Identification of Somatic Proteins in Haemonchus Contortus Infective Larvae (L3) and Adults.

Haemonchus contortus is considered the most pathogenic nematode in sheep production systems based on grazing. Comparing infective larvae (L3) with adult parasites can lead to the identification of proteins that play an important role in parasite-host interactions. In this study, we report a list of H. contortus somatic proteins and made a comparative analysis of somatic proteins of L3 and adult worms. L3 and adult parasites were subjected to protein extraction and subsequently to peptide fractionation. Peptides were analysed by mass spectrometry and LC-MS/MS data analysis. Data analysis and search on SEQUEST and MASCOT against H. contortus from the WormBase ParaSite database resulted in the identification of 775 unique peptide sequences corresponding to 227 proteins at 1 % FDR. From these, 18 proteins were specific to L3 and 63 to adult parasites. The gene ontology (GO) enrichment analysis of the proteins specific to L3 and adult worms to gain insight into cellular components, molecular functions and biological processes that affect the parasite-host interaction showed some differences between the two parasite stages. The list of proteins found provides a database to identify target proteins that could be useful as biomarkers of the infection or in the generation of anthelmintic drugs that inhibit proteins essential for the establishment of the infection and the survival of adult parasites. They can also serve as new candidates for vaccine research.

Green Roots: Photosynthesis and Photoautotrophy in an Underground Plant Organ.

The potential for photosynthetic and photoautotrophic growth was studied in hairy root cultures of Asteraceae and Solanaceae species. Upon transfer to light, initially heterotrophic root cultures of Acmella oppositifolia and Datura innoxia greened rapidly, differentiated chloroplasts, and developed light-dependent CO2 fixation in the cortical cells. Photosynthetic potential was expressed in root cultures of all the Asteraceae genera examined (Acmella, Artemisia, Rudbeckia, Stevia, and Tagetes). Hairy roots of A. oppositifolia and D. innoxia were further adapted to photoautotrophy by growing in the presence of light and added CO2 (1-5%) and by direct or sequential transfers into media containing progressively lower sugar concentrations. The transition to photoautotrophy was accompanied by an increase in CO2 fixation and in the specific activity of 1,5-ribulose-bisphosphate carboxylase/ oxygenase (Rubisco). During the adaptation of A. oppositifolia roots to photoautotrophy, the ratio of Rubisco to phosphoenolpyruvate carboxylase increased significantly, approaching that found in the leaves. The levels and patterns of alkaloids and polyacetylenes produced by Solanaceae and Asteraceae hairy roots, respectively, were dramatically altered in photomixotrophic and photoautotrophic cultures. Photoautotrophic roots of A. oppositifolia have been mainitained in vitro for over 2 years.

Pointing to potential reference areas to assess soil mutagenicity.

Several have been performed to evaluate the mutagenicity of soil samples in urban and industrial areas. The use of uncontaminated reference areas has been an obstacle to the study of environmental mutagenesis. The study aimed to indicate a methodology to define reference areas in studies of environmental contamination based on "Ambient Background Concentration" of metallic elements associated with the Salmonella/microsome assay. We looked at three potential reference areas, two of them close by the industrial sources of contamination (São Jerônimo reference, near the coal-fired power plant, and Triunfo reference, near the wood preservative plant), but not directly influenced by them and an area located inside a protected area (Itapuã reference). We also carried out chemical analyses of some metals to plot the metal profile of these potential reference areas and define basal levels of these metals in the soils. After examining the mutagenicity of the inorganic extracts using strains TA98, TA97a, and TA100, in the presence and absence of S9 mix, we indicated the São Jerônimo reference and the Itapuã reference as two sites that could be used in future studies of mutagenicity of soils in southern Brazil. The association between a mutagenicity bioassay and the "Ambient Background Concentration" seems to be a useful method to indicate the reference areas in studies of contamination by environmental mutagens, where these results were corroborated by canonical correspondence analysis.

Differential gene expression during somatic embryogenesis in Coffea arabica L., revealed by RT-PCR differential display.

Molecular and biochemical studies of somatic embryogenesis may help to shed light on the mechanisms governing this phenomenon. In this article, a differential display analysis approach was employed to investigate the changes taking place during the induction of somatic embryogenesis in leaf explants and suspension cultures of coffee. Cloned fragments show homologies to several proteins reported in databases, but only one has previously been described as regulated during somatic embryogenesis. By a reverse dot blot modification, the expression pattern of such fragments was evaluated.

Cholinergic facilitation of erection and ejaculation in spinal cord-transected rats.

Penile reflexes (PRs) were monitored in chronic spinal cord-transected rats by identifying them visually, and at the same time they were recorded as the electromyographic activity of bulbospongiosus muscles. Intraperitoneal injection of the agonist muscarine (10 microg) produced a facilitation of PRs. A decrease in the latency, an increase in the number of clusters and often an increase in the duration of cups were found after muscarine. In addition, 66% (six out of nine) of the animals ejaculated after muscarine. These results suggest that cholinergic receptor stimulation may be involved in erectile and ejaculatory mechanisms mediated by the spinal cord.

Inhibition of L-aromatic amino acid decarboxylase by polychlorinated biphenyls.

PC12 cells were used to examine the mechanisms by which polychlorinated biphenyls (PCBs) reduce cellular levels of dopamine (DA). In cells treated 3 days with Aroclor 1254, 2,2',5,5'-tetrachlorobiphenyl (2,2',5,5'-TCB), or 2,2',3,3',4,4'-hexachlorobiphenyl (2,2',3,3',4,4'-HCB), the PCB-mediated reduction in 3H-tyrosine uptake was observed only at high PCB concentrations that produced a reduction in DNA levels. The PCB congener, 2,2',4,4',5,5'-hexachlorobiphenyl (2,2',4,4',5,5'-HCB) did not produce a reduction in 3H-tyrosine uptake at any concentration tested. Thus, there were PCB concentrations at which a reduction in DA levels did not coincide with a decrease in 3H-tyrosine uptake, suggesting that inhibition of tyrosine uptake was not the primary mechanism by which PCBs reduce DA levels. Aroclor 1254-treated cells also exhibited elevated levels of DOPA, further supporting the conclusion that tyrosine levels were not limiting. Incubation of Aroclor 1254-pretreated cells with 3H-tyrosine resulted in a dose-dependent increase in cellular levels of 3H-DOPA and decrease in cellular levels of 3H-DA, suggesting a PCB-mediated inhibition of the conversion of 3H-DOPA to 3H-DA. When the media was supplemented with DOPA, Aroclor 1254-treated cells still exhibited reduced levels of DA, compared to control cells, even though the control and PCB-treated cells had similar cellular levels of DOPA. Thus, one mechanism by which PCBs may reduce cellular levels of DA is by inhibiting L-aromatic amino acid decarboxylase-mediated conversion of DOPA to DA. The PCB congeners, 2,2',4,4'-TCB, 2,2',5,5'-TCB, and 2,2',4,4',5,5'-HCB, also produced dose-dependent increases in DOPA levels. The congener 2,2',3,3',4,4'-HCB did not produce an increase in DOPA levels, although it did mediate reductions in cellular DA levels. However, when PC12 cells were supplemented with DOPA, all four PCB congeners produced a similar reduction in DA levels, suggesting that the conversion of DOPA to DA was inhibited by the PCBs.

Use of two short-term tests to evaluate the genotoxicity of river water treated with different concentration/extraction procedures.

The genotoxicity of river water samples was evaluated by the Salmonella mutagenicity assay and by the microscreen phage-induction assay. Different processes of sample treatment were compared using the following assays: different volumes of a non-concentrated sample (direct method); concentrated sample fractionated into portions with acid, basic and neutral activity (liquid-liquid extraction method); sample submitted to extraction of volatile substances (volatile extraction method). Samples that were positive to the Salmonella assay by the direct concentration method lost this activity after liquid-liquid extraction. This difference was related to the loss of substances that volatilize during the extraction process. The study of volatile product concentrates confirmed the role of these compounds in inducing activity present in some samples. The microscreen phage-induction assay proved to be a good screening assay for genotoxic compounds present in small concentration in environmental samples. We conclude that, whenever possible, samples should be treated by the direct method in different volumes to prevent the loss of genotoxic substances.

Mutagenic activity detected by the Ames test in river water under the influence of petrochemical industries.

The present study was carried out on the waters of the Caí River (Rio Grande do Sul, Brazil) in an area under the influence of a petrochemical industrial complex, as the continuation of a study in which the mutagenic activity of water samples was evaluated in the internal area of this complex. In the previous study, the release of inducing substances was detected, revealing the need for a full analysis of the real ecological impact of the industrial complex on the river. Water samples from different sites along the Caí River were subjected to the Ames test during a study of 20 months duration for the detection of possible mutagens. Strains TA100 and TA98 were used for initial sample screening in the presence and absence of the S9 mix at a standard dose of 2000 microliters/plate. When positive activity (values equal to twice the spontaneous mutation rate) and/or cytotoxic activity (cell survival below 60%) was detected, the dose-response relationship was studied. Thirty-four percent of the samples tested were mutagenic, with different values according to collection site. Of the total number of positive responses, 6% were obtained for samples collected at the blank site upstream from the area studied, 82% at sites closest to the industrial complex, and 12% in downstream areas. Strain TA98 was the most sensitive in assays with no metabolic activation. A low frequency of induction (2%) was observed for strain TA102. Application of the Ames test permitted the delimitation of three areas of influence of the petrochemical industrial complex, and the test proved to be adequate for the detection of contaminants from the petrochemical industry.

Mutagenic and genotoxic effects of aqueous extracts of Achyrocline satureoides in prokaryotic organisms.

Aqueous extracts of Achyrocline satureoides (Marcela and/or Macela) were tested for the presence of genotoxic activity in microorganisms. This species belongs to the family Compositae and is used on a large scale by the population of South Brazil. The extracts showed genotoxic activity in the presence of S9 mix in the Ames test TA100, TA98 and TA102 strains, 'SOS' spot chromotest and Microscreen phage-induction assay. The positive results were related to the presence of quercetin and caffeic acid in the aqueous extracts.

Genotoxicity of the boldine aporphine alkaloid in prokaryotic and eukaryotic organisms.

The aporphine alkaloid boldine, present in Peumus boldus (boldo-do-Chile) widely used all over the world, was tested for the presence of genotoxic, mutagenic and recombinogenic activities in microorganisms. This alkaloid did not show genotoxic activity with or without metabolic activation in the SOS chromotest and Ames tester strains TA100, TA98 and TA102. It was not able to induce point and frameshift mutations in haploid Saccharomyces cerevisiae cells. However, mitotic recombinational events such as crossing-over and gene conversion were weakly induced in diploid yeast cells by this alkaloid. Also, boldine was able to induce weakly cytoplasmic 'petite' mutation in haploid yeast cells.

Genotoxicity assessment in aquatic environments under the influence of heavy metals and organic contaminants.

The genotoxicity of river water and sediment including interstitial water was evaluated by microscreen phage-induction and Salmonella/microsome assays. Different processes used to fractionate the sediment sample were compared using solvents with different polarities. The results obtained for mutagenic activity using the Salmonella/microsome test were negative in the water and interstitial water samples analysed using the direct concentration method. The responses in the microscreen phage-induction assay showed the presence of genotoxic or indicative genotoxic activity for at least one water sample of each site analysed using the same concentration method. Similar results were obtained for interstitial water samples, i.e. absence of mutagenic activity in the Salmonella/microsome test and presence of genotoxic activity in the microscreen phage-induction assay. Metal contamination, as evidenced by the concentrations in stream sediments, may also help explain some of these genotoxic results. Stream sediment organic extracts showed frameshift mutagenic activity in the ether extract detected by Salmonella/microsome assay. The concentrates evaluated by microscreen phage-induction assay identified the action of organic compounds in the non-polar, medium polar and polar fractions. Thus, the microscreen phage-induction assay has proven to be a more appropriate methodology than the Salmonella/microsome test to analyse multiple pollutants in this ecosystem where both organic compounds and heavy metals are present.

Chemicals from roots, hairy roots, and their application.

Plants produce thousands of different compounds through the secondary metabolism pathways. Since many of these products are obtained by direct extraction from plants that are cultivated in the field or some times even collected in their original habitat several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution to our understanding of secondary metabolism. Furthermore, a new alternative has arisen: transformed root cultures. Until now, hairy roots have been obtained from more than 100 different species. The products that they are able to produce range from alkaloids to aromatic compounds and dyes. These kinds of cultures have turned out to be an invaluable tool to study the biochemistry and the gene expression of the metabolic pathways in order to elucidate the intermediaries and enzymes involved in the biosynthesis of secondary metabolites.

Screening of plants used in south Brazilian folk medicine.

Thirty-seven species of medicinal plants used in folk phytotherapy were chemically screened for alkaloids, coumarins, flavonoids, anthraquinones, saponins, sterols and/or triterpenes and tannins. Seven of these were also screened for the presence of mutagenic activity using the Ames test (Salmonella/microsome).

Occurrence of the bat tick Ornithodoros (Alectorobius) Kelleyi Cooley & Kohls (Acari: Argasidae) in Costa Rica and its relation to human bites.

Several persons from two localities in Costa Rica (San Rafael de Coronado, 1510 m and Dulce Nombre de Tres Ríos, 1445 m) were bitten by ticks, subsequently identified as Ornitohodoros (A.) Kelleyi (Argasidae), commonly associated with bats. In the attics of their homes, live adults, larvae, nymphs and skins were found in the bat guano. Since there are no records of adult argasids in this country, a laboratory colony was established to observe the biology of these ticks and their association with bats. In this particular case, the bats were Molossidae and Vespertilionidae, both insectivorous. Adults and last nymphal ticks easily fed on suckling white mice in the dark; feeding lasts from 20 to 40 minutes, when abundant coxal fluid is produced; oviposition occurs from 14 to 27 days later. Comparison of the most common structures of both whole larvae and larval skins revealed no significant differences, taxonomically important, since according to the habits of the Argasidae, it is easier to find larval molts than live specimens.

Effects of bacterial infestation caused by human wastes on the skin structures of Mugil platanus Günther, 1880 (Mugilidae).

Manifestation of infectious pathologies in fishes usually increases in environments where organic wastes are disposed. Specimens of Mugil platanus Günther, 1880 and water samples collected at three points of the Tramandaí river were analyzed during a one year period. The macroscopic observation revealed ulcerations in the caudal peduncle area covered with a mass of amorphous and whitened tissues. Histopathologic analysis showed the presence of negative gram bacteria, probably responsible for alterations of the normal structure of the epidermic tissues. Non-parametric statistical analysis for ammonia concentration showed a significant variation among the three collected spots as well as in the multiple comparison between two spots. In this study, we describe cutaneous lesions observed in Mugil platanus specimens and tested their correlation with environmental ammonia concentration.

Genotoxicity of plant extracts.

Aqueous extracts of seven species used in Brazilian popular medicine (Achyrocline satureoides, Iodina rhombifolia, Desmodium incanum, Baccharis anomala, Tibouchina asperior, Luehea divaricata, Maytenus ilicifolia) were screened to the presence of mutagenic activity in the Ames test (Salmonella/microsome). Positive results were obtained for A. satureoides, B. anomala and L. divaricata with microsomal activation. As shown elsewhere (Vargas et al., 1990) the metabolites of A. satureoides extract also show the capacity to induce prophage and/or SOS response in microscreen phage induction assay and SOS spot chromotest.

Mutagenicity of medicinal plant extracts in Salmonella/microsome assay.

Aqueous extracts of medicinal plants used in south Brazilian folk medicine (Myrciaria tenella, Smilax campestris, Tripodanthus acutifolius and Cassia corymbosa) were screened for the presence of mutagenic activity in the Salmonella/microsome assay. Signs of an increased mutagenic induction below revertant rates, which is two times the spontaneous yields, were detected for all extracts. The extracts of C. corymbosa showed signs of mutagenic activity in the following strains: TA100 with and without metabolization and TA98 after metabolization. These mutagenic signs were observed in the M. tenella extracts without metabolization. S. campestris and T. acutifolius infusions also showed signs indicative of direct mutagenic activity in the TA98 strain. The positive results may be related to the presence of flavonoids, tannins and anthraquinones in the extracts.

Mutagenic and antimutagenic potential of the medicinal plants M. laevigata and C. xanthocarpa.

Aqueous extracts of medicinal plants (Mikania laevigata and Campomanesia xanthocarpa) were screened for the presence of mutagenic activity in the Salmonella/microsome assay. The extracts of Campomanesia xanthocarpa showed frameshift (TA97a strain) signs of mutagenic activity without exogenous metabolism (S9 fraction). The infusions of Mikania laevigata, negative for mutagenic activity, showed high percentages of inhibition of mutagenesis induced by mutagens 2AF (2-amino fluorene), in the presence of exogenous metabolism (S9 fraction), for frameshift (TA98) and base pair substitution (TA100) lesions. In addition, these inhibitions were observed against mutagen SAZ (sodium azide) in assays with the TA100 strain, without exogenous metabolism (S9 fraction). A synergistic effect was also observed in frameshift mutagenic events, with direct action in the presence of 4NQO (4-oxide-1-nitroquinoline) and a tendency to a low percentage of action enhancement, in the presence of the 2AF mutagen. The variable responses observed in the extract assays show the potentials for interaction of the different active principles in genetic material.

Erythrocyte deformability in the fetus, preterm, and term neonate.

Deformability of red blood cells (RBC) is an important determinant of microcirculation, of oxygen transport and release to the tissues, and of RBC life span. Deformability of RBC from five fetuses, 20 preterm infants, 20 term neonates, and 20 adults was determined by direct microscopic observation of RBC subjected to shear stresses of 6 to 85 dyn/cm2 using a counter-rotating rheoscope. There was no significant difference in deformability among RBC from the fetuses, the preterm and term neonates, and the adults at any shear stress. More than 95% of fetal, neonatal, and adult RBC were capable of tank-tread motion. Compared to adults, the frequency distribution of RBC deformability was slightly broader in the fetuses and neonates because of the presence of more highly and poorly deformable RBC. The increased number of rigid RBC may contribute to the shortened life span of fetal RBC.

Relationship between atmospheric and urinary nickel in workers manufacturing electrical resistances using nickel oxide: role of the bioavailability of nickel.

The daily concentrations of nickel in total (ie inhalable) and respirable airborne dust (personal sampling) and in post-shift and pre-shift urine samples were monitored during five consecutive work days in 20 workers exposed to NiO in a workshop manufacturing electrical resistances. The individual daily atmospheric nickel concentrations ranged from 0.5 to 9586 micrograms Ni/m3 (geometric mean 22.9) for total dust and from 0.2 to 332 micrograms Ni/m3 (geometric mean 3.5) for respirable dust. The results of the urinary excretion of nickel suggested that the occupationally-related systemic absorption of nickel strongly differed in one subject (worker E) compared to the other 19 workers. In the latter group the nickel concentration in urine never exceeded 5 micrograms Ni/g creatinine, it did not differ between post-shift and pre-shift samples (geometric means: 1.1 versus 1.2 micrograms Ni/g creatinine), and it was only slightly higher than that measured in a group of 17 non-exposed subjects (mean 0.5 micrograms Ni/g creatinine; range 0.1-1.7); furthermore their nickel elimination in urine did not change during the days off or after two weeks of holiday. In worker E, the nickel concentration ranged from 21 to 101 micrograms Ni/g creatinine in post-shift urine, the next morning (after 16 h) it had dropped on average by 50 per cent, it decreased further during the days off, and amounted still to 4.4 micrograms Ni/g creatinine after two weeks of holiday. These divergent patterns of elimination of nickel in urine are most likely related to differences in the nature of exposure to airborne nickel involving both particle size and bioavailability of nickel. Worker E was exposed to NiO powder of 1-8 microns particle size resulting in nickel levels of the respirable fraction on average about 50 times that measured for the 19 other workers (3 micrograms Ni/m3). Transformation of the initial NiO powder into particles of 150 to 600 microns size and associated changes in physicochemical properties of NiO in the particles of the respirable fraction may explain why the urinary excretion of nickel in the 19 workers is hardly influenced by their occupational exposure to this metal. The pattern of urinary nickel elimination in worker E, however, most likely reflects very recent exposure to NiO, suggesting that the degree of bioavailability of nickel from this particular physicochemical form of NiO powder is much higher than that usually accepted for poorly soluble nickel compounds.