Ecotoxicological effect of zinc pyrithione in the freshwater fish Gambusia holbrooki.

Currently diverse biocidal agents can be used for distinct applications, such as personal hygiene, disinfection, antiparasitic activity, and antifouling effects. Zinc pyrithione is an organometallic biocide, with bactericidal, algicidal and fungicidal activities. It has been recently incorporated in antifouling formulas, such as paints, which prevent the establishment of a biofilm on surfaces exposed to the aquatic environment. It has also been used in cosmetics, such as anti-dandruff shampoos and soaps. Previously reported data has shown the presence of this substance in the aquatic compartment, a factor contributing to the potential exertion of toxic effects, and there is also evidence that photodegradation products of zinc pyrithione were involved in neurotoxic effects, namely by inhibiting cholinesterases in fish species. Additional evidence points to the involvement of zinc pyrithione in alterations of metal homeostasis and oxidative stress, in both aquatic organisms and human cell models. The present work assesses the potential ecotoxicity elicited by zinc pyrithione in the freshwater fish Gambusia holbrooki after an acute (96 h) exposure. The oxidative stress was assessed by the quantification of the activities of specific enzymes from the antioxidant defense system, such as catalase, and glutathione-S-transferases; and the extent of peroxidative damage was quantified by measuring the thiobarbituric acid reactive substances levels. Neurotoxicity was assessed through measurement of acetylcholinesterase activity; and a standardized method for the description and assessment of histological changes in liver and gills of was also used. Zinc pyrithione caused non-specific and reversible tissue alterations, both in liver and gills of exposed organisms. However, histopathological indices were not significantly different from the control group. In terms of oxidative stress biomarkers, none of the tested biomarkers indicated the occurrence of pro-oxidative effects, suggesting that the oxidative pathway is not the major toxicological outcome of exposure to zinc pyrithione.

Acute effects of tetracycline exposure in the freshwater fish Gambusia holbrooki: antioxidant effects, neurotoxicity and histological alterations.

A large body of evidence was compiled in the recent decades showing a noteworthy increase in the detection of pharmaceutical drugs in aquatic ecosystems. Due to its ubiquitous presence, chemical nature, and practical purpose, this type of contaminant can exert toxic effects in nontarget organisms. Exposure to pharmaceutical drugs can result in adaptive alterations, such as changes in tissues, or in key homeostatic mechanisms, such as antioxidant mechanisms, biochemical/physiological pathways, and cellular damage. These alterations can be monitored to determine the impact of these compounds on exposed aquatic organisms. Among pharmaceutical drugs in the environment, antibiotics are particularly important because they include a variety of substances widely used in medical and veterinary practice, livestock production, and aquaculture. This wide use constitutes a decisive factor contributing for their frequent detection in the aquatic environment. Tetracyclines are the individual antibiotic subclass with the second highest frequency of detection in environmental matrices. The characterization of the potential ecotoxicological effects of tetracycline is a much-required task; to attain this objective, the present study assessed the acute toxic effects of tetracycline in the freshwater fish species Gambusia holbrooki by the determination of histological changes in the gills and liver, changes in antioxidant defense [glutathione S-transferase (GST), catalase (CAT), and lipoperoxidative damage] as well as potential neurotoxicity (acetylcholinesterase activity). The obtained results suggest the existence of a cause-and-effect relationship between the exposure to tetracycline and histological alterations (more specifically in gills) and enzymatic activity (particularly the enzyme CAT in liver and GST in gills) indicating that this compound can exert a pro-oxidative activity.

Ecotoxicological effects of salicylic acid in the freshwater fish Salmo trutta fario: antioxidant mechanisms and histological alterations.

The presence of pharmaceutical drugs in aquatic ecosystems has been widely reported during the past years. Salicylic acid (SA) is mainly used in human medicine as an analgesic and antipyretic drug, being also active in preventing platelet aggregation. To study the ecotoxicological effects potentially elicited by SA in freshwater fish, brown trout individuals (Salmo trutta fario) were chronically exposed (28 days) to this drug, in order to evaluate the enzymatic and histological effects, in both gills and liver. A qualitative and semi-qualitative evaluation of the gills and liver was performed, and also a quantitative evaluation of various lamellar structures. Oxidative stress was quantified trough the determination of glutathione S-transferases (GSTs), glutathione reductase (GRed), total and selenium-dependent glutathione peroxidase (GPx) and Catalase (Cat) activities. Lipid peroxidative damage was also assessed by the quantification of thiobarbituric acid reactive substances (TBARS) in the liver. The here-obtained data showed the occurrence of oxidative stress, reflected by an increased activity of GPx and GRed in the liver; additionally, it was possible to observe non-specific histological changes in gills. The global significance of the entire set of results is discussed, giving emphasis to the ecological relevance of the responses.

Substitution of crude cell wall for neutral detergent fibre in the equations of the Cornell Net Carbohydrate and Protein System that predict carbohydrate fractions: application to sunflower ( Helianthus annuus L.).

Prediction of carbohydrate fractions using equations from the Cornell Net Carbohydrate and Protein System (CNCPS) is a valuable tool to assess the nutritional value of forages. In this paper, these carbohydrate fractions were predicted using data from three sunflower (Helianthus annuus L.) cultivars, fresh or as silage. The CNCPS equations for fractions B2 and C include measurement of ash and protein-free neutral detergent fibre (NDF) as one of their components. However, NDF lacks pectin and other non-starch polysaccharides that are found in the cell wall (CW) matrix, so this work compared the use of a crude CW preparation instead of NDF in the CNCPS equations. There were no differences in the estimates of fractions B1 and C when CW replaced NDF; however, there were differences in fractions A and B2. Some of the CNCPS equations could be simplified when using CW instead of NDF. Notably, lignin could be expressed as a proportion of DM, rather than on the basis of ash and protein-free NDF, when predicting CNCPS fraction C. The CNCPS fraction B1 (starch + pectin) values were lower than pectin determined through wet chemistry. This finding, along with the results obtained by the substitution of CW for NDF in the CNCPS equations, suggests that pectin was not part of fraction B1 but present in fraction A. We suggest that pectin and other non-starch polysaccharides that are dissolved by the neutral detergent solution be allocated to a specific fraction (B2) and that another fraction (B3) be adopted for the digestible cell wall carbohydrates.

Programmed cell death in the larval salivary glands of Apis mellifera (Hymenoptera, Apidae).

The morphological and histochemical features of degeneration in honeybee (Apis mellifera) salivary glands were investigated in 5th instar larvae and in the pre-pupal period. The distribution and activity patterns of acid phosphatase enzyme were also analysed. As a routine,the larval salivary glands were fixed and processed for light microscopy and transmission electron microscopy.Tissue sections were subsequently stained with haematoxylin -eosin,bromophenol blue,silver,or a variant of the critical electrolyte concentration (CEC) method.Ultrathin sections were contrasted with uranyl acetate and lead citrate.Glands were processed for the histochemical and cytochemical localization of acid phosphatase,as well as biochemical assay to detect its activity pattern. Acid phosphatase activity was histochemically detected in all the salivary glands analysed.The cytochemical results showed acid phosphatase in vesicles, Golgi apparatus and lysosomes during the secretory phase and,additionally, in autophagic structures and luminal secretion during the degenerative phase. These findings were in agreement with the biochemical assay. At the end of the 5th instar, the glandular cells had a vacuolated cytoplasm and pyknotic nuclei, and epithelial cells were shed into the glandular lumen.The transition phase from the 5th instar to the pre-pupal period was characterized by intense vacuolation of the basal cytoplasm and release of parts of the cytoplasm into the lumen by apical blebbing; these blebs contained cytoplasmic RNA, rough endoplasmic reticule and, occasionally, nuclear material. In the pre-pupal phase, the glandular epithelium showed progressive degeneration so that at the end of this phase only nuclei and remnants of the cytoplasm were observed.The nuclei were pyknotic,with peripheral chromatin and blebs. The gland remained in the haemolymph and was recycled during metamorphosis. The programmed cell death in this gland represented a morphological form intermediate between apoptosis and autophagy.

Oviposition sequence and offspring of mated and virgin females of Cotesia flavipes (hymenoptera: braconidae) parasitizing Diatraea saccharalis larvae (lepidoptera: crambidae)

Large scale mass rearing of natural enemies has been a mean of improving biological control in the sugarcane intensive agriculture. Among them, Cotesia flavipes, a gregarious koinobiont endoparasitoid, was imported by Brasil to control caterpillars of the sugarcane borer Diatraea saccharalis. The C. flavipes larval development depends on its association with polydnavirus, which blocks the host defense reaction. To verify if the oviposition sequence (1st, 2nd or 3rd) and the female condition (mated or virgin) interfere in the number of C. flavipes descendents, 4th instar caterpillars of D. saccharalis were parasitized. Analysis of the data showed that: a) there is an inverse correlation between the parasitism efficiency and the host reaction (encapsulation); b) the number of caterpillars parasitized by virgin females that released parasitoid larvae in the period from 12 to 15 days was higher than that of caterpillars parasitized by mated females; c) a slight difference between mated and virgin females in relation to the parasitim success was observed; and d) the number of encapsulated parasitoid larvae was higher than that of eggs, suggesting that eggs have a better capacity to overcome the host reaction. In this study, the viability of C. flavipes eggs and larvae in the non-specific host D. saccharalis could be correlated with the oviposition sequence and the female condition

A new firefly luciferase with bimodal spectrum: identification of structural determinants of spectral pH-sensitivity in firefly luciferases.

Fireflies emit flashes in the green-yellow region of the spectrum for the purpose of sexual attraction. The bioluminescence color is determined by the luciferases. It is well known that the in vitro bioluminescence color of firefly luciferases can be shifted toward the red by lower pH and higher temperature; for this reason they are classified as pH-sensitive luciferases. However, the mechanism and structural origin of pH sensitivity in fireflies remains unknown. Here we report the cloning of a new luciferase from the Brazilian twilight active firefly Macrolampis sp2, which displays an unusual bimodal spectrum. The recombinant luciferase displays a sensitive spectrum with the peak at 569 nm and a shoulder in the red region. Comparison of the bioluminescence spectra of Macrolampis, Photinus and Cratomorphus firefly luciferases shows that the distinct colors are determined by the ratio between green and red emitters under luciferase influence. Comparison of Macrolampis luciferase with the highly similar North American Photinus pyralis luciferase (91%) showed few substitutions potentially involved with the higher spectral sensitivity in Macrolampis luciferase. Site-directed mutagenesis showed that the natural substitution E354N determines the appearance of the shoulder in the red region of Macrolampis luciferase bioluminescence spectrum, helping to identify important interactions and residues involved in the pH-sensing mechanism in firefly luciferases.

Haemolymph electrophoretic pattern of Ascia monuste orseis larvae (Lepidoptera: Pieridae) parasitized by Cotesia glomerata (Hymenoptera: Braconidae)

Cotesia glomerata is a natural enemy of the vegetable plague Ascia monuste orseis and preferably parasites 2nd, 3rd and 4th instar larvae. Parasitism effects on the haemolymph protein profile of Ascia monuste orseis larvae from the 2nd to 7th days were analyzed both qualitatively and quantitatively by SDS-PAGE and Coomassie-Blue binding methods. Quantitative analysis showed a progressive increase in the protein content of about 6.5 and 12.5 times in parasitized and non-parasitized larvae from the 2nd to 5th days, respectively. On the 6th day, a decrease in protein content was observed in both groups, although this decrease was significantly less than the control group that continued to metamorphosis. Meanwhile, parasitized larvae had one more day (7th day) in their larval period to complete parasitoid development, justified by the fact that parasitoid is koinobiont and allows host feeding. On this day, a drastic increase in protein content was detected when the parasitoids left the host. The SDS-PAGE showed proteins of high molecular weight (>120 kDa) on the 5th day of the non-parasitized larvae when they entered pre-pupa stage and on the 7th day of parasitized larvae. Proteins with MW lower than 62 kDa and higher than 27 kDa were absent on the 5th day in control larvae (pre-pupa phase), but present in parasitized larvae. This could indicate a possible relation between these proteins and the host juvenile hormone. Therefore, the presence of C. glomerata influences Ascia monuste orseis development, but its own physiological development is apparently independent of the host, which tends to die when parasitism succeeds.

Hymenoptera venom review focusing on Apis mellifera

Hymenoptera venoms are complex mixtures containing simple organic molecules, proteins, peptides, and other bioactive elements. Several of these components have been isolated and characterized, and their primary structures determined by biochemical techniques. These compounds are responsible for many toxic or allergic reactions in different organisms, such as local pain, inflammation, itching, irritation, and moderate or severe allergic reactions. The most extensively characterized Hymenoptera venoms are bee venoms, mainly from the Apis genus and also from social wasps and ant species. However, there is little information about other Hymenoptera groups. The Apis venom presents high molecular weight molecules - enzymes with a molecular weight higher than 10.0 kDa - and peptides. The best studied enzymes are phospholipase A2, responsible for cleaving the membrane phospholipids, hyaluronidase, which degrades the matrix component hyaluronic acid into non-viscous segments and acid phosphatase acting on organic phosphates. The main peptide compounds of bee venom are lytic peptide melittin, apamin (neurotoxic), and mastocyte degranulating peptide (MCD).

Protein requirements in larvae and adults of Scaptotrigona postica (Hymenoptera: Apidae [correction of Apidia], Meliponinae): midgut proteolytic activity and pollen digestion.

The number and degree of digestion of pollen grains in the midgut and rectum, the midgut proteolytic activity and the time of pollen grain passage through the digestive tract in the stingless bee Scaptotrigona postica (Latreille) have been analyzed. The results show similar protein requirements among larvae, nurse bees and queens, as well as between forager bees and old males, but these requirements are higher in individuals from the former groups than in those from the latter. Although protein requirements have been demonstrated to vary according to a bee's activity in the colony, they are similar among bees from different castes or sexes. These changes in feeding behavior are related to the bee's function and to less competition for nourishment among individuals of the colony. It is also noted that pollen grains took between 6 and 28 h to pass through the digestive tract. Pollen grains are irregularly accumulated in the various regions of the midgut, which may reflect functional differentiation throughout the midgut.

Extracellular inulinases from Penicillium janczewskii, a fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae).

Extracellular inulinases from Penicillium janczewskii were obtained from the filtrate of 12 day-old cultures supplemented with inulin from Vernonia herbacea. Crude filtrates and partially-purified enzyme preparations (peaks I and II) were active on inulin, sucrose and raffinose. The apparent M(r) of the enzymes from peaks I and II were 48 and 66 kDa, respectively. The apparent K(m) (mmol l-1) values of peak I were 0.43 for inulin and 18.7 for sucrose; for peak II they were 0.87 and 18.5 for inulin and sucrose, respectively. Their temperature and pH optima were 55 degrees C and 5.0, respectively. Both peaks catalysed the hydrolysis of beta-(2,1) fructans more rapidly than beta-(2,6) fructans. Free fructose was the predominant product released from inulin, indicating that these enzymes display exo-inulinase activity. In view of these characteristics, the yield and the high specific activity towards beta-(2,1) fructans, inulinases from P. janczewskii can be utilized for the preparation of fructose syrup from inulin.

[Oral communication with hematologic patients: facilitating and blocking behaviors]. / Comunicação oral com pacientes hematológicos: comportamentos facilitadores e bloqueadores.

In this descriptive study we adopted the model of FORREST (1983) for the categorization of oral utterances by the members of the nursing team when caring for hematological patients with oncological alterations. In order to identify the frequency of facilitating(F) and blocking(B) categories of communication and of their subcategories, we videotaped and observed the interactions between 8 patients and 14 members of the Nursing team in a Teaching Hospital in the city of Ribeirão Preto-SP. We recorded 8822 categories, 56.9% of which were facilitating. The most frequent of these were: providing information (50.3%), clarifying (28.0%) and recognizing the presence (11.2%). The most frequent blocking categories were: closed questions (64.6%), giving advice (13.9%) and approving or agreeing (10%). The remaining F and B subcategories were observed at frequencies of less than 4.5%. We suggest the more frequent use of other facilitating categories and the use of closed questions in special situations. We believe that, for a more profound relationship between nursing team and patients, it is of fundamental importance to reduce the "giving advice" and "approval" categories as they are being used now.

Molecular exclusion chromatography of crude venom as an auxiliary tool to identify hybrid honeybee populations

A comparison among the profiles of molecular exclusion chromatography in Sephadex G 100 column of venoms from Apis mellifera adansonii and Africanized honeybees revealed unique peaks which might be used to identify these populations. The venoms from hybrid populations resulting from the reciprocal mating of Apis mellifera adansonii and Africanized honeybees presented unique peaks, probably resulting from a synergistic effect between the parental genomes. The occurrence of characteristic peaks in venoms of hybrid populations might be used to identify these populations as well as to distinguish them from their parents.

The effect of a polydnavirus of Apanteles Galleriae on the hemolymph proteins of host Galleria Mellonela

The presence of a polydnavirus in the ovary of the microhymennoptera Apanteles galleriae was detected. This wasp parasites the larvae of the wax moth Galleria mellonella. To study the possible viral effects on the total protein of G. mellonella, hemolymph extracts of the ovary calyxes of parasitized A. galleriae were injected into 6th and 7th instar cells of G. mellonella larvae. Protein profiles on polyacrilamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE) of host cell extracts showed a suppression of several protein bands when infection occurred in the 6th instar. However, in the 7th instar, parasitism caused an amplification in the synthesis of most proteins with some changes in the electrophoretic profile.

Biochemical properties and study of antigenic cross-reactivity between Africanized honey bee and wasp venom.

Africanized honey bees and the wasp Polistes versicolor are common insects in Brazil; their venoms are composed of a complex mixture of components which present several biological activities. Stinging accidents are very frequent and are generally followed by important clinical reactions, and even deaths are not uncommon. In the present study, venom was extracted from Africanized honey bees and P. versicolor, and it was biochemically characterized and the antigenic cross-reactivity was investigated by Western blot analysis and specific IgE determination by ELISA in the sera of subjects allergic to each venom. The honey bee venom presented higher phospholipase A2 and hyaluronidase activities than P. versicolor venom, which in turn presented higher lipase, acid phosphatase and esterase activities. A high incidence of false-negatives was also observed during determinations of specific IgE for P. versicolor venom when the kits with venoms from wasps of temperate climates were used, suggesting that the diagnosis of allergy to neotropical wasp venom must take into consideration the clinical history and skin tests.

Partial purification and characterization of lysine-ketoglutarate reductase in normal and opaque-2 maize endosperms.

Lysine-ketoglutarate reductase catalyzes the first step of lysine catabolism in maize (Zea mays L.) endosperm. The enzyme condenses l-lysine and alpha-ketoglutarate into saccharopine using NADPH as cofactor. It is endosperm-specific and has a temporal pattern of activity, increasing with the onset of kernel development, reaching a peak 20 to 25 days after pollination, and there-after decreasing as the kernel approaches maturity. The enzyme was extracted from the developing maize endosperm and partially purified by ammonium-sulfate precipitation, anion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Blue-Sepharose CL-6B. The preparation obtained from affinity chromatography was enriched 275-fold and had a specific activity of 411 nanomoles per minute per milligram protein. The native and denaturated enzyme is a 140 kilodalton protein as determined by polyacrylamide gel electrophoresis. The enzyme showed specificity for its substrates and was not inhibited by either aminoethyl-cysteine or glutamate. Steady-state product-inhibition studies revealed that saccharopine was a noncompetitive inhibitor with respect to alpha-ketoglutarate and a competitive inhibitor with respect to lysine. This is suggestive of a rapid equilibrium-ordered binding mechanism with a binding order of lysine, alpha-ketoglutarate, NADPH. The enzyme activity was investigated in two maize inbred lines with homozygous normal and opaque-2 endosperms. The pattern of lysine-ketoglutarate reductase activity is coordinated with the rate of zein accumulation during endosperm development. A coordinated regulation of enzyme activity and zein accumulation was observed in the opaque-2 endosperm as the activity and zein levels were two to three times lower than in the normal endosperm. Enzyme extracted from L1038 normal and opaque-2 20 days after pollination was partially purified by DEAE-cellulose chromatography. Both genotypes showed a similar elution pattern with a single activity peak eluted at approximately 0.2 molar KCL. The molecular weight and physical properties of the normal and opaque-2 enzymes were essentially the same. We suggest that the Opaque-2 gene, which is a transactivator of the 22 kilodalton zein genes, may be involved in the regulation of the lysine-ketoglutarate reductase gene in maize endosperm. In addition, the decreased reductase activity caused by the opaque-2 mutation may explain, at least in part, the elevated concentration of lysine found in the opaque-2 endosperm.

Phytoalexin induction in rubiaceae.

Phytoalexin responses were measured by modified drop-diffusate and facilitated diffusion techniques after fungal inoculation of leaves of 32 Rubiaceae species from Brazilian forest and savanna. Such responses presented a trend similar to that previously observed for a broad sample of dicotyledonous plants and are more frequently positive for the more primitive (or slower growing) trees than for the advanced (or faster growing) herbs. Fifteen of these species analyzed during a one-year period showed that positive phytoalexin responses are stronger for the rainy (and hotter) than for the dry (and cooler) season. Species that contain relatively large quantities of phenolics gave invariably negative responses. Positive responses are not necessarily associated with the appearance of new substances within leaf tissue and are thus caused by inhibitins rather than by phytoalexins. These results are discussed recognizing that the tested plants are subject to the multifarious influences of their natural environment and of a possible conjugate-caused compartmentation of plant metabolites.

[Blastomycotic granuloma of the cervical spine. Report of a case]. / Granuloma blastomicotico na medula cervical. Registro de um caso

A case of blastomycotic granuloma of the spinal cord is reported. The patient had a Brown-Séquard syndrome. He was submitted to myelography and he was underwent a cervical laminectomy. The mass was removed being lately confirmed by histology as a blastomycotic granuloma. Despite the medical treatment with Amphotericin B and physical therapy there was no improvement of the condition.