Purification and subunit structure of RNA polymerases I and II from Dictyostelium discoideum vegetative cells

Summary A purification procedure to obtain RNA polymerases I (or A) and II (or B) from Dictyostelium discoideum amoeba has been developed. The enzymes were solubilized from purified nuclei and separated by DEAF-Sephadex chromatography. RNA polymerases I and II were further purified by a second chromatography on DEAE-Sephadex followed by chromatographies on phosphocellulose and heparin-sepharose. The specific activities of purified RNA polymerases I and II are 92 units/ mg protein and 70 units/ mg protein, respectively. The subunit structure of both RNA polymerases were analyzed by polyacrylamide gel electrophoresis under denaturing conditions after glycerol gradient centrifugation of the enzymes. The putative subunits of RNA polymerase I have molecular weights of 180 000,125 000,43 000,40 000,34 000, 31 000, 25 000,19 000, 17 000 and 14 000. The putative subunits of RNA polymerase II have molecular weights of 200 000 (170 000), 130 000, 33 000, 25 000, 19 000, 17 000, 15 000, 13 000. There are three polypeptides with common molecular weight in Dictyostelium RNA polymerases I and 11. The subunit of 25 000 daltons of both enzymes has common immunological determinants with RNA polymerase II from crustacean Artemia.Abbreviations TLCK tosyl-lysine-chloromethyl-ketone - DPT diazophenylthioether     

Revisiting the dimensionality of biological diversity

Biodiversity can be represented by different dimensions. While many diversity metrics try to capture the variation of these dimensions they also lead to a ‘fragmentation’ of the concept of biodiversity itself. Developing a unified measure that integrates all the dimensions of biodiversity is a theoretical solution for this problem, however, it remains operationally impossible. Alternatively, understanding which dimensions better represent the biodiversity of a set of communities can be a reliable way to integrate the different diversity metrics. Therefore, to achieve a holistic understand of biological diversity, we explore the concept of dimensionality. We define dimensionality of diversity as the number of complementary components of biodiversity, represented by diversity metrics, needed to describe biodiversity in an unambiguously and effective way. We provide a solution that joins two components of dimensionality – correlation and the variation – operationalized through two metrics, respectively: evenness of eigenvalues (EE) and importance values (IV). Through simulation we show that considering EE and IV together can provide information that is neglected when only EE is considered. We demonstrate how to apply this framework by investigating the dimensionality of South American small mammal communities. Our example evidenced that, for some representations of biological diversity, more attention is needed in the choice of diversity metrics necessary to effectively characterize biodiversity. We conclude by highlighting that this integrated framework provides a better understanding of dimensionality than considering only the correlation component.     

Comparative effects of desiccation,heat shock and high temperatures on seed germination of savanna and forest tree species

Although forest and savanna biomes predominate in tropics regions, the factors that control their distribution remain unclear. South American savannas occur in regions that are considered warm and humid enough to support forests, indicating that agents other than climate determine the occurrence of one or the other physiognomy. Herbivory, fire and water deficit have been considered environmental filters that limit the forest species encroachment in savanna physiognomies, but the effects of these filters on the capability of these species to recruit from seeds remain poorly understood. In this study we investigated how stress factors characteristic of savanna environments, such as soil desiccation, heat shocks and high temperatures affect the survival and germination of seeds from savanna and forest tree species. We found that desiccation (to 5%) reduced the germination percentage of forest seeds, but had no effect on the germination of savanna seeds. Forest seeds were less tolerant to heat shocks of 140°C and 200°C, and showed lower germination percentage at temperatures of 35 and 40°C, when compared with savanna seeds. Savanna seeds presented longer germination times and higher germination variance than forest seeds, indicating a risk‐spreading germination strategy among savanna species. The low tolerance of forest seeds to desiccation, heat shock and high temperatures may explain the low recruitment of forest trees into savanna physiognomies. Climate change models predict lower soil moisture, higher temperatures and higher fires frequency for South America biomes. Our results suggest that savanna species are likely to be more capable of withstanding the effects of these changes than forest species.     

Depressed Pollination of Lapageria rosea Ruiz et Pav. (Philesiaceae) in the Fragmented Temperate Rainforest of Southern South America

We studied the pollination and reproductive success in continuous and fragmented populations of Lapageria rosea, a self-compatible plant endemic to temperate forests of Chile. Pollinator abundance, visitation rates, flower abundance, nectar volume and concentration, pollen germination and fruit and seed production, were compared between continuous forest of 145 ha and four forest fragments of 6, 3, 3, and 1 ha respectively, surrounded by mature pine plantations of Pinus radiata. Flower abundance was lower in three out of four forest fragments relative to continuous forest. Nectar volume and sugar concentration did not differ between flowers in the two habitats. Pollinators of L. rosea, the hummingbird Sephanoides sephaniodes and bumblebee Bombus dahlbomii were less abundant and visited flowers of L. rosea at lower rates in fragments than in continuous forest. In addition, in vitro rates of pollen germination were lower for flowers in forest fragments. The number of seeds per fruit was also lower in forest fragments. We suggest that fragmentation affects the reproductive success of L. rosea, lowering the total numbers of seeds produced and possibly compromising long term persistence of fragmented populations.     

In vitro activity of propyl gallate-azole drug combination against fluconazole- and itraconazole-resistant Candida albicans strains

AIMS: The influence of an antioxidant, propyl gallate (PG), on the in vitro antifungal activity of itraconazole and fluconazole, was investigated to determine whether PG could increase the antifungal activity and reduce strain resistance. METHODS AND RESULTS: Susceptibility tests were performed against azole-resistant isolates of Candida albicans by the microbroth dilution method in the presence of PG at 400 microg ml-1. PG-triazole combination brought about a marked reduction of inhibitory azole concentration. In particular, the MIC90 for itraconazole and fluconazole dropped from 1 microg ml-1 to 0.125 microg ml-1 and from > 64 microg ml-1-8 microg ml-1, respectively. CONCLUSION: It is likely that more than one mechanism is involved in the above synergistic interaction, including effects of PG on ATP synthesis, thus reducing the ABC transporters activity, or an effect on the target of azole, i.e. the P-450 cytochrome. SIGNIFICANCE AND IMPACT OF THE STUDY: The PG-triazole combination may have a role in future topical antifungal strategies but other studies are warranted.     

Enzymatic activity and degradation of different kinds of organic wastes by Saccobolus saccoboloides (Fungi,Ascomycotina)

The ability to degrade organic solid wastes by the fungus Saccobolus saccoboloides was studied. The organism, unusual in such studies, was cultivated in synthetic liquid media with agitation, and on day 8 of growth the mycelium was passed to flasks with trimming. On day 16 of growth, the trimming degradation was assesed by carboxymethylcellulase, xylanase, and amylase activities evaluation, and NaOH 1% hydrolysis. Later on, the type of waste was modified (trimming, filter paper, newspaper, cardboard, sawdust and wood shaving were used) as well as the mass (300-1800 mg/flask). In these cases the enzymatic activities increased between 300 and 600 mg/flask. The total separation of the cellular components in all types of paper and cardboard was observed, together with a high loss of weight. S. saccoboloides was not able to degrade the wood wastes     

Novel model for “calcium paradox” in sympathetic transmission of smooth muscles: Role of cyclic AMP pathway

It is well established that reduction of Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channel (L-type VDCC), or increase of cytosolic cAMP concentration ([cAMP]c), inhibit contractile activity of smooth muscles in response to transmitters released from sympathetic nerves. Surprisingly, in this work we observed that simultaneous administration of L-type VDCC blocker (verapamil) and [cAMP]c enhancers (rolipram, IBMX and forskolin) potentiated purinergic contractions evoked by electrical field stimulation of rat vas deferens, instead of inhibiting them. These results, including its role in sympathetic transmission, can be considered as a “calcium paradox”. On the other hand, this potentiation was prevented by reduction of [cAMP]c by inhibition of adenylyl cyclase (SQ 22536) or depletion of Ca²⁺ storage of sarco-endoplasmic reticulum by blockade of Ca²⁺ reuptake (thapsigargin). In addition, cytosolic Ca²⁺ concentration ([Ca²⁺]_c) evaluated by fluorescence microscopy in rat adrenal medullary slices was significantly reduced by verapamil or rolipram. In contrast, simultaneous incubation of adrenal slices with these compounds significantly increased [Ca²⁺]_c. This effect was prevented by thapsigargin. Thus, a reduction of [Ca²⁺]_c due to blockade of Ca²⁺ influx through L-type VDCC could stimulate adenylyl cyclase activity increasing [cAMP]c thereby stimulating Ca²⁺ release from endoplasmic reticulum, resulting in augmented transmitter release in sympathetic nerves and contraction.     

Analysis of ascorbate peroxidase genes expressed in resistant and susceptible wheat lines infected by the cereal cyst nematode, Heterodera avenae

Changes in ascorbate peroxidase (APX) enzyme activity in response to nematode (Heterodera avenae) attack were studied in roots of three hexaploid wheat lines carrying Cre2, Cre5, or Cre7 nematode resistance genes and the susceptible Triticum aestivum cv. Anza. A spectrophotometric analysis was carried out with root extracts of infected plants 4, 7, 11, and 14 days after nematode inoculation using uninfected plant as control. APX induction in infected resistant genotypes was similar and higher than in the susceptible control. The introgression wheat/Aegilops ventricosa H-93-8 line, carrying the Cre2 gene, and its parental line H-10-15 as susceptible control were used to analyze whether this increase of activity was correlated with the induction of APX gene expression. Genes encoding cytosolic forms of APX were induced in roots of both lines in response to nematode infection. This induction took place both earlier and with greater intensity in the resistant line than in the susceptible one, and it was also higher in the root area at the site of nematode attachment.     

Boron neutron capture synovectomy (BNCS) as a potential therapy for rheumatoid arthritis: boron biodistribution study in a model of antigen-induced arthritis in rabbits

Boron neutron capture synovectomy (BNCS) is explored for the treatment of rheumatoid arthritis (RA). The aim of the present study was to perform boron biodistribution studies in a model of antigen-induced arthritis (AIA) in female New Zealand rabbits, with the boron carriers boronophenylalanine (BPA) and sodium decahydrodecaborate (GB-10) to assess the potential feasibility of BNCS for RA. Rabbits in chronic phase of AIA were used for biodistribution studies employing the following protocols: intra-articular (ia) (a) BPA-f 0.14 M (0.7 mg ¹⁰B), (b) GB-10 (5 mg ¹⁰B), (c) GB-10 (50 mg ¹⁰B) and intravenous (iv), (d) BPA-f 0.14 M (15.5 mg ¹⁰B/kg), (e) GB-10 (50 mg ¹⁰B/kg), and (f) BPA-f (15.5 mg ¹⁰B/kg) + GB-10 (50 mg ¹⁰B/kg). At different post-administration times (13–85 min for ia and 3 h for iv), samples of blood, pathological synovium (target tissue), cartilage, tendon, muscle, and skin were taken for boron measurement by inductively coupled plasma mass spectrometry. The intra-articular administration protocols at <40 min post-administration both for BPA-f and GB-10, and intravenous administration protocols for GB-10 and [GB-10 + BPA-f] exhibited therapeutically useful boron concentrations (>20 ppm) in the pathological synovium. Dosimetric estimations suggest that BNCS would be able to achieve a therapeutically useful dose in pathological synovium without exceeding the radiotolerance of normal tissues in the treatment volume, employing boron carriers approved for use in humans. Radiobiological in vivo studies will be necessary to determine the actual therapeutic efficacy of BNCS to treat RA in an experimental model.     

Seed predation and seedling recruitment in plants: the effect of the distance between parents

We present a graphic model that explores the effect of distance between parent plants on seed predation and seedling recruitment. Based on the assumption that distance between parents may affect the shape of the seed shadow, the model predicts that seed predators may affect seedling recruitment curves under isolated plants but they are unable to affect these curves under close parent plants. The predictions of the model are tested experimentally in Cryptocarya alba (Lauraceae), a common tree of the Mediterranean forest, Central Chile. Results show that predictions are not met under isolated parent plants. Although seed density decreases significantly away from parent plants, this effect is not relevant for seed predation and seedling recruitment. The biotic/abiotic contrast existing under the canopy vs outside the canopy, plus the shade-tolerance of this tree, better explains the seedling recruitment observed under isolated parent plants. Nevertheless, the predictions of the model are corroborated under close parent plants. Seed shadows overlap to the extent that they generate a homogeneous seed distribution, homogeneous seed predation and homogeneous seedling recruitment as well. We discuss the implications of the model in terms of the spatial pattern of seedlings and the benefits of dispersal from isolated and close parent plants.