Phenylpropanoid compounds in primary leaf tissues of rye (Secale cereale). Light response of their metabolism and the possible role in UV-B protection

The present study was undertaken in order to investigate the suitability of certain markers for UV plant response. In addition, we attempted to link the internal tissue distribution of specific UV-absorbing compounds to profiles of radiation gradients within intact primary rye leaves ( Secale cereale L. cv. Kustro). Etiolated rye seedlings irradiated with low visible light (LL) and/or UV radiation were used to study enzyme activities of the two key enzymes, phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS), together with the tissue-specific accumulation of soluble phenylpropanoid products. Plants grown under relatively high visible light (HL) with or without supplementary UV-B radiation were used for further characterization. Apparent quantum yield and fluorescence quenching parameters were monitored to assess potential physiological changes due to UV-B exposure in HL-grown seedlings. A quartz fibreoptic microprobe was used to characterize the internal UV-B gradient of the leaf. The response of the phenylpropanoid metabolism to UV radiation was similar in primary leaves of both etiolated and HL-treated green plants. The epidermis-specific flavonoids together with CHS activity turned out to be suitable markers for assessing the effect of UV on the phenolic metabolism. The functional role of phenylpropanoid compounds was strongly implicated in protecting rye from UV-B radiation.     

Isolation of Arabidopsis genes that differentiate between resistance responses mediated by the RPS2 and RPM1 disease resistance genes.

The Arabidopsis disease resistance gene RPS2 is involved in recognition of bacterial pathogens carrying the avirulence gene avrRpt2, and the RPM1 resistance gene is involved in recognition of pathogens carrying avrRpm1 or avrB. We identified and cloned two Arabidopsis genes, AIG1 and AIG2 (for avrRpt2-induced gene), that exhibit RPS2- and avrRpt2-dependent induction early after infection with Pseudomonas syringae pv maculicola strain ES4326 carrying avrRpt2. However, ES4326 carrying avrRpm1 or avrB did not induce early expression of AIG1 and AIG2. Conversely, ES4326 carrying avrRpm1 or avrB induced early expression of the previously isolated defense-related gene ELI3, whereas ES4326 carrying avrRpt2 did not. The induction patterns of the AIG genes and ELI3 demonstrate that different resistance gene-avr gene combinations can elicit distinct defense responses. Furthermore, by examining the expression of AIG1 and ELI3 in plants infiltrated with a mixed inoculum of ES4326 carrying avrRpt2 and ES4326 carrying avrRpm1, we found that there is interference between the RPS2- and RPM1-mediated resistance responses.     

Regulation of Rhizobium meliloti exo genes in free-living cells and in planta examined by using TnphoA fusions.

The exo loci of Rhizobium meliloti are necessary for the production of an acidic exopolysaccharide, EPS I, that is needed for alfalfa nodule invasion by strain Rm1021. We have isolated and characterized alkaline phosphatase fusions made with TnphoA in several exo loci of R. meliloti and used these fusions to examine the subcellular localization of exo gene products and the regulation of exo genes in free-living cells and in planta. In the course of this work, we isolated a new exo locus, exoT. We have obtained evidence that several of the exo loci may encode membrane proteins. The activity of TnphoA fusions in several exo loci is increased two- to fivefold in the presence of the regulatory mutations exoR95 and exoS96. While examining the regulation of the exo gens by exoR95 and exoS96, we found that certain classes of exo mutations are lethal in an exoR95 or exoS96 background unless a plasmid complementing the exo mutation is present. This result has possible implications for the role of these exo loci in EPS I biosynthesis. We have developed a method for staining nodules specifically for the alkaline phosphatase activity present in the inducing bacteria and used this method to show that an exoF::TnphoA fusion is expressed mainly in the invasion zone of the nodule.     

Life history of frogs of the Brazilian semi-arid (Caatinga), with emphasis in aestivation

The semi-arid region (Caatinga), that corresponds to 18.26% (1,540,000 km²) of Brazil, occupies most the northeast region. Rainfall is irregular and concentrated within the first 3 months of the year. Prolonged periods of drought, with low total rainfall, may extend for two or more years. Most of the rivers of this biome are temporary, remaining totally dry during periods of drought. We collected and observed the natural history and biology of the species Proceratophrys cristiceps, Pleurodema diplolister and Physalaemus spp, both in the rainy and dry seasons, in ten field expeditions to the State of Rio Grande do Norte (Brazil). We focused on the morphology of the skin and cutaneous glands, specifically in their defence against desiccation. During the dry season, they form concentrations in the bed of temporary rivers, burrowing themselves in favourable locations within the sand, at depths that may exceed 1.50 m. No morphological evidence was found that there are specific cutaneous adaptations against water loss. We suggest that the cutaneous fragility per se is a cutaneous adaptation to water balance. The behaviour of such anuran species, together with their physiological characteristics, should be the main tools to face the challenge of living in xeric conditions.     

Tissue localization of phenolic compounds in plants by confocal laser scanning microscopy

Phenolic compounds are involved in many interactions of plants with their biotic and abiotic environment. These substances accumulate in different plant tissues and cells during ontogenesis and under the influence of various environmental stimuli, respectively. Studies on the tissue localization of phenolic compounds provide a fundamental prerequisite for understanding the ecological functions of these compounds. The present work shows the localization of various phenolics in cell walls, vacuoles, and associated with cell nuclei, in leaves of a monocotyledonous and a dicotyledonous plant, in a gymnosperm as well as in rhizomes of a horsetail by confocal laser scanning microscopy (CLSM). Using fresh plant material, it compares in detail the tissue localization of autofluorescent styrylpyrones and hydroxycinnamic acids and the visualization of epidermal flavonoid compounds using shift reagents like ammonia, and fluorescence-inducing reagents like Naturstoffreagenz A (diphenyl-boric acid 2-aminoethyl ester). The comparison of microscopic data obtained from different plant species shows the advantages and limitations of confocal laser scanning microscopy in ecological biochemistry of phenolic plant metabolites.     

Histopathology of carcinomas of the liver in mice ingesting heptachlor or heptachlor epoxide.

C3H male and female mice, ingesting 10 ppm of the pesticides heptachlor or heptachlor epoxide in the diet, developed highly significant incidences of carcinomas of the liver. The carcinomas varied from well-differentiated to poorly differentiated and undifferentiated and were capable of invasion and metastasis.     

Determination of thyroxine in the hair of newborns by radioimmunoassay with high-performance liquid chromatographic confirmation

Hair analysis is often used in forensic toxicology to study, retrospectively, chronic exposure of individuals to drugs, and consequently newborn hair may become an ideal sample to study intrauterine exposure to xenobiotics as well as to endogenous compounds. As a tool to investigate a supposed maternal thyroxine (T₄) supply to the congenital hypothyroid fetus, we devised to use the analysis of T₄ extracted from newborn hair. In the present paper, the analytical method based on T₄ extraction from hair followed by a radioimmunoassay is described. To verify the nature of the T₄-like immunoreactive material present in newborn hair, it was further studied by HPLC fractionation with radioimmunoassay of the eluted fractions. On the basis of a clear correspondence between retention times of T₄ standard and T₄-immunoreactive compound extracted from hair, we assigned this immunoreactive material to T₄. Then, we determined T₄ hair concentrations in 19 control newborns at birth and 12 congenital hypothyroid infants at 22 days of life. Values obtained from hypothyroid infants (31.47±8.8 pg/mg_hair, mean±S.D.) were not significantly lower than those obtained from healthy newborns at birth (36.10±13.2 pg/mg_hair). Such results are in agreement with the hypothesis of a maternal supply of thyroxine to the fetus through placental crossing.     

Antimicrobial peptides isolated from Phyllomedusa nordestina (Amphibia) alter the permeability of plasma membrane of Leishmania and Trypanosoma cruzi

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC₅₀ values in the range concentration of 0.25–0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC₅₀ value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC₅₀ = 34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX® Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.     

Endogenous excretion and true absorption of cobalt as affected by the oral supply of cobalt

At the end of a 49-d experiment with 32 growing male rats, a period of 8 d was used to determine endogenous excretion and true absorption as well as apparent absorption and retention of cobalt with the aid of the isotope dilution technique. For this purpose, a single im dose of⁵⁸Co was applied at d 35 of the experiment. After that, urine and feces were collected separately from d 8 to 15 after injection of the isotope. The specific cobalt activity of the liver was used as an endogenous reference source. The basal diet provided 5.9 ppb cobalt, the different treatment groups were obtained by supplementing the diet with 0, 10, 50, 250, or 1250 ppb cobalt. The different diets were offered from the beginning of the experiment. In the balance period, apparent and true absorption as well as fecal excretion behaved similar to cobalt intake, whereas urinary excretion increased more rapidly with increasing cobalt supply. Endogenous fecal excretion accounted for 3.5 ng Co/d in the groups fed the diets without and with 10 ppb cobalt. An increase was not observed until supplementing the diet with 50 ppb cobalt. This increase between 250 and 1250 ppb cobalt was higher than the corresponding increase in the dietary cobalt supply. This indicates that endogenous fecal excretion might be more important for homeostatic regulation at a higher dietary cobalt concentration. Endogenous renal excretion as calculated from the results of the isotope dilution technique showed a similar kind of response to increasing cobalt supply as endogenous fecal loss. Nevertheless, the elimination of excessive cobalt mainly took place by adjusting urinary excretion, whereas the variations in true absorption and endogenous fecal excretion had no quantitative importance. Apparent and true absorption were on average 28.0 and 29.8%, respectively, of the cobalt intake. In the case of retention, a marked decline was observed from 19% in the depletion group to 3% with 1250 ppb cobalt, again demonstrating the importance of urinary excretion for controlling the cobalt content of the organism.     

Morphological and biochemical characterization of the cutaneous poison glands in toads (<Emphasis Type="Italic">Rhinella marina</Emphasis> group) from different environments

Background

Amphibian defence against predators and microorganisms is directly related to cutaneous glands that produce a huge number of different toxins. These glands are distributed throughout the body but can form accumulations in specific regions. When grouped in low numbers, poison glands form structures similar to warts, quite common in the dorsal skin of bufonids (toads). When accumulated in large numbers, the glands constitute protuberant structures known as macroglands, among which the parotoids are the most common ones. This work aimed at the morphological and biochemical characterization of the poison glands composing different glandular accumulations in four species of toads belonging to group Rhinella marina (R. icterica, R. marina, R. schneideri and R. jimi). These species constitute a good model since they possess other glandular accumulations together with the dorsal warts and the parotoids and inhabit environments with different degrees of water availability.

Results

We have observed that the toads skin has three types of poison glands that can be differentiated from each other through the morphology and the chemical content of their secretion product. The distribution of these different glands throughout the body is peculiar to each toad species, except for the parotoids and the other macroglands, which are composed of an exclusive gland type that is usually different from that composing the dorsal warts. Each type of poison gland presents histochemical and biochemical peculiarities, mainly regarding protein components.

Conclusions

The distribution, morphology and chemical composition of the different types of poison glands, indicate that they may have different defensive functions in each toad species.