The use of fluorescein for labeling genomic probes in the checkerboard DNA-DNA hybridization method

Molecular methods that permit the simultaneous detection and quantification of a large number of microbial species are currently employed in the evaluation of complex ecosystems. The checkerboard DNA-DNA hybridization technique enables the simultaneous identification of distinct bacterial. species in a large number of dental samples. The original technique employed digoxigenin-labeled whole genomic DNA probes which were detected by chemiluminescence. In this study, we present an alternative protocol for labeling and detecting whole genomic DNA probes in the Checkerboard DNA-DNA hybridization method. Whole genomic DNA was extracted from five bacterial species and labeled with fluorescein. The fluorescein labeled whole genomic DNA probes were hybridized against whole genomic DNA or subgingival plaque samples in a checkerboard hybridization format, followed by chemiluminescent detection. Our results reveal that fluorescein is a viable and adequate alternative labeling reagent to be employed in the checkerboard DNA-DNA hybridization technique. (c) 2007 Elsevier GmbH. All rights reserved.

Morphological differentiation between S. mutans and S. sobrinus on modified SB-20 culture medium

Due to the major role of Streptococcus mutans and Streptococcus sobrinus in the etiology of dental caries, it is important to use culture media that allow for differentiating these bacterial species. The aim of this study was to evaluate the suitability of a modified SB-20 culture medium (SB-20M) for the isolation and morphological differentiation of S. mutans and S. sobrinus, compared to biochemical identification (biotyping). Saliva samples were collected using the spatula method from 145 children, seeded on plates containing the SB-20M, in which sucrose was replaced by coarse granular cane sugar, and incubated in microaerophilia at 37 degrees C during 72 h. Identification of the microorganisms was performed under stereomicroscopy based on colony morphology of 4904 colonies. The morphological identification was examined by biochemical tests of 94 randomly selected colonies with the macroscopic characteristic of S. mutans and S. sobrinus using sugar fermentation, resistance to bacitracin and production of hydrogen peroxide. There was no statistically significant difference (p> 0.05) between morphological identification in the SB-20M medium and biochemical identification (biotyping). Biotyping confirmed that S. mutans and S. sobrinus colonies were correctly characterized in the SB-20M in 95.8% and 95.5% of the cases, respectively. Of the mutans streptococci detected in the children 98% were S. mutans and 2% S. sobrinus. The SB-20M medium is reliable for detection and direct morphological identification of S. mutans and S. sobrinus. (C) 2010 Elsevier GmbH. All rights reserved.

Baroreflex sensitivity and oxidative stress in the LDL receptor knockout mice

This study alms at observing the effect of low-density lipoprotein (LDL) receptor deficiency in cholesterol blood levels, baroreflex sensitivity (BRS), nitric oxide (NO) bioavailability, and oxidative stress. The lack of LDL receptors in mice significantly increased the cholesterol blood levels (179 +/- 35 vs. 109 +/- 13 mg/dL) in the knockout (KO) mice compared to control. There was no difference in basal mean arterial pressure and heart rate between the groups. However, in KO mice the BRS was significantly attenuated and the antioxidant enzyme activities, measured in erythrocytes and heart, were significantly decreased. On the other hand, the oxidative damage measured by chemiluminescence and carbonyls was increased, while total plasma nitrate levels were lower in KO mice, indicating a decrease in NO availability. In conclusion, these results indicate that the lack of LDL receptor increased cholesterol blood levels, induced oxidative stress and decreased BRS. (C) 2008 Elsevier GmbH. All rights reserved.

Morphological architecture of Actinocephalus (Koern.) sano (Eriocaulaceae-Poales)

Actinocephalus exhibits perhaps more diversity in habit than any other genus of Eriocaulaceae. This variation is largely a result of differences in the arrangement of the paraclades. Based on the analysis of stem architecture of all 25 species of Actinocephalus, the following patterns were established: (1) leaf rosette, with no elongated axis, instead the axillary paraclades originating directly from the short aerial stem, (2) rosette axis continuing into an elongated axis with spirally arranged paraclades, (3) an elongated axis originating from a rhizome, with ramified paraclades, and (4) an elongated axis originating from a short aerial stem, with paraclades arranged in a subwhorl. The elongated axis exhibits indeterminate growth only in pattern 4. Patterns 3 and 4 are found exclusively in Actinocephalus; pattern I occurs in many other genera of Eriocaulaceae, while pattern 2 is also found in Syngonanthus and Paepalanthus. Anatomically, each stem structure (i.e., paraclade, elongated axis, short aerial stem, rhizome) is thickened in a distinctive way and this can be used to distinguish them. Specifically, elongated axes and paraclades lack thickening, thickening of short aerial stems results from the primary thickening meristem and/or the secondary thickening meristem. Thickening of rhizomes results from the activity of the primary thickening meristem. (c) 2008 Elsevier GmbH. All rights reserved.

Phylogeny of South American Pogonieae (Orchidaceae, Vanilloideae) based on sequences of nuclear ribosomal (ITS) and chloroplast (psaB, rbcL, rps16, and trnL-F) DNA, with emphasis on Cleistes and discussion of biogeographic implications

Tribe Pogonieae (Orchidaceae), as Currently known, comprises live genera distributed from South to North America and Eastern Asia. Phylogenetic inferences within Cleistes and among genera of tribe Pogonieae were made based oil nrDNA (ITS) and cpDNA (trnL-F, rps16, rbcL, and psaB) Sequence data and maximum parsimony. Eighteen species of Cleistes, members of all other genera of Pogonieae, and outgroups were sampled. Analyses based oil individual DNA regions provided similar topologies. All evidence indicates that Cleistes is paraphyletic. The North American C. divaricata and C bifaria are more closely related to the temperate genera Isotria and Pogonia than to their Central and South American congeners, the latter Constituting a monophyletic group characterized by the production of nectar as reward, tuberous roots, and their distribution in Central and South America. The Amazonian Duckeella is sister to the remainder of Pogonieae. Taxonomic and biogeographic implications are discussed, and morphological synapomorphies are given For clades obtained in the inferred molecular phylogeny. (C) 2008 Gesellschaft fur Biologische Systematik. Published by Elsevier GmbH. All rights reserved.

Morphology and anatomy of inflorescence and inflorescence axis in Paepalanthus sect. Diphyomene Ruhland (Eriocaulaceae, Poales) and its taxonomic implications

Paepalanthus sect. Diphyomene has inflorescences arranged in umbels. The underlying bauplan seems however to be more complex and composed of several distinct subunits. Despite appearing superficially very similar, the morphology and anatomy of the inflorescences can supply useful information for the understanding of the phylogeny and taxonomy of the group. Inflorescences of Paepalanthus erectifolius, Paepalanthus flaccidus, Paepalanthus giganteus, and Paepalanthus polycladus were analyzed in regard to branching pattern and anatomy. In P. erectifolius, P. giganteus and P. polycladus the structure is a tribotryum, with terminal dibotryum, and with pherophylls bearing lateral dibotrya. In P. flaccidus, the inflorescence is a pleiobotryum, with terminal subunit, and without pherophylls. Secondary inflorescences may occur in all species without regular pattern. Especially when grown in sites without a pronounced seasonality, the distinction between enrichment zone (part of the same inflorescence) and new inflorescences may be obscured. The main anatomical features supplying diagnostic and phylogenetic information are as follows: (a) in the elongated axis, the thickness of the epidermal cell walls and the cortex size; (b) in the bracts, the quantity of parenchyma cells (c) in the scapes, the shape and the presence of a pith tissue. Therefore, P. sect. Diphyomene can be divided in two groups; group A is represented by P. erectifolius, P. giganteus and P. polycladus, and group B is represented by P. flaccidus. The differentiation is based in both, inflorescence structure and anatomy. Group A presents a life cycle and anatomical features similar to species of Actinocephalus. Molecular trees also point that these two groups are closely related. However, inflorescence morphology and blooming sequence are different. Species of group B present an inflorescence structure and anatomical features shared with many genera and species in Eriocaulaceae. The available molecular and morphology based phylogenies still do not allow a precise allocation of the group in the bulk of basal species of Paepalanthus collocated in P. sect. Variabiles. The characters described and used here supply however important information towards this goal. (C) 2009 Elsevier GmbH. All rights reserved.

Correlation between citric acid and nitrate metabolisms during CAM cycle in the atmospheric bromeliad Tillandsia pohliana

Crassulacean acid metabolism (CAM) confers crucial adaptations for plants living under frequent environmental stresses. A wide metabolic plasticity can be found among CAM species regarding the type of storage carbohydrate, organic acid accumulated at night and decarboxylating system. Consequently, many aspects of the CAM pathway control are still elusive while the impact of this photosynthetic adaptation on nitrogen metabolism has remained largely unexplored. In this study, we investigated a possible link between the CAM cycle and the nitrogen assimilation in the atmospheric bromeliad Tillandsia pohliana by simultaneously characterizing the diel changes in key enzyme activities and metabolite levels of both organic acid and nitrate metabolisms. The results revealed that T. pohliana performed a typical CAM cycle in which phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase phosphorylation seemed to play a crucial role to avoid futile cycles of carboxylation and decarboxylation. Unlike all other bromeliads previously investigated, almost equimolar concentrations of malate and citrate were accumulated at night. Moreover, a marked nocturnal depletion in the starch reservoirs and an atypical pattern of nitrate reduction restricted to the nighttime were also observed. Since reduction and assimilation of nitrate requires a massive supply of reducing power and energy and considering that T. pohliana lives overexposed to the sunlight, we hypothesize that citrate decarboxylation might be an accessory mechanism to increase internal CO(2) concentration during the day while its biosynthesis could provide NADH and ATP for nocturnal assimilation of nitrate. Therefore, besides delivering photoprotection during the day, citrate might represent a key component connecting both CAM pathway and nitrogen metabolism in T. pohliana: a scenario that certainly deserves further study not only in this species but also in other CAM plants that nocturnally accumulate citrate. (C) 2010 Elsevier GmbH. All rights reserved.

Dark-induced hormone changes coincide with the resumption of light-inhibited shoot growth in Catasetum fimbriatum (Orchidaceae)

Catasetum fimbriatum plants cultivated in the absence of light exhibit continuous shoot growth leading to the formation of nodes and internodes. On the other hand, when these plants are incubated in the presence of light, shoot longitudinal growth is inhibited and pseudobulbs develop just below the shoot apical meristem. These facts provide evidence of a possible influence of light on mitotic cell division in the shoot apex as well as on pseudobulb initiation. The effects of light and dark on the interruption and/or maintenance of shoot apex mitotic activity and the subsequent formation of pseudobulbs in the sub-meristematic regions were investigated by means of histological and hormonal studies. The interruption of shoot apex development occurred around the 150th d of light incubation and seems to have resulted from the establishment of a strong storage sink in the region of the future pseudobulb, in detriment to the continuous activity of the shoot apical meristem. The reduced total cytokinins/IAA ratio in the apex, mainly due to high levels of IAA, could be a key factor in the interruption of cell divisions. Transfer to the dark brings about the resumption of shoot apex development of plants through the re-entrance of cells in the cell cycle which coincides with a significant increase in the total cytokinins/IAA ratio. (C) 2009 Elsevier GmbH. All rights reserved.

Nitrogen Metabolism in leaves of a tank epiphytic bromeliad: Characterization of a spatial and functional division

The leaf is considered the most important vegetative organ of tank epiphytic bromeliads due to its ability to absorb and assimilate nutrients. However, little is known about the physiological characteristics of nutrient uptake and assimilation. In order to better understand the mechanisms utilized by some tank epiphytic bromeliads to optimize the nitrogen acquisition and assimilation, a study was proposed to verify the existence of a differential capacity to assimilate nitrogen in different leaf portions. The experiments were conducted using young plants of Vriesea gigantea. A nutrient solution containing NO(3)(-)/NH(4)(+) or urea as the sole nitrogen source was supplied to the tank of these plants and the activities of urease, nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (NADH-GDH) were quantified in apical and basal leaf portions after 1, 3, 6, 9, 12, 24 and 48 h. The endogenous ammonium and urea contents were also analyzed. Independent of the nitrogen sources utilized, NR and urease activities were higher in the basal portions of leaves in all the period analyzed. On the contrary. GS and GDH activities were higher in apical part. It was also observed that the endogenous ammonium and urea had the highest contents detected in the basal region. These results suggest that the basal portion was preferentially involved in nitrate reduction and urea hydrolysis, while the apical region could be the main area responsible for ammonium assimilation through the action of GS and GDH activities. Moreover, it was possible to infer that ammonium may be transported from the base, to the apex of the leaves. In conclusion, it was suggested that a spatial and functional division in nitrogen absorption and NH(4)(+) assimilation between basal and apical leaf areas exists, ensuring that the majority of nitrogen available inside the tank is quickly used by bromeliad`s leaves. (C) 2011 Elsevier GmbH. All rights reserved.

Strong quenching of chlorophyll fluorescence in the desiccated state in three poikilohydric and homoiochlorophyllous moss species indicates photo-oxidative protection on highly light-exposed rocks of a tropical inselberg

The three poikilophydric and homoiochlorophyllous moss species Campylopus savannarum (C. Muell.) Mitt., Racocarpus fontinaloides (C. Muell.) Par. and Ptychomitrium vaginatum Besch. grow on sun-exposed rocks of a tropical inselberg in Brazil subject to regular drying and wetting cycles. Effective photo-oxidative protection in the light-adapted desiccated state in all three species is achieved by a reduction of ground chlorophyll fluorescence, F, to almost zero. Upon rewatering, the kinetics of the recovery of F in air dry cushions to higher values is very fast in the first 5min, but more than 80min are needed until an equilibrium is reached gradually. The kinetics were not different between the three species. The three moss species, have a distinct niche occupation and form a characteristic zonation around soil vegetation islands on the rock outcrops, where C. savannarum and R. fontinaloides form an inner and outer belt, respectively, around vegetation islands and P vaginatum occurs as small isolated cushions on bare rock. However, they were not distinguished by the reduction of F in the dry state and the rewetting recovery kinetics and only slightly different in their photosynthetic capacity. Stable isotope ratios (delta C-13, delta N-15) indicate that liquid films of water limiting diffusion of CO2 are important in determining carbon acquisition and suggest that limitation of CO2 fixation by water films must be more pronounced over time in P vaginatum than in the latter species. This is determined by both the micro site occupied and the form of the moss cushions. (c) 2007 Elsevier GmbH. All rights reserved.

How does a riverine setting affect the lifestyle of shellmound builders in Brazil?

The contact of inland and coastal prehistoric groups in Brazil is believed to have been restricted to regions with no geographical barrier, as is the case in the Ribeira de Iguape valley. The inland osteological collection from the riverine shellmound Moraes (5800-4500 BP) represents a unique opportunity to test this assumption for this region. Despite cultural similarities between riverine and coastal shellmounds, important ecological and site distribution differences are expected to impact on lifestyle. The purpose of this study is thus to document and interpret health and lifestyle indicators in Moraes in comparison to coastal shellmound groups. Specifically we test if the rare evidence of fish and mollusc remains in the riverine shellmound led to (a) higher caries rates and (b) lower auditory exostosis frequency and (c) if the small size of the riverine shellmound translates into reduced demographic density and thus rarity of communicable infectious diseases. Of the three hypotheses, (a) was confirmed, (b) was rejected and (c) was partly rejected. Bioanthropological similarities between Moraes and coastal shellmounds include auditory exostoses with equally high frequencies; significantly more frequent osteoarthritis in upper than in lower limbs; cranial and dental morphological affinities and low frequencies of violent trauma. However, there are also important differences: Moraes subsisted on a much broader protein diet and consumed more cariogenic food, but showed a stature even shorter than coastal groups. Thus, despite the contact also suggested by treponematoses in both site types, there was enough time for the people at the riverine site to adapt to local conditions. (c) 2008 Elsevier GmbH. All rights reserved.

On spermiogenesis, sperm cell morphology and accompanying secretions of Copidognathus (Acari: Halacaridae)

The genus Copidognathus includes one-third of the species of Halacaridae described to date. This article describes spermiogenesis, sperm cell morphology and accompanying secretions from three species of Copidognathus. Initial spermatids have electron-dense cytoplasm with scattered mitochondria, a well-developed Golgi body, and nuclei with patches of heterochromatin. The cytoplasm and nuclei of these cells undergo intense swelling. The second spermatids are large electron-translucent cells, with small mitochondria in row along the remains of the endoplasmatic reticulum. In the succeeding stage, most of the cytoplasmatic structures and mitochondria have disappeared or have undergone profound transformations. Nuclei and cells elongate and chromatin begins to condense near the nuclear envelope. An acrosomal complex appears at the tip of the nucleus. The acrosomal filament is thick and runs the entire length of the nucleus. Plasmalemmal invaginations at the cell surface give rise to tubules filled with an electron-dense material. Sperm cell maturation is completed in the ventral portion of the germinal part, near the testicular lumen. As a final step in spermiogenesis, cytoplasm of the last spermatid undergoes a moderate condensation and the cariotheca disappears. Mature sperm cells were found in a matrix of ""simple"" and ""complex"" corpuscles, the latter consisting of flattened, spindle-shaped secreted bodies. Rather than in individual sperm aggregates, spermatozoa were contained in a single droplet inside the vas deferens, on a large secretion mass, structured as rows of platelets sunk in a fine grained matrix. Each mature sperm cell is covered by a thick secreted coat. In contrast to the genera Rhombognathus and other Actinotrichida, Copidognathus displays a set of features that must be regarded as apomorphic. The absence of usual mitochondria, the presence of electro-dense tubules and secretions similar to those present in Thalassarachna and Halacarellus, and the pattern of nuclear condensation are possibly shared apomorphies with these latter genera. (C) 2010 Elsevier GmbH. All rights reserved.

Scale architecture of the palmar and plantar epidermis of Polychrus acutirostris Spix, 1825 (Iguania, Polychrotidae) and its relationship to arboreal locomotion

In tetrapod squamates, the diversity of micro-ornamentations of the epidermis of the contact areas of hands and feet is generally associated with constraints and modalities related to locomotion. Polychrus acutirostris is a medium-sized lizard that occurs in open heterogeneous habitats in South America, such as the cerrados, caatingas, and fallow lands. It progresses slowly on branches of various diameters in its arboreal environment. It can also move more rapidly on the ground. The hands and feet are prehensile and may be considered an adaptation for grasping and climbing. Epidermal surfaces from the palmar and plantar areas of the hands and feet of P. acutirostris were prepared for SEM examination, and studied at various magnifications. They show three major levels of complexity: (1) scale types, organized in gradients of size and imbrication, (2) scalar ornamentations, organized by increasing complexity and polarity, and (3) presence of Oberhautchen showing typically iguanian honeycomb micro-ornamentations. The shape and surface structure of the scales with their pattern of micro-ornamental peaks, which improve grip, and the grasping hands and feet indicate that P. acutirostris is morpho-functionally specialized for arboreality. (C) 2009 Elsevier GmbH. All rights reserved.

Locomotion patterns in two South American gymnophthalmid lizards: Vanzosaura rubricauda and Procellosaurinus tetradactylus

We quantified gait and stride characteristics (velocity, frequency, stride length, stance and swing duration, and duty factor) in the bursts of locomotion of two small, intermittently moving, closely related South American gymnophthalmid lizards: Vanzosaura rubricauda and Procellosaurinus tetradactylus. They occur in different environments: V rubricauda is widely distributed in open areas with various habitats and substrates, while P. tetradactylus is endemic to dunes in the semi-arid Brazilian Caatinga. Both use trot or walking trot characterised by a lateral sequence. For various substrates in a gradient of roughness (perspex, cardboard, sand, gravel), both species have low relative velocities in comparison with those reported for larger continuously moving lizards. To generate velocity, these animals increase stride frequency but decrease relative stride length. For these parameters, P. tetradactylus showed lower values than V rubricauda. In their relative range of velocities, no significant differences in stride length and frequency were recorded for gravel. However, the slopes of a correlation between velocity and its components were lower in P. tetradactylus on cardboard, whereas on sand this was only observed for velocity and stride length. The data showed that the difference in rhythmic parameters between both species increased with the smoothness of the substrates. Moreover, P. tetradactylus shows a highly specialised locomotor strategy involving lower stride length and frequency for generating lower velocities than in V. rubricauda. This suggests the evolution of a central motor pattern generator to control slower limb movements and to produce fewer and longer pauses in intermittent locomotion. (c) 2008 Elsevier GmbH. All rights reserved.

The gynoecium of male Anchietea pyrifolia (Violaceae): Preserved structure with a new function

Structure of inflorescences and flowers and flowering behaviour are reported for the woody liana Anchietea pyrifolia (Violaceae) from Brazil. The specimen studied is grown for some decades now in the greenhouses of Halle Botanical Garden and turned out unisexually male, which adds a further example of dioecism to the family Violaceae, in which this type of sex distribution is rarely encountered. The flowers are exceptional also for the strongly asymmetric anterior petal, which represents a rare case of a species with enantiomorphic flowers pollinated by Lepidoptera. They have a fully developed gynoecium with a complicated architecture comparable to the pistil of bisexual Violaceae flowers, though without ovules. The style head is capable to release viscose liquid on tactile stimulation or pressure, which is known to act as pollen-gathering mechanism in bisexual Violaceae species with usually dry pollen and buzz-pollination. This function has switched in male A. pyrifolia to a mechanism for efficient pollen release mediated by insect pollinators from its short-lived flowers. (C) 2009 Elsevier GmbH. All rights reserved.