ecosystem. The specificity of the designed primers was evaluated in three steps. First, in silico tests were performed which demonstrated that all designed primers 100% matched with database sequences of their respective groups, whereas lower matches with other non-target bacterial groups were found. Second, PCR amplification with the different primer sets was performed on genomic DNA extracts from target and from non-target bacteria. This test demonstrated specificity of the designed primers for the target groups, as single amplicons of expected sizes were found only for the target bacteria. Third, the qPCR systems were tested for specific amplifications from soil DNA extracts and 48 amplicons from each primer system were sequenced. All sequences were > 97% similar to database sequences of the respective target groups. Estimated cell numbers based on Holophagae-, Luteolibacter/Prosthecobacter- and unclassified Verrucomicrobiaceae subdivision 1-specific qPCRs from leek rhizosphere compartments and bulk soils demonstrated higher preference for one or both rhizosphere compartments above bulk soil for all three bacterial groups.     

β-Galactosidases of Lilium pollen

Lily (Lilium auratum) pollen contains very high levels of β-galactosidase. There are three forms: β-galactosidase I and II differ in M_r, while β-galactosidase III is firmly bound in the pollen wall. The two cytoplasmic forms were separated and partially purified using a combination of chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose 6B. Forms I and II appear to be glycoprotein in nature as shown by binding to Con A-Sepharose. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. The wall-bound enzyme, β-galactosidase III effectively hydrolysed nitrophenyl β-galactosidase but not lactose, and could not be released from the wall polysaccharide matrix by high salt concentrations or detergents. The total β-galactosidase activity of lily pollen remained constant during in vitro germination. A possible role for this enzyme may be in degradation of stylar arabinogalactans providing a carbon source for pollen tube nutrition.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.     

In Vivo and in Vitro binding of platinum to metallothionein

The in vivo binding of platinum to metallothionein (MT) has been observed in rat tissues following injections of the cis and trans isomers of DDP (dichlorodiammine-platinum(II)). Platinum in either cis-DDP or trans-DDP does not directly induce MT; platinum-MT is produced by the replacement of previously bound zinc in the protein. The binding of Pt(II) to MT depends on the availability of SH groups in MT. Preinjection with CdCl₂ significantly enhances the association of Pt(II) with MT fractions compared to the degree of association resulting from injections with either cis-DDP or trans-DDP without CdCl₂ pretreatment. In vitro experiments in which tissue extracts including a known (Cd,Zn)-MT were incubated with either cis-DDP or trans-DDP show that these isomers differ with respect to the transfer of Pt to MT; the equilibrium in both cases was reached when approximately 40% of the available Pt is bound to MT but with this equilibrium value attained in 2 h in the case of trans-DDP and only after 72 h in the case of cis-DDP. Pt-MTs were also formed by a series of incubation steps in which a native MT was used to prepare the apoprotein which was subsequently incubated with either cis-DDP or trans-DDP. Spectrophotometry established that a shoulder occurs at 285 nm for the Pt-MTs resulting from the incubation with either isomer. A competitive double-antibody radioimmunoassay for MT demonstrated that these Pt-MTs had complete cross-reactivity with a native (Cd,Zn)-MT. Gel filtration of tissue extracts after either in vivo or in vitro treatment with DDP showed that Pt was bound to a molecular species with properties characteristic of MT. These results were verified by atomic absorption spectrophotometry and polyacrylamide gel electrophoresis assays.     

Evaluation of the antigenic value of recombinant Toxoplasma gondii HSP20 to detect specific immunoglobulin G antibodies in Toxoplasma infected humans

Recombinant Toxoplasma gondii small heat shock protein HSP20, surface antigen SAG1 and dense granule GRA7 were analyzed by IgG-ELISA with serum samples of Toxoplasma infected humans grouped as I (IgG+, IgM+), II (IgG+, IgM−) and III (IgG−, IgM−). rHSP20 reacted against 80% and 62.5% of serum samples from groups I and II, respectively. rSAG1 was recognized by 85% of the samples from group I and 70.8% from group II, whereas rGRA7 was recognized by 85% and 66.6% of the serum samples from groups I and II, respectively. When a combination of two or three recombinant antigens was used, the sensitivity values improved to 85-95% for group I and 87.5-91.7% for group II. All combinations tested produced similar reactivity profiles. None of the recombinant proteins reacted against group III serum samples. In conclusion, we demonstrated that T. gondii HSP20 elicits an important B-cell response during human infection, and could be suitable for the development of serodiagnosis tools.     

Distribution of ent-kaurene synthetase in Helianthus annuus and Marah macrocarpus

Kaurene synthetases catalyse the biosynthesis of ent-kaurene, a precursor of the gibberellins. In 4-day-old dark- or light-grown Helianthus annuus seedlings, the cotyledons contained over 90% of the synthetase activity. The low enzyme activity in the seedling hypocotyls and roots is not a consequence of inhibitory factors in these tissues. The cotyledons not only have the highest kaurene synthetase activity, but also have the highest inhibitory activity. The differences in kaurene synthetase activities in the different tissues cannot be explained on the basis of the levels of inhibitor(s) in the extracts. The mature perennial root of Marah macrocarpus has very low kaurene synthetase activity, in contrast to the liquid endosperm of immature seeds of the same plant which is a rich source of the enzyme.     

Plasmodium chabaudi: Expression of active recombinant chabaupain-1 and localization studies in Anopheles sp.

Plasmodium cysteine proteases have been shown to be immunogenic and are being used as malaria potential serodiagnostic markers and vaccine targets. Genes encoding two Plasmodium chabaudi cysteine proteases chabaupain-1 (CP-1) and chabaupain-2 (CP-2) were identified and further expressed in Escherichia coli. Solubilisation of recombinant CP-1 and CP-2 was achieved by decreasing the temperature of induction.Anopheles gambiae tissues infected with Plasmodium were analyzed by Western blotting using the anti-CP-1 antibody showing that CP-1 is only present in the A. gambiae midguts being absent from other infected mosquito biological material. Anti-CP-1 anti-serum recognized a 30 kDa band in P. chabaudi, Plasmodium berghei and Plasmodium yoelii lysates but does not recognize the recombinant CP-2 extracts suggesting high antibody specificity.     

Rapid decarboxylation of the products of dark fixation of CO2 in roots of pisum and Plantago

Seedlings of Pisum sativum and excised roots of Plantago major and P. lanceolata were given, in the dark, a pulse of ¹⁴CO₂ in air followed by a chase in ¹²CO₂ in air. A very substantial proportion of the ¹⁴C fixed into organic compounds in the pulse was lost from the tissues in the chase. The activity of NAD malic enzyme in extracts of roots of all three species exceeded their rate of respiration. Azide, 2-n-butylmalonate, and salicylhydroxamic acid each inhibited CO₂ fixation by excised roots of pea. The first two compounds inhibited respiratory gas exchange, but the third stimulated it. Arguments are presented for the widespread diversion of phosphoenolpyruvate from glycolysis to oxaloacetate and thence to malate in the cytosol followed by transport of the malate into the mitochondria for conversion to pyruvate via NAD malic enzyme. No differences, in the above respects, were found between the two species of Plantago.     

Isoperoxidase pattern and internode length genotype in Pisum

Inbred Pisum sativum lines of known constitution for the intemode length genes Le, La and Cry, and representing four height phenotypes, were grown to the 7-intemode stage in the light. Six cationic isoperoxidases, making up ca. 90% of the activity of stem extracts, were resolved by concave gradient elution from Dowex 50 columns and shown to run as single peroxidase bands on starch gel electrophoresis. They were all able to oxidise IAA in the presence of 2,4-dichlorophenol, but fell into two groups with widely differing IAA oxidase/peroxidase ratios. The isoperoxidase patterns were independent of both genotype and phenotype for internode length, thus making it unlikely that these loci exert their effect on internode extension via control of synthesis of a particular isoperoxidase. Amongst the lines screened polymorphism was detected involving two of the isoperoxidases, and limited F₂ data suggest that these two variants fire determined by alleles of a single gene. Isoperoxidase patterns of stem extracts of 6 other Pisum species did not differ significantly from the two found in P. sativum.     

毛竹浸提液对苦槠幼苗生长的化感效应

为探讨毛竹(Phyllostachys edulis)扩张过程中潜在的化感作用,选择苦槠(Castanopsis sclerophylla(Lindl)Schott)为研究对象。采用水浸提的方法,用毛竹茎叶、枯落物和土壤3部分浸提液浇灌苦槠幼苗,以蒸馏水处理作为对照,对比分析质量浓度分别为0.1、0.05、0.02 g/mL的3个浓度梯度浸提液处理下苦槠幼苗生长指标及各项光合生理指标的差异。结果表明,毛竹浸提液对苦槠幼苗苗高、地径和叶绿素相对含量的影响大体上呈现高浓度抑制低浓度促进的双重浓度效应。不同来源毛竹浸提液的化感效应不尽相同,土壤浸提液对苦槠幼苗生长和光合生理均呈现抑制作用,而茎叶、枯落物浸提液低浓度时为促进作用。毛竹潜在的化感作用,在其扩张过程中可能会干扰森林主要树种更新,从而对森林群落产生威胁。     

Cysteine Protease Gene Expression and Proteolytic Activity During Floral Development and Senescence in Ethylene-insensitive Gladiolus grandiflora

This paper reports the changes in protein content and protease activity in the petals of ethylene-insensitive Gladiolus flowers, during development and senescence. A partial cDNA for putative cysteine protease (GgCyP) was cloned from Gladiolus using degenerate PCR primers and the expression pattern of this gene was determined semi-quantitatively by RT-PCR. There was a dramatic up-regulation in the expression of GgCyP at the incipient senescent stage of flower development indicating that this gene may encode an important enzyme for the proteolytic process in Gladiolus.     

Seasonal variation in antifouling activity of crude extracts of the brown alga Bifurcaria bifurcata (Cystoseiraceae) against cyprids of Balanus amphitrite and the marine bacteria Cobetia marina and Pseudoalteromonas haloplanktis

Previous studies have demonstrated that macroalgae from Brittany (France) contain products with antifouling activity against marine bacteria, fungi, diatoms, seaweeds and mussels. Little is known regarding the ecological function of these compounds and insufficient attention has been paid to evaluating the possible temporal variation in antifouling activity. Studies of chemical defenses in both terrestrial and marine organisms suggest that organisms vary widely in the production of chemical defenses associated with physical (temperature, light) and biological (e.g. grazing pressure) factors, season and geographical location. The present study aimed to investigate the antifouling activity of crude extracts of monthly collections of the brown alga, Bifurcaria bifurcata, against two marine bacteria, Cobetia marina and Pseudoalteromonas haloplanktis, and cypris larvae of the barnacle, Balanus amphitrite. The toxicity of the extracts was determined with a B. amphitrite nauplius assay.The antimicrobial activity of the extracts was found to be subject to seasonal variation, with the highest level of activity recorded from samples collected between April and September. Results of the anti-settlement experiments showed that the extracts of B. bifurcata (when tested from 0 to 100 μg/ml) can be divided into three groups on the basis of their minimum inhibitory concentrations (MICs): (1) extracts from plants collected from September to March reduced settlement at nontoxic concentrations (50-100 μg/ml); (2) extracts from plants collected from April to July (which were the most active extracts) reduced settlement significantly when tested at >5 μg/ml, but were toxic at 100 μg/ml; (3) the extract prepared from plants harvested in August was inhibitory at >25 μg/ml, but was toxic at 100 μg/ml. Toxicity tests on nauplii showed that LC₅₀ values of samples from the September to March collections were >100 μg/ml, demonstrating that they were nontoxic to nauplii. In contrast, samples obtained from the April to August collections were toxic to nauplii; the most toxic ones being from algae collected in May (LC₅₀=55.6 μg/ml) and in June (LC₅₀=38.3 μg/ml).The antifouling activity of extracts thus reached a peak in summer corresponding to maximal values for water temperature, light intensity and fouling pressure. It remains to be investigated whether this activity has an ecological role in the alga.     

Immunoglobulin isotype distribution of malaria-specific antibodies produced during infection with Plasmodium chabaudi adami and Plasmodium yoelii

The antibody response of mice to Plasmodium chabaudi adami and Plasmodium yoelii has been compared using a solid phase isotype-specific radioimmunoassay and sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Serological cross-reactivity between these parasites was substantial. Studies using a radioimmunoassay detecting all classes of malaria-specific antibody demonstrated that during the early part of infection it was not possible to distinguish between homologous and heterologous reactions. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that 50% or more of the protein antigens detected were apparently shared by both parasites although the intensity of bands was always greater with homologous reactions. However, the distribution of isotypes in the antibody (Ab) response differed in the two infections. P. chabaudi infections were characterized by a predominant and persistent IgM response, moderate IgG₂ and IgG₃ and little significant IgG₁ response during a primary infection. By contrast, IgM antibodies were transient in P. yoelii infection, IgG₂ was the predominant isotype, and both IgG₁ and IgG₃ antibodies were present during a primary infection. These differences in isotypes were also detected when sera were tested on the heterologous antigen extracts suggesting that antigens shared by P. chabaudi and P. yoelii do not necessarily induce similar antibody responses in the two infections.     

Experimental Neospora caninum infection in chickens (Gallus gallus domesticus) with oocysts and tachyzoites of two recent isolates reveals resistance to infection

The importance of birds in the biological cycle of Neospora caninum is not clear. We report unsuccessful Neospora infection in chickens (Gallus gallus domesticus) using two isolates of N. caninum. In experiment #1, 30 White Leghorn chickens were orally inoculated with viable N. caninum oocysts (NC-SP1 isolate, 200 oocysts per bird) via the crop at 21 days of age. Groups of three birds were euthanised at intervals of 7 days (a total of 9 weeks) and one group was challenged with the same oocyst dose at 37 days p.i. and observed for 11 weeks. Blood samples were collected weekly, and sera were tested using IFAT. Chicken tissues were collected for PCR, quantitative PCR and immunohistochemistry. Two dogs approximately 45 days of age were fed with tissues from chickens euthanised at 138 and 159 days p.i. The results indicated that the chickens were resistant to neosporosis as revealed by failure to seroconvert, to detect parasite DNA or N. caninum antigen by immunohistochemistry in inoculated bird tissues, and by no oocyst excretion by the dogs fed avian tissues. Similar results were obtained in experiment #2, in which 34 1-week-old chickens were each s.c. inoculated with 100,000 tachyzoites of the NcWTDMn1 isolate of N. caninum. The chickens were euthanised on days 7, 15, 22, 28, 36 and 60 p.i. At necropsy, all tissues and serum from each bird were collected. All chickens remained asymptomatic, and N. caninum antigen was not detected by immunohistochemistry. Seven chickens euthanised at day 60 p.i. demonstrated low (1:25 dilution) levels of antibodies by using the Neospora agglutination test. Two 12-week-old dogs fed tissues pooled from 10 inoculated chickens euthanised at day 60 p.i. did not excrete N. caninum oocysts. This investigation indicates that chickens are resistant to experimental infection by N. caninum.     

Peppermint (Mentha piperita) inhibits microbial biofilms in vitro

Microbial biofilms have become increasingly problematic in the food processing and medical industries where they cause food and surface contamination. Biofilms have also been implicated as the cause of serious infections in humans as their occurrence makes it difficult to treat common infections and the likelihood of recurrent infections is high. Due to emerging resistance, conventional control methods are fast becoming ineffective. In this study, the use of a selection of commercial plant extracts is investigated. The inhibitory effects of eight herbal extracts on the development of microbial biofilms was investigated against clinical and reference strains of Pseudomonas aeruginosa and Candida albicans. The antimicrobial activity was investigated on the planktonic forms using the minimum inhibitory concentration assay. The extracts that showed the highest antimicrobial activity against the two test organisms were Echinacea angustifolia (cone flower), Mentha piperita (peppermint) and Rosmarinus officinalis (rosemary) with minimum inhibitory concentration values between 0.38 and 2.5 mg/ml. The crystal violet assay was used to assess the effect of pre-treating a surface with plant extracts on cell attachment and the extent of biofilm development following exposure to extracts (biofilm biomass). Most of the extracts reduced microbial colonization by at least 50%. In contrast, preformed biofilms were less responsive to the majority of extracts, thus growth inhibition was more difficult to achieve. Mentha piperita was the only extract that showed some antibiofilm activity against both pathogens.     

Schistosoma mansoni: Lysozyme activity in Biomphalaria glabrata during infection with two strains

Levels of lysozyme activity were determined in the hemolymph, digestive gland, and headfoot extracts of M-line stock of snails, Biomphalaria glabrata, during infection with the PR-1 and Lc-1 strains of the trematode, Schistosoma mansoni. At 3 hr postexposure there was a 10-fold increase in the levels of enzyme activity in the hemolymph of snails infected with the Lc-1 strain to which the snail is resistant. This increase was considerably higher when compared to the threefold increase in the PR-1-infected snails. The infection also induced a gradual depletion of lysozyme activity in the headfoot muscles of the two groups of infected snails. There were no changes in the levels of enzyme activity in the digestive gland extracts of the control and the two groups of infected snails. Similar changes in the levels of enzyme activity in the hemolymph and headfoot extracts of infected snails suggest a nonspecific response to a parasite infection and do not indicate that lysozyme is primarily responsible for the destruction of schistosome parasite in a resistant snail host.     

Dianthramides A and B,two N-benzoylanthranilic acid derivatives from elicited tissues of Dianthus caryophyllus

Two new phenolic derivatives, dianthramide A and B, were isolated from Dianthus caryophyllus tissues elicited with mycelial extracts of Phytophthora parasitica. The purified substances were identified on the basis of their spectral data and were characterized as N-salicyl-4-methoxyanthranilic acid (dianthramide A) and N-salicyl-4-hydroxyanthranilic acid methyl ester (dianthramide B). Dianthramides A and B co-occur in carnation tissues with the known phytoalexin dianthalexin.     

浒苔干粉末提取物对东海原甲藻和中肋骨条藻的克生作用

研究不同溶剂的浒苔干粉末组织提取液对两种赤潮藻—东海原甲藻和中肋骨条藻生长的克生效应。结果表明, 浒苔提取物中确实含有可以影响赤潮藻类生长的克生物质,克生作用具有较明显的浓度效应,添加浓度低时可能会表现为一定的促进生长的作用,添加浓度较高时表现为抑制作用,添加浓度越大,抑制作用越强,即“低促高抑”的特点,这与浒苔提取物对其他微藻的作用相似。其中,蒸馏水提取物对这两种赤潮藻的克生作用小于有机溶剂提取物,有机溶剂中,甲醇和乙酸乙酯提取物对这两种赤潮藻的克生效果最好,正己烷相对较差。根据相似相溶原理,可以初步推断最有效的克生物质应为具有相对较高的极性的有机物。两种赤潮藻对克生物质的敏感程度不同。东海原甲藻对克生物质的敏感性高于中肋骨条藻。蒸馏水、甲醇、乙酸乙酯、正己烷的浒苔干粉末提取物影响东海原甲藻生长的致死作用阈值浓度分别为5.00 g/L、0.50 g/L、0.50 g/L、0.63 g/L（相当于浒苔新鲜藻体浓度为22.00 g-wet/L、2.20 g-wet/L、2.20 g-wet/L、2.75 g-wet/L）;影响中肋骨条藻生长的致死作用阈值浓度则分别为20.00 g/L、1.25 g/L、1.25 g/L、2.50 g/L（相当于新鲜藻体浓度为88.00 g-wet/L、5.50 g-wet/L、5.50 g-wet/L、11.00 g-wet/L）。     

Volatile cephalic substances of the stingless bees, Trigona mexicana and Trigona pectoralis

Extracts of the heads of the stingless bees, Trigona mexicana and T. pectoralis, contain mixtures of compounds that are identifiable by gas chromatography and mass spectrometry. These compounds form homologous series of aliphatic alcohols and ketones with an odd number of carbon atoms and functional groups at the 2-position. The alcohols and the ketones range from 7 to 17 carbon atoms. Benzaldehyde and a nitrogen containing compound are also present in the mixtures. The series of compounds from the two species are nearly identical qualitatively. They differ in the absence of 2-undecanol and 2-pentadecanol from the extracts of T. mexicana and T. pectoralis, respectively. The highest concentration of material is found in the 7-carbon fraction in T. mexicana and in the 13 to 15 carbon range in T. pectoralis. There is a major difference in the relative concentration of 2-heptanol and 2-heptanone in the two species with the concentration of the alcohol being one-fourth that of 2-heptanone in T. mexicana and ten times greater than the ketones in T. pectoralis.Both the alcohols and ketones are alarm pheromones. The alcohols are more active in inducing attack by the bees than are the ketones, but a mixture of the ketones and benzaldehyde was very active.