A phylogenetic analysis of species in the Bufo crucifer group (Anura: Bufonidae), based on indolealkylamines and proteins from skin secretions

This research tested the utility of two classes of skin secretion compounds to the phylogeny of the Bufo crucifer group. Skin secretions from specimens of nine populations of B. crucifer group were obtained and submitted to qualitative analysis. We observed a clear difference in the composition of the skin secretion molecules obtained from the species of Bufo studied. Fifty-nine molecules, 16 indolealkylamines and 43 proteins, were used as characters, and 39 of these were parsimonious informative. The tree topology of the skin secretion combined data showed areas of congruence and conflict when compared to an mtDNA phylogeny of the B. crucifer group. We used the Templeton test to evaluate the heterogeneity between the skin secretion and mtDNA data. Although not recommended, we performed a combined analysis with the two partitions. The skin secretion characters from the species of Bufo studied have phylogenetic signal. These data are indicative, at least as a preliminary study, of the phylogenetic relationships among the B. crucifer group taxa.     

Effect of microwave treatment on the shear bond strength of different types of commercial teeth to acrylic resin

doi:10.1111/j.1741‐2358.2009.00333.x
Effect of microwave treatment on the shear bond strength of different types of commercial teeth to acrylic resin Objective: The purpose of this study was to verify the effect of microwave treatment on the shear bond strength of commercial types of teeth to acrylic resin, when the glossy ridge laps were unmodified (groups 1 and 5), bur abraded (groups 2 and 6), bur grooved (groups 3 and 7) or etched by monomer (groups 4 and 8). Background: Controversial findings have shown that mechanical or chemical changes in ridge‐lap surface of the tooth increase or decrease the bond strength between tooth and acrylic resin, and the microwave disinfection may cause different changes on this bond strength. Materials and methods: Eighty specimens (n = 10) were made with the acrylic resin bonded to tooth glossy ridge lap, polymerised in water at 74°C for 9 h, and deflasked after flask cooling. Specimens of the groups 5, 6, 7 and 8 were individually immersed in 150 ml of water and submitted to microwave treatment in an oven at 650 W for 3 min. Control specimens (groups 1, 2, 3 and 4) were not microwave treated. Shear bond strength test was performed in an Instron machine with a cross‐speed of 1 mm/min. Collected data were submitted to anova and Tukey’s test (α = 0.05). Results: Microwave treatment decreased the shear bond strength values of the tooth/resin bond. In the microwaved and non‐microwaved procedures, mechanical retention improved the shear bond strength when compared with the control and monomer treatments. Conclusion: Shear bond strength of the tooth/resin bond was influenced by the microwave treatment and different commercial teeth association, and was lower for the Biotone tooth.     

The importance of location and visual cues during foraging in the German wasp (Vespula germanica F.) (Hymenoptera: Vespidae)

Abstract

The way in which foraging wasps use cues for prey location and choice appears to depend on both the context and on the type of prey. Vespula germanica is an opportunistic, generalist prey forager, and individual wasp foragers often return to hunt at sites of previous hunting success. In this paper, we studied which cues are used by this wasp when relocating a food source. Particularly we analysed the response to a displaced visual cue versus a foraging location at which either honey or cat food had been previously presented. We conclude that location is used over a displaced visual cue for directing wasp hovering, although the landing response is directed differently according to bait type. When wasps are exploiting cat food, location also elicits landing, but if they are exploiting honey, a displaced visual cue elicits landing more frequently than location.     

Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.     

PHYLOGENY AND SYSTEMATICS OF THE MARINE ALGAL FAMILY GRACILARIACEAE (GRACILARIALES,RHODOPHYTA) BASED ON SMALL SUBUNIT rDNA AND ITS SEQUENCES OF ATLANTIC AND PACIFIC SPECIES1

We sequenced the small subunit rDNA and internal transcribed spacer region of Gracilariaceae from the tropical Atlantic and Pacific, with emphasis on flattened or compressed species. Sequence comparisons confirmed three main lineages of Gracilariaceae: Curdiea/Melanthalia, Gracilariopsis/Gracilariophila, and Gracilaria. The Curdiea/Melanthalia diverged early in the family. Gracilariopsis was paraphyletic, because at least one Gracilariophila species evolved from it. The Atlantic Gracilariopsis were monophyletic and separated from the Pacific lineages. The Gracilaria included all species referable to its own species and to Hydropuntia, which was paraphyletic, formed by distantly related lineages. The new combination Gracilaria pauciramosa (N. Rodríguez Ríos) Bellorin, M. C. Oliveira et E. C. Oliveira is proposed for Polycavernosa pauciramosa N. Rodríguez Ríos. Recognition of subgenera within Gracilaria, based on spermatangial arrangement, was not supported. Instead, infrageneric groups were delineated by geographic origins and combinations of reproductive characters. Most Pacific species with either “textorii” or “verrucosa” type spermatangia were deeply separated from Atlantic species. Within the Atlantic Gracilaria, a lineage encompassing mostly tropical cylindrical species with “henriquesiana” type spermatangia and distinctive cystocarp anatomy was recognized. A lineage was also retrieved for cold water stringy species with verrucosa type spermatangia. Several species from the western Atlantic are closely related to Gracilaria tikvahiae McLachlan with nearly identical morphology. On the other hand, most flattened species from the tropical Atlantic were closely related despite their diverse morphologies. The interpretation of our data in addition to the literature indicates that more populations from the Indo‐Pacific must be studied before a general picture of Gracilariaceae evolution can be framed.     

Immunofluorescence colocalization of the 90-kDa heat-shock protein and microtubules in interphase and mitotic mammalian cells

A mouse monoclonal antibody (AC88) that was raised against the 88-kDa heat-shock protein of the water mold, Achlya ambisexualis, and that cross-reacts with the 90-kDa mammalian heat-shock protein (hsp90), and an antibody against tubulin were used to localize hsp90 and microtubules, respectively, in the same cultured rat endothelial and PtK1 epithelial cells by indirect immunofluorescence. AC88 and tubulin antibodies labeled the same structures in cells at all stages of the cell cycle, regardless of whether cells were permeabilized before or after fixation. Labeling of cell structures by both AC88 and anti-tubulin antibodies was identically affected by treating cells with colcemid. Double labeling with AC88 and anti-tubulin antibodies in interphase and mitotic cells is consistent with the conclusion that all microtubules are labeled and that no subclass of microtubules is preferentially labeled. Fluorescent labeling by AC88 was prevented by preabsorption of the antibody with purified rat hsp90 but was unaffected by preabsorption with purified 6S tubulin dimer. In contrast to AC88, fluorescent labeling by an anti-tubulin antibody was prevented by preabsorption with tubulin dimer but was unaffected by preabsorption with rat hsp90. Western-blot analysis demonstrated no cross-reactivity of AC88 for tubulin and no cross-reactivity of the anti-tubulin antibody for hsp90. A polyclonal antiserum fraction from a rabbit immunized with the 89-kDa heat-shock protein from chicken also labeled the mitotic apparatus in dividing cells and, somewhat less distinctly, fibrous structures in interphase cells. Labeling by hsp89 anti-serum was prevented by absorption with hsp90. AC88 also labeled microtubules in cultured mouse (L929 and 3T3), rat (endothelium and TRST), hamster (CHO) and primate (BSC, COS-1 and HeLa) cell lines. The demonstration of colocalization of hsp90 with microtubules should provide a valuable clue to eventual understanding of the cellular function of this ubiquitous, conserved and abundant stress-response protein.     

Reversible inhibition of osteoclastic activity by bone-bound gallium (III).

Gallium(III) is a new therapeutic agent for hypercalcemia. Ga3+ reduces osteoclast action, but how it inhibits the cell's physiology is unknown. In vivo, 7-12 microM Ga(III) reduces calcium release from bone, but surprisingly, 10-100 microM Ga3+ added to isolated avian osteoclasts did not reduce their degradation of L-(5-3H)-proline bone. 3H-proline labels bone collagen specifically, and collagenolysis is an excellent indicator of bone dissolution because collagen is the least soluble component of bone. Ga(III) greater than 100 microM inhibited osteoclasts in vitro, but also killed the cells. To resolve this apparent conflict, we measured 67Ga distribution between bone, cells, and media. Gallium binds avidly but slowly to bone fragments. One hundred micrograms of bone clears 60% of 1 microM gallium from 500 microliters of tissue culture medium, with steady state at greater than 24 h. Osteoclasts on bone inhibited gallium binding capacity approximately 40%, indicating a difference in available binding area and suggesting that osteoclasts protect their substrate from Ga binding. Less gallium binds to bone in serum-containing medium than in phosphate-buffered saline; 30% reduction of the affinity constant suggests that the serum containing medium competes with bone binding. Consequently, the effect of [Ga] on bone degradation was studied using accurately controlled amounts of Ga(III) pre-bound to the bone. Under these conditions, gallium sensitivity of osteoclasts is striking. At 2 days, 100 micrograms of bone pre-incubated with 1 ml of 1 microM Ga3+, with 10 pmoles Ga3+/micrograms bone, was degraded at 50% the rate of control bone; over 50 pM Ga3+/micrograms bone, resorption was essentially zero. In contrast, pre-treatment of bone with [Ga3+] as high as 15 microM had no significant effect on bone resorption rate beyond 3 days, indicating that gallium below approximately 150 pg/micrograms bone acts for a limited time and does not permanently damage the cells. We conclude that bone-bound Ga(III) from medium concentrations less than 15 microM inhibits osteoclasts reversibly, while irreversible toxicity occurs at solution [Ga3+] greater than 50 microM.